*********A*********
By Authority Of
THE UNITED STATES OF AMERICA
Legally Binding Document
By the Authority Vested By Part 5 of the United States Code § 552(a) and
Part 1 of the Code of Regulations § 51 the attached document has been duly
INCORPORATED BY REFERENCE and shall be considered legally
binding upon all citizens and residents of the United States of America.
HEED THIS NOTICE : Criminal penalties may apply for noncompliance.
Document Name: AOAC: Official Methods of Analysis (Volume 1)
CFR Section(s): 9 CFR 318.19(b)
Standards Body: AOAC International
OFFICIAL
ETHODS of
ANALYSIS
15 th Edition, 1990
ASSOCIATION
of OFFICIAL
ANALYTICAL
CHEMISTS
Agricultural Chemicals;
Contaminants; Drugs
VOLUME ONE
OFFICIAL
METHODS OF ANALYSIS
OF THE
ASSOCIATION OF OFFICIAL
ANALYTICAL CHEMISTS
Edited by Kenneth Helrich
FIFTEENTH EDITION, 1990
Published by the
Association of Official Analytical Chemists, Inc.
Suite 400
2200 Wilson Boulevard
Arlington, Virginia 22201 USA
Copyright © 1920, 1925, 1931, 1936, 1940, 1945, 1950, 1955, 1960, 1965
by the Association of Official Agricultural Chemists
and 1970, 1975, 1980, 1984, 1990
by the Association of Official Analytical Chemists
The methods of the Association were also copyrighted in 1916, when they were
published in the Journal of the Association of Official Agricultural Chemists,
All rights reserved. No part of this book may be reproduced in any form or by
any means without the written permission of the Association.
Printed in the United States of America
Printed on acid-free paper
Library of Congress Catalog Card Number 85-647589
ISBN 0-935584-42-0
ISSN 0066-96 IX
A copy of the 15th edition of this publication is on file with the Office of the
Federal Register. U.S. Government Agencies may apply to the Director of the
Office of the Federal Register for approval to incorporate this edition by reference
in their regulations. The procedures that Federal agencies must follow in applying
for the Director's approval are in Title 1, Part 51, of the Code of Federal
Regulations .
iv
Important Notices
to Librarians and ASI Users
of this Edition
FREE SUPPLEMENTS
Purchasers of this volume will receive the annual supplements of Changes in Official Methods of Analysis without
additional charge only by removing the card inserted after p. 40, filling in the specific name and address to which
the supplements should be sent, and returning this card to the Association. Address must include postal code or delivery
cannot be assured.
SURPLUS METHODS
Neither the 10th, 11th, 12th, 13th, or 14th editions should be destroyed upon appearance of the 15th edition. They
contain surplus methods which are not reprinted in the 15th edition. See page xvii for the definition of surplus methods.
If methods marked with this symbol, *, continue to be used and therefore should be retained in full, please notify
the Association.
USE OF METHODS
Analytical methods and procedures included in this volume are those which AOAC members have evaluated and
validated through collaborative studies and appropriate similar techniques to give accurate and reproducible analytical
results on the matrix and analyte to which the method is stated to apply, provided the analysis is conducted by a
competent analyst as written. No warranty, implied or expressed, is made by the Association on methods described
or products mentioned. The mention of commercial trade names does not imply endorsement by AOAC or its members
over similar products that might be suitable. AOAC and its members who have aided in development and validation
of methods enclosed assume no responsibility for any economic, personal injury, or other damage that may occur to
individuals or organizations because of use of such methods.
EQUIVALENT COMMERCIAL PRODUCTS
AOAC recognizes that the instrumentation and laboratory supplies stated in these published methods may have
equivalents which will produce analytical results having the same statistical performance. Names of manufacturers
and suppliers and trade names are furnished solely as a matter of identification and convenience within the context
of the way each method was developed within the originator's laboratories, without implication of AOAC promotion,
approval, endorsement, or certification. The same or equivalent products, instruments, supplies, apparatus, or reagents
available from suppliers other than those named or other brands from other sources may serve equally well if proper
validation indicates that their use is satisfactory.
INQUIRIES
Inquiries regarding procedures published in this book should be directed to AOAC, Executive Director, 2200 Wilson
Blvd, Arlington, VA 22201-3301 USA. Telephone (703) 522-3032. Facsimile (703) 522-5468.
Inquiries regarding purchase of Official Methods of Analysis, supplements ("Changes in Official Methods"), Journal
of the AOAC, or other AOAC publications should be directed to AOAC, Fulfillment Coordinator, 2200 Wilson Blvd,
Arlington; VA 22201-3301 USA. Telephone (703) 522-3032. Facsimile (703) 522-5468.
COMMENTS ON METHODS
AOAC adopts methods that show by their performance data, obtained through the collaborative study, what can be
expected of them. As analysts use AOAC methods, they generate additional information and data concerning the
applicability, specificity, sensitivity, and accuracy of the methods. Analysts are requested to advise AOAC about their
experiences with official methods published in this book. In particular, analysts should notify AOAC of problems in
the performance of any method that indicate the method should be revised or restudied. Direct comments to AOAC,
Assistant Executive Director, Technical Services and Development, 2200 Wilson Blvd, Arlington, VA 22201-3301
USA. Telephone (703) 522-3032. Facsimile (703) 522-5468.
Preface to the 15th Edition
The most obvious change in this new edition of Official Methods of Analysis of the AOAC is the new format,
splitting the original single volume into two volumes containing Agricultural Chemicals, Contaminants, and Drugs in
Volume I, and Food Composition in Volume II. Extensive discussions, user polls, and committee deliberations re-
garding the most useful and desirable form for publication of the 15th Edition led to the decision to make this change.
While this has necessitated the repetition of a few items such as the index and the safety chapter, the convenience of
smaller volumes with a logical division of subject matter is a definite advantage. The two volume arrangement also
allows for more manageable growth as the number of validated methods increases.
In actual content, the most striking change in this new edition is the assignment of permanent numbers to all official
methods. This tedious and time consuming task was undertaken primarily because, as Editor William Horwitz stated
in the preface to the Thirteenth Edition in 1980, "Users expressed a desire for a system that will keep the same
reference number of a given method from edition to edition." There are significant advantages to a permanent num-
bering system. Since AOAC methods are cited worldwide in laws and regulations at every level of government, in
definitions of standards of identity, and in public and private specifications and contracts, it is practical and highly
desirable to have a single, unchanging number for any method. Permanent numbers will reduce citation errors and
simplify citation by eliminating the necessity to specify editions in some instances. The publication of future editions
will not be encumbered by the need to keep track of changing numbers for existing, unchanged methods. There is
also the advantage of desirable consistency for electronic databases.
Permanent numbers are based on the year the method first appeared in "Changes in Official Methods of Analysis"
in the Journal. The year determines the first three numbers with the next digits being simply the sequence in which
the methods were adopted in a given year. For example, the first method adopted in 1988 and published in "Changes
in Official Methods of Analysis" in 1989 would be given the number 989.01. The year of adoption was not researched
for methods adopted before 1960. For those, the numbers are based on the date of the first reference, or, if that was
not available, on the year the method first appeared in Official Methods of Analysis of the AOAC. An index to the
new numbers is included to facilitate locating methods when only the method number is known. References for the
more recent methods in the 15th Edition have been verified, corrected, and brought up to date so that the user can
more readily find the original work that resulted in adoption of the method. The list of suppliers, as well as supplier
references in each method, has also been revised and updated.
Method performance data appear at the beginning of methods adopted as part of "Changes in Official Methods of
Analysis" in 1989. Previously published method performance data (14th Edition) have been deleted because of a
change in the procedure for calculation. All future new methods will have the method performance section included,
using the performance parameters that were adopted by the AOAC Board of Directors in 1988. Method performance
data are generated from the collaborative study results.
The addition of about 150 new methods to the 15th Edition continues to respond to the AOAC mandate to keep
pace with the practical needs of regulatory and research chemists and microbiologists. Some previously adopted meth-
ods have been expanded in scope; some have had efficiency or accuracy improved. Additional methods have been
declared surplus and omitted from the present Edition in instances where they were no longer sufficiently used to
warrant reprinting. Again, the user is asked to preserve previous editions for the rare instance when a surplus method
may be needed.
Liquid chromatography (LC) and gas chromatography (GC) have continued to be the most popular and useful
techniques of analysis. A variety of detectors are still being utilized, along with internal standards. In addition to
sophisticated modern instrumentation, such classical techniques as gravimetric analysis, distillation and physical sep-
arations, and Kjeldahl nitrogen determinations are still yielding new, needed methods.
Among the most innovative techniques are the additions to the chapter on Microbiological Methods. Over twenty
new methods have been added to this chapter. These include DNA colony hybridization, enzyme immunoassay, mi-
crobial receptor assay, and immunodiffusion methods. Many new methods utilize beads, pretreated pectin gel films,
dry rehydratable films, and hydrophobic grid plates. Sources of the kits and pertinent information on components are
provided. In some instances, generic substitutions are possible for kit components.
As always, thanks are due to the many individuals who worked so diligently and carefully to maintain the quality
of the Official Methods of Analysis of the AOAC. These include the Associate Referees, General Referees, collabo-
rators, and Methods Committee members who researched, perfected, validated, and reviewed each method. The Gen-
eral Referees, beyond the call of duty, contributed their services as Chapter Associate Editors, arranging and reviewing
chapters where their expertise was an invaluable asset. The AOAC Official Methods Board, Editorial Board, and
Board of Directors provided the guidance throughout the duration of this project. The AOAC Scientific Publications
staff did a heroic job from the very beginning in editing methods, keeping the methods publication on schedule,
handling the unending details of actual publication, and renumbering over 1,000 pages of methods with innumerable
cross references.
Kenneth Helrich
Editor, Official Methods of Analysis
Contents
Volume S
PAGE
Important Notices v
Preface vii
About the Association xiii
Guide to Method Format xiv
Definitions of Terms and Explanatory
Notes xv
Collaborative Study Procedures xxii
CHAPTER
1. Agricultural Liming Materials 1
Calcium Silicate Slags 3
Gravimetric Elemental Analyses 4
Chelometric Elemental Analyses 5
Colorimetric Elemental Analyses 6
2. Fertilizers 9
Water 11
Phosphorus 12
Nitrogen 17
Potassium 23
Other Elements 27
Peat 36
3. Plants 40
Metals 40
Individual Metals 43
Nonmetals 50
Other Constituents 58
Pigments 62
Tobacco 64
4. Animal Feed 69
Protein 70
Urea 76
Nitrogen 77
Fat 79
Fiber 80
Sugars 83
Minerals 84
Microscopy 88
5. Drugs in Feeds 91
Chemical Methods 91
Microbiological Methods
for Antibiotics 115
Chemical Methods for Antibiotics 129
PAGE
Common and Chemical Names
of Drugs 132
6. Disinfectants 133
Phenol Coefficient Methods 133
Use-Dilution Methods 135
Other Tests 137
7. Pesticide Formulations 147
General Methods 147
Inorganic and Organometallic
Pesticides and Adjuvants 153
Pesticides Related to Natural
Products and Their Synergists 164
Organohalogen Pesticides 174
Thiophosphorus and Other
Organophosphorus Pesticides 197
Carbamate, Substituted Urea,
and Triazine Pesticides 212
Miscellaneous Pesticides 223
Common and Chemical Names
of Pesticides 230
8. Hazardous Substances 232
9. Metals and Other Elements
at Trace Levels in Foods 237
Multielement Methods 237
Single Element Methods 242
10. Pesticide and Industrial
Chemical Residues 274
Multiresidue General
Considerations 274
Organochlorine Residues 283
Organophosphorus Residues 286
Fumigant Residues 290
Carbamate Residues 291
Individual Residues 294
Common and Chemical Names
of Pesticides 310
11. Waters; and Salt 312
Waters 312
Salt 334
12. Microchemical Methods 337
PAGE
13. Radioactivity 349
14. Veterinary Analytical Toxicology 356
15. Cosmetics 359
General Methods 359
Deodorants and Antiperspirants 361
Dipilatories 365
Face Powders 365
Hair Preparations 366
Suntan Preparations 367
16. Extraneous Materials: Isolation 369
General 369
Beverages and Beverage
Materials 373
Dairy Products 375
Nuts and Nut Products 379
Grains and Their Products 380
Baked Goods 385
Breakfast Cereals 387
Eggs and Egg Products 388
Poultry, Meat, and Fish and Other
Marine Products 389
Fruits and Fruit Products 391
Snack Food Products 393
Sugars and Sugar Products 393
Vegetables and Vegetable
Products 394
Spices and Other Condiments 397
Miscellaneous 406
Animal Excretions 407
Mold and Rot Fragments 416
Fruits and Fruit Products 418
Vegetables and Vegetables
Products 419
General References 423
17. Microbiological Methods 425
Cross Reference Tables 425
Eggs and Egg Products 427
Chilled, Frozen, Precooked, or
Prepared Foods, and Nutmeats 429
Coliforms 434
Escherichia coll 438
Staphylococcus 449
Sterility (Commercial) of Foods
(Canned, Low Acid) 455
Clostridium 459
Bacillus 464
Salmonella 467
Vibrio 492
Miscellaneous 494
Somatic Cells 496
18. Drugs: Part 1 498
Solvents 498
Halogenated Drugs 499
PAGE
Inorganic Drugs 501
Antihistamines 512
Alkanolamines 515
Phenethylamines 520
Aminobenzoates 521
Synthetics 525
Microchemical Tests 533
Microscopy 541
Miscellaneous 542
Common and Chemical
Names of Drugs 545
19. Drugs: Part II 548
Acids 548
Phenolic Drugs 549
Analgesics and Antipyretics 553
Hypnotics and Sedatives 558
Anticoagulants 565
Sulfonamides 567
Thiazides 570
Other Sulfur-Containing Drugs 573
Common and Chemical
Names of Drugs 578
20. Drugs: Part III 579
Opium Alkaloids 579
Tropane Alkaloids 583
Xanthine Alkaloids 584
Ipecac Alkaloids 584
Ephedra Alkalids 586
Ergot Alkaloids 588
Physostigmine Alkaloids 590
Chinchona Alkaloids 592
Rauwolfia Alkaloids 593
Other Alkaloids 598
Digitalis 599
Other Natural Products 602
Common and Chemical
Names of Drugs 606
21. Drugs: Part IV 607
Natural Estrogens 607
Synthetic Estrogens 609
Progestational Steroids 612
Adrenocortico Steroids 613
Thyroid 618
Common and Chemical
Names of Drugs 619
22. Drugs: Part V 620
Common and Chemical Names of Drugs 624
23. Drugs and Feed Additives in Animal
Tissues 625
Common and Chemical Names of
Drugs 636
PAGE
24. Forensic Sciences 637
Appendix: Standard Solutions and Certified
Reference Materials 640
Appendix: Laboratory Safety 649
Appendix: Reference Tables 656
PAGE
Appendix: Guidelines for Collaborative Study
Procedure to Validate
Characteristics of a Method
of Analysis 673
Subject Index 1-1
Method Number Index 1-55
Volume II
PAGE
Important Notices v
Definitions of Terms and Explanatory
Notes xi
Guide to Method Format xviii
CHAPTER
25. Baking Powders and Baking Chemicals . . 685
26. Distilled Liquors 690
Spirits 690
Cordials and Liqueurs . . . . 704
27. Malt Beverages and Brewing Materials . . 708
Beer 708
Barley 723
Malt 723
Cereal Adjuncts 730
Hops . . .! 731
Brewing Sugars and Sirups 733
Wort 734
Yeast 735
Brewers' Grains 737
28. Wines 739
General 739
Preservatives 749
Flavors 750
29. Nonalcoholic Beverages and Concentrates 751
30. Coffee and Tea 757
Green Coffee 757
Roasted Coffee 757
Tea 761
31. Cacao Bean and Its Products 763
General 763
Shell 764
Chocolate Liquor 770
Fat 770
Other Constituents 772
PAGE
32. Cereal Foods 777
Wheat Flour 777
Wheat, Rye, Oats, Corn, Buckwheat,
Rice, Barley, and Soybeans and Their
Products Except Cereal Adjuncts 788
Bread 790
Baked Products 794
Macaroni, Egg Noodles,
and Similar Products 796
33. Dairy Products . . 802
Sampling 802
Milk 804
Cream 831
Evaporated and Condensed Milk 833
Dried Milk, Nonfat Dry Milk,
and Malted Milk 834
Butter 836
Cheese 840
Ice Cream and Frozen Desserts 850
34. Eggs and Egg Products 853
35. Fish and Other Marine Products 864
36. Flavors 890
Vanilla Extract and Its Substitutes 890
Lemon, Orange, and Lime Extracts,
Flavors and Oils 898
Almond Extract 903
Cassia, Cinnamon, and Clove Extracts. . . 905
Flavor Extracts and Toilet Preparations . . 905
37. Fruits and Fruit Products 910
38. Gelatin, Dessert Preparations, and Mixes 929
39. Meat and Meat Products 931
40. Nuts and Nut Products 949
PAGE
41. Oils and Fats 951
42. Vegetable Products, Processed 987
Canned Vegetables 987
Dried Vegetables 994
Frozen Vegetables 995
43. Spices and Other Condiments 999
44. Sugars and Sugar Products 1010
Sugars and Sirups 1010
Molasses and Molasses Products 1021
Confectionary 1024
Honey 1025
Maple Sap, Maple Sirup, Maple
Sirup Products 1034
Sugar Beets 1039
Corn Sirups and Sugars 1039
45. Vitamins and Other Nutrients 1045
Chemical Methods 1045
Microbiological Methods 1080
Bioassay Methods 1091
Nutritionally Related Components 1099
Infant Formula 1 106
46. Color Additives 1115
Separation and Identification of Color
Additives in Foods, Drugs,
and Cosmetics 1115
Intermediates 1 126
Subsidiary and Lower Sufonated Dyes ... 1132
Metals and Other Elements 1132
PAGE
Halogens 1135
Miscellaneous 1 1 36
47. Food Additives: Direct 1 137
Antioxidants 1 137
Chemical Preservatives 1 141
Emulsifying Agents . 1 163
Enzymes 1 1 64
Miscellaneous 1 165
48. Food Additives: Indirect 1 176
49. Natural Poisons 1 1 84
Mycotoxins-General 1 1 84
Aflatoxins 1185
Aflatoxin M 1199
Deoxynivalenol 1205
Ochratoxins 1207
Patulin 1209
Sterigmatocystin 1210
Zearalenone 1211
Marine Toxins 1213
Phytotoxins 1213
Appendix: Standard Solutions and Certified
Reference Materials 1214
Appendix: Laboratory Safety 1223
Appendix: Reference Tables 1230
Subject Index 1-1
Method Number Index 1-55
About the Association
PURPOSE AND FUNCTION
AWARDS
The primary objectives of the Association of Official Ana-
lytical Chemists (AOAC) are to obtain, improve, develop, test,
and adopt uniform, precise, and accurate methods for the anal-
ysis of foods, drugs, feeds, fertilizers, pesticides, water, or
any other substances affecting public health and safety, eco-
nomic protection of the consumer, or quality of the environ-
ment; to promote uniformity and reliability in the statement of
analytical results; to promote, conduct, and encourage research
in the analytical sciences related to agriculture and public health
and the regulatory control of commodities in these fields; and
to afford opportunity for discussion of matters of interest to
scientists engaged in relevant pursuits.
AOAC itself maintains no laboratories, conducts no anal-
yses, performs no tests. The actual work of devising and test-
ing methods is done by members of AOAC in their official
and professional capacities as staff scientists of federal, state,
provincial, and municipal regulatory agencies, experiment sta-
tions, colleges and universities, commercial firms, and con-
sulting laboratories.
AOAC coordinates these scientific studies, receives and
evaluates the results, gives official sanction to acceptable
methods, and publishes and disseminates the methods.
The reliability of methods of analysis is more important than
ever before. Regulatory agencies need reliable, reproducible,
and practical methods to enforce laws and regulations. Indus-
try needs reliable methods to meet compliance and quality con-
trol requirements. Few organizations in the world are devoted
primarily to testing and validating analytical methods through
interlaboratory collaborative studies — as is AOAC.
MEETINGS
The Annual International Meeting is the focal point of
AOAC's yearly work. Here, members have opportunities to
exchange ideas with colleagues from all over the world, and
to update their technical knowledge at scientific sessions and
symposia, exhibits, and short courses.
The regional section program provides AOAC-affiliated lo-
cal or regional scientific meetings, workshops, short courses,
and other activities for laboratory analysts. Each regional sec-
tion is organized by a local volunteer committee.
COOPERATIVE ACTIVITIES
AOAC has established joint committees, liaison, and rep-
resentation with numerous scientific organizations worldwide .
Thus, methods are often developed in cooperation with other
standards- setting bodies. AOAC liaison representatives for
contact outside North America are the following: Derek Ab-
bott, 33 Agates Ln, Ashtead, Surrey KT21 2ND, England;
Lars Appelqvist, Swedish University of Agricultural Sciences,
Dept of Food Hygiene, S 750 07 Uppsala, Sweden; and Mar-
greet Lauwaars, PO Box 153, 6720 AD Bennekom, The Neth-
erlands.
The awards program of AOAC includes the following: The
Scholarship Award is given each year to a student intending
to do further study or work in an area important to public health
or agriculture.
The Fellow of the AOAC Award is given to selected mem-
bers in recognition of at least 10 years of meritorious service
to the Association as referees and/or committee members.
The Harvey W. Wiley Award, honoring the "father" of the
U.S. Pure Food and Drug Act and a founder of AOAC, is
presented each year to a scientist or group of scientists who
have made outstanding contributions to analytical methodol-
ogy in an area of interest to AOAC. The $2500 award is sup-
ported by the Wiley Fund.
PUBLICATIONS
Official Methods of Analysis includes full details of official
methods but no descriptive or interpretative material or tables
of data. However, AOAC publishes the Journal of the AOAC,
which contains research articles and reports of the develop-
ment, validation, and interpretation of analytical methods, and
all collaborative study results. Journal contributors and its
readers represent the worldwide analytical science community.
The Journal is a forum for the exchange of information among
methods researchers. The Journal also records the transactions
of the Annual International Meeting, including committee and
referee reports, lists of officers, referees, and committee mem-
bers, and all official actions of the Association, including newly
adopted methods. The Association publishes a variety of other
books, manuals, video tapes, and symposium proceedings of
interest to analytical scientists.
MEMBERSHIP
The organization of AOAC consists of the members; the Board
of Directors, a governing body concerned with administration
and policy making; Official Methods Board; Editorial Board;
special standing committees and other groups concerned with
development of methods and general activities; and the head-
quarters staff which carries out the publications program and
manages the Association.
The AOAC Bylaws provide for individual members and sus-
taining members. Chemists, microbiologists, and other sci-
entists engaged in analysis or analytical research related to ag-
riculture and public health, and employed by a college or
university, any agency of a local, state, provincial, or national
government, or firm or industry concerned with commodities
or substances of interest to AOAC may be members. Sustain-
ing members are government agencies or private industries that
provide financial support to AOAC.
The Referee, published 12 times yearly, is sent free to all
members, and contains news about methods, collaborative
studies, meetings, publications, AOAC people, board and
committee activities, regional sections, and other items of in-
terest. All members also receive the membership directory,
issued annually.
XIII
GUIDE TO METHOD FORMAT*
Unique number identifies
method by year of
adoption or first
appearance in Official
Methods of Analysis
(older methods).
980= first action 1980;
.06= sequence of
adoption in 1980.
Chemical names of
pesticides and drugs are
given at end of pertinent
chapter.
Cautionary notes refer
to Safety Chapter.
Addresses for suppliers
frequently cited
throughout book are
listed in "Definitions of
Terms and Explanatory
Notes."
Letters identify main
sections for ease of
citation and cross-
referencing.
Calculation symbols are
identified and show
correct units.
Chemical Abstracts
Service Registry
Number. A unique
identifier that may be
used to search a number
of data-retrieval systems,
-980.06 Captan in Pesticide Formulations
Liquid Chromatographic Method -
First Action 1980
Final Action 1982
AOAC-CIPAC Method
(Method is suitable for tech. captan and formulations with captan as-
only active ingredient.)
A. Principle
— Captan is extd from inerts with soln of diethyl phthalate in CH 2 Cl 2 .
Soln ischromatgd on microparticulate silica gel column, using CH 2 C1 2 _
as mobile phase. Ratio of captan peak ht to diethyl phthalate peak ht is
calcd from UV response and compared to std material for quantitation .
(Caution: See safety notes on pipets and pesticides.)
8. Apparatus and Reagents
(a) Liquid chromato graph. — Able to generate over 1000 psi and ■
measured at 254 nm,
— (d) Diethyl phthalate.— EM Science No. 1295.
(e) Reference std captan. — Chevron Chemical Co., PO Box 4010,
Richmond, CA 94804.
C. Preparation of Standard
(a) Internal std soln. — 0.312 mg diethyl phthalate/mL. Weigh
ca 156 mg diethyl phthalate and transfer to 500 mL vol. flask. Dil. to
vol. with same CH 2 C1 2 to be used for mobile . . . within 20%.
-D. Preparation of Sample
Accurately weigh sample expected to contain 40 mg captan into
glass bottle. Pipet in 50 mL internal std soln. Place on mech. shaker
15 min. Centrf. and filter supernate thru glass fiber paper. Prep, fresh
sample daily.
£. Determination
Adjust operating parameters to cause captan to elute in 4-6 min.
Maintain all parameters const thruout analysis. Typical values are:
flow rate, 2.5 rnLCH 2 Cl 2 /min, max.; pressure, ca 800 psi; chart speed -
0.2'7min; mobile phase . . . immediately preceding and following
sample injections must agree to within ± 2% of their mean. If not,
repeat detn.
F. Calculation
Measure peak hts to 3 significant figures, and calc. ratio for each
injection. Average 4 std ratios, and the 2 sample ratios.
% Captan = (R/R') x (W'(W) x P
where R ~ av. sample ratio (captan peak ht/diethyl phthalate peak ht);
R' = av. std ratio (captan peak ht/diethyl phthalate peak ht); W — mg
sample; W ~ mg std, andP - % purity of std.
Ref.: JAOAC 63, 1231(1980).
-CAS-133-06-2 (captan)
Method head may
include analyte and
matrix, type of method,
official status,
cooperating organization.
Applicability
statement— limitations
on use of method or other
information.
Scientific basis for
method of analysis.
Specifications for
necessary laboratory
apparatus and reagent
preparations. See also
"Definitions of Terms and
Explanatory Notes."
Method may be divided
into several descriptive
sections.
Abbreviations used
throughout method are
defined in "Definitions of
Terms and Explanatory
Notes."
References direct the
user to the published
collaborative study and
any subsequent revisions
in the method. Other
informative references
may be included.
* Method shown is incomplete to allow space for description.
Definition of Terms and Explanatory Notes
Official Methods
(1) Official methods are designated first action or final ac-
tion, and, in a few cases, procedures. A first action method
has undergone collaborative study, has been recommended by
the appropriate General Referee and Methods Committee, has
been approved interim first action by the chairman of the Of-
ficial Methods Board, and has been adopted official by the
Association members at an annual meeting. A method may be
adopted final action a minimum of 2 years after it has been
adopted first action, and, again, after it has been recommended
by the appropriate General Referee and Methods Committee
and voted on by the Association members at an annual meet-
ing.
A sampling or sample preparation procedure or other type
of procedure for which an inter! aboratory collaborative study
is impractical may be adopted, as above, as a procedure.
All methods in this book — first action, final action, or pro-
cedure — are official methods of AOAC.
Reagents
(2) Term "H 2 CT means distilled water, except where oth-
erwise specified, and except where the water does not mix
with the determination, as in "H 2 bath."
(3) Term "alcohol" means 95% ethanol by volume. Alco-
hol of strength x% may be prepared by diluting x mL 95%
alcohol to 95 mL with H 2 0. Absolute alcohol is 99.5% by
volume. Formulas of specially denatured alcohols (SDA) used
as reagents are as follows:
SDA No.
100
parts alcohol plus
1
5
wood alcohol
2-B
0.5
benzene or rubber
hydrocarbon solv.
3-A
5
MeOH
12-A
5
benzene
13-A
10
ether
23-A
10
acetone
30
10
MeOH
"Reagent" alcohol is 95 parts SDA 3-A plus 5 parts isopro-
panol.
(4) Term "ether" means ethyl ether, peroxide-free by fol-
lowing test: To 420 mL ether in separator add 9.0 mL 1%
NH4VO3 in H 2 S0 4 (1 + 16). Shake 3 min and let separate.
Drain lower layer into 25 mL glass-stoppered graduate, dilute
to 10 mL with H 2 S0 4 (1 + 16), and mix. Any orange color
should not exceed that produced by 0.30 mg H 2 2 (1 mL of
solution prepared by diluting 1 mL 30% H 2 2 to 100 mL with
H 2 0) and 9.0 mL 1% NH4VO3 in H 2 S0 4 (1 + 16). Peroxides
may be eliminated by passing <700 mL ether through 10 cm
column of Woelm basic alumina in 22 mm id tube.
(5) Reagents listed below, unless otherwise specified, have
approximate strength stated and conform in purity with Rec-
ommended Specifications for Analytical Reagent Chemicals of
the American Chemical Society:
Assay
Sulfuric acid 95.0-98.0% H 2 S0 4
Hydrochloric acid 36.5-38.0% HCI
Nitric acid 69.0-71 .0% HNQ 3
Fuming nitric acid >90% HN0 3
Acetic acid >99.7% HC 2 H 3 2
Hydrobromic acid 47.0-49.0% HBr
Ammonium hydroxide 28-30% NH 3
Phosphoric acid >85% H 3 P0 4
Where no indication of dilution is given, reagent concentration
is the concentration given above.
(6) All other reagents and test solutions, unless otherwise
described in the text, conform to requirements of the American
Chemical Society. Where such specifications have not been
prepared, use highest grade reagent. When anhydrous salt is
intended, it is so stated; otherwise the crystallized product is
meant.
(7) Unless otherwise specified, phenolphthalein (phthln) used
as indicator is 1% alcohol solution; methyl orange is 0.1%
aqueous solution; methyl red is 0.1% alcohol solution.
(8) Directions for standardizing reagents are given in the
chapter on Standard Solutions and Certified Reference Mate-
rials.
(9) Unusual reagents not mentioned in reagent sections or
cross referenced, other than common reagents normally found
in laboratory, are italicized the first time they occur in a method.
(10) Commercially prepared reagent solutions must be
checked for applicability to specific method. They may con-
tain undeclared buffers, preservatives, chelating agents, etc.
(11) In expressions (1 + 2), (5 + 4), etc., used in connec-
tion with name of reagent, first numeral indicates volume re-
agent used, and second numeral indicates volume of H 2 0. For
example, HCI (1 + 2) means reagent prepared by mixing 1
volume of HCI with 2 volumes of H 2 0. When one of the re-
agents is a solid, expression means parts by weight, first nu-
meral representing solid reagent and second numeral H 2 0. So-
lutions for which the solvent is not specified are aqueous
solutions.
(12) In making up solutions of definite percentage, it is
understood that x g substance is dissolved in H 2 and diluted
to 100 mL. Although not theoretically correct, this convention
will not result in any appreciable error in any methods given
in this book.
(13) Chromic acid cleaning solution is prepared by (7) add-
ing 1 L H 2 S0 4 to approx. 35 mL saturated aqueous Na 2 Cr 2 7
solution; or (2) adding 2220 mL (9 lb) H 2 S0 4 to approx. 25
mL saturated aqueous Cr0 3 solution (170 g/100 mL). Re-
agents may be technical high grade. Use only after first clean-
ing by other means (e.g., detergent) and draining. Mixture is
expensive and hazardous. Use repeatedly until it is diluted or
has a greenish tinge. Discard carefully with copious amounts
of H 2 0.
(14) All calculations are based on table of international atomic
weights.
xv
Apparatus
(15) Burets, volumetric flasks, and pipets conform to the
following U.S. Federal specifications (available from General
Services Administration, Specification Activity 3F1, Wash-
ington Navy Yard, Building 197, Washington/ DC 20407):
Buret
NNN-B-00789a
May 19, 1965
Flask, vol.
NNN-F-289d
Feb. 7, 1977
Pipet, vol.
NNN-P-395d
Feb. 24, 1978
Pipet, measuring
NNN-P-35UC
July 16, 1973
See also Appendix V, "Testing of Glass Volumetric Appara-
tus," in NIST Specification Publication 260-54, "Certification
and Use of Acidic Potassium Dichromate Solutions as an Ul-
traviolet Absorbance Standard SRM935" (available from NIST,
Office of Standard Reference Materials, B3I6 Chemicals,
Gaithersburg, MD 20899).
(16) Standard taper (I) glass joints may be used instead of
stoppers where the latter are specified or implied for connect-
ing glass apparatus.
(17) Sieve designations, unless otherwise specified, are those
described in U.S. Federal Specification RR-S-366e, Novem-
ber 9, 1973 (available from General Services Administration).
Designation "100 mesh" (or other number) powder (material,
etc.) means material ground to pass through standard sieve No.
100 (or other number). Corresponding international standard
and U.S. standard sieves are given in Table 1.
(18) Term "paper" means filter paper, unless otherwise
specified.
(19) Term "high-speed blender" designates mixer with 4
canted, sharp-edge, stainless steel blades rotating at the bottom
of 4-lobe jar at 10,000-12,000 rpm, or with equivalent shear-
ing action. Suspended solids are reduced to fine pulp by action
of blades and by lobular container, which swirls suspended
solids into blades. Waring Blender, or equivalent, meets these
requirements.
(20) "Flat-end rod" is glass rod with one end flattened by
heating to softening in flame and pressing vertically on flat
surface to form circular disk with flat bottom at end.
(21) Designation and pore diameter range of fritted glass-
ware are: extra coarse, 170-220 jxm; coarse, 40-60; medium,
10-15; fine, 4-5.5; Jena designations and pore diameter are:
1, 110 ujn; 2, 45; 3, 25; 4, 8.
(22) Unless otherwise indicated, temperatures are expressed
as degrees Centigrade.
Standard Operations
(23) Operations specified as "wash (rinse, extract, etc.) with
two (three, four, etc.) 10 mL (or other volumes) portions of
H 2 (or other solvent)" mean that the operation is to be per-
formed with indicated volume of solvent and repeated with
same volume of solvent until number of portions required have
been used.
(24) Definitions of terms used in methods involving spec-
trophotometry are those given in JAOAC 37, 54(1954). Most
important principles and definitions are:
(a) More accurate instrument may be substituted for less
accurate instrument (e.g., spectrophotometer may replace col-
orimeter) where latter is specified in method. Wavelength
specified in method is understood to be that of maximum ab-
sorbance (A), unless no peak is present.
(b) Absorbance(s) (A). — Negative logarithm to base 10 of
ratio of transmittance (T) of sample to that of reference or
standard material. Other names that have been used for quan-
tity represented by this term are optical density, extinction,
and absorbancy.
(c) Absorptivity(ies) (a). — Absorbance per unit concentra-
Table 1. Nominal Dimensions of Standard Test Sieves (USA
Standard Series)
Sieve Designation
Nominal
Nominal
International
Sieve
Wire
Standard 3 U.S.A.
Opening,
Diameter,
(ISO) Standard
inches
mm
12.5
mm 6 V2 i
n. &
0.500
2.67
11.2
mm 7 / i6
in.
0.438
2.45
9.5
mm 3 /s i
n.
0.375
2.27
ao
mm 5 / ]6
in.
0.312
2.07
6.7
mm 0.265 in.
0.265
1.87
6.3
mm" 'a i
n b
0.250
1.82
5.6
mm No.
3Va
0.223
1.68
4.75
mm No.
4
0.187
1.54
4.00
mm No.
5
0.157
1.37
3.35
mm No.
6
0.132
1.23
2.80
mm No.
7
0.111
1.10
2.38
mm No.
8
0.0937
1.00
2.00
mm No.
10
0.0787
0.900
1.70
mm No.
12
0.0661
0.810
1.40
mm No.
14
0.0555
0.725
1.18
mm No.
16
0.0469
0.650
1.00
mm No.
18
0.0394
0.580
850
ixm c No.
20
0.0331
0.510
710
M,m No.
25
0.0278
0.450
600
fxm No.
30
0.0234
0.390
500
ixm No.
35
0.0197
0.340
425
ixm No.
40
0.0165
0.290
355
ixm No.
45
0.0139
0.247
300
|jim No.
50
0.0117
0.215
250
ixm No.
60
0.0098
0.180
212
^m No.
70
0.0083
0.152
180
jam No.
80
0.0070
0.131
150
fxm No.
100
0.0059
0.110
125
fxm No.
120
0.0049
0.091
106
ixm No.
140
0.0041
0.076
90
|xim No.
170
0.0035
0.064
75
jxm No.
200
0.0029
0.053
63
ixm No.
230
0.0025
0.044
53
M,m No.
270
0.0021
0.037
"These standard designations correspond to the values for test sieve ap-
ertures recommended by the International Organization for Standardization,
Geneva, Switzerland.
fa These sieves are not in the standard series but they have been included
because they are in common usage.
c 1000 ixm = 1 mm.
tion and cell length, a = A /be, where b is cm and c is g/L,
or a - (A J be) x 1000, if c is mg/L. Other names that have
been used for this or related quantities are extinction coeffi-
cient, specific absorption, absorbance index, and Ej^.
(d) Transmittance(s) (T). — Ratio of radiant power trans-
mitted by sample to radiant power incident on sample, when
both are measured at same spectral position and with same slit
width. Beam is understood to be parallel radiation and incident
at right angles to plane parallel surface of sample. If sample
is solution, solute transmittance is quantity usually desired and
is detected directly as ratio of transmittance of solution in cell
to transmittance of solvent in an equal cell. Other names that
have been used for this quantity are transmittancy and trans-
mission.
(e) Standardization. — Spectrophotomer may be checked for
accuracy of wavelength scale by referring to Hg lines: 239.94,
248.3, 253.65, 265.3, 280.4, 302.25, 313.16, 334.15, 365.43,
404.66, 435.83, 546.07, 578.0, and 1014.0 nm. To check
consistency of absorbance scale, prepare solution of 0.0400 g
K 2 Cr0 4 /L 0.05N KOH and determine absorbance at following
wavelengths in 1 cm cell: 230 nm, 0.171; 275, 0.757; 313.2,
0.043; 375, 0.991; 400, 0.396. See NIST Spec. Pub. 378,
"Accuracy in Spectrophotometry and Luminescence Measure-
ments," 1973 (available from NIST, Office of Standard Ref-
erence Materials, B316, Chem., Gaithersburg, MD 20899).
(25) Least square treatment of data and calculation of
regression lines.-— This technique finds the best fitting straight
line for set of data such as standard curve. It calculates that
straight line for which sum of squares of vertical deviations
(usually A) of observations from the line is smaller than cor-
responding sum of squares of deviation from any other line.
Equation of straight line is:
Y = a + bX
where a is intercept at Y axis (X = 0), and b is slope of line.
Least square estimates of constants are:
b = -
a = Y
2(XXd - [<JX£Yi)/n\
2X1
- bX
axf/n
where X = "sum of" the n individual values of indicated op-
eration, and X and Y are the averages of the X and Y points.
Example: To find "best" straight line relating A (Y) to con-
centration (X):
Observation
Concn
Absorbance
No. (,-)
K
Ys
x?
XX,
1
80
1.270
6400
101.6
2
60
1.000
3600
60.0
3
40
0.700
1600
28.0
4
30
0.550
900
16.5
5
20
0.250
400
5.0
6
10
0.100
100
1.0
7
0.050
0.0
Totals:
n - 7
2X, = 240
2Y; = 3.92
2X?
= 13000
2(XXi)
= 212.1
X - IX; /n - 240/7 - 34.29
Y=2Yj/n = 3.92/7 = 0.56
212.1 - (240)(3.92)/7 77.7
b = = = 0.0163
13000- (240) 2 /7 4771
a - 0.56 - 0.0163(34.29) = 0.001
Best equation is then:
If for sample, A
be:
Y = 0.00 + 0.01 63X
: 0.82, corresponding concentration (X) would
X = (Y - 0.00)/0.0163
Many scientific and
0.82/0.0163 - 50.3
statistical calculators are prepro-
grammed to perform this calculation.
(26) Recovery (R) of analyte from fortified sample by a
method of analysis. — Fraction of an analyte added to a sample
(fortified sample) prior to analysis, which is measured (re-
covered) by the method. When the same analytical method is
used to analyze both the unfortified and fortified samples, cal-
culate %R as follows:
%R - [(C F - Cu)/C A ] X 100
where C F = concentration of analyte measured in fortified
sample;
Cu = concentration of analyte measured in unfortified
sample;
C A — concentration of analyte added in fortified sam-
ple.
(Note: C A is a calculated value, not a value measured by the
method being used.)
Concentration of added analyte should be no less that con-
centration of analyte in unfortified sample. Sum of concentra-
tion of added analyte plus analyte present before fortification
should be in same range as analyte concentration sought in
actual samples. Addition of analyte must not cause measuring
instrument to exceed linear dynamic range of standard curve.
Both fortified and unfortified samples must be treated identi-
cally during analysis to minimize experimental bias.
(27) Common safety precautions are given in the safety
chapter.
Method Performance
(28) Efforts are being made to standardize the symbols and
associated definitions for the statistical parameters that will ac-
company approved methods. Users of the method should con-
sult the report of the collaborative study (reference given with
the method) for complete details.
Beginning with methods published in "Changes in Official
Methods of Analysis" (1989) JAOAC 72, 188, the following
statistical parameters are shown. Data from some studies may
not be amenable to provide these measures of evaluation.
Within -laboratory precision:
s r repeatability standard deviation
s R reproducibility standard deviation
Among-laboratories precision:
RSD r repeatability relative standard deviation
RSD R reproducibility relative standard deviation
Surplus Methods
(29) * This symbol indicates a method which has been de-
clared surplus. Such methods are satisfactory methods, having
been subjected to collaborative study and review. They are
thought not to be in current use for various reasons: The pur-
pose for which the method was developed no longer exists; the
product for which the method was developed is no longer mar-
keted; the method has been replaced by other methods; etc.
These methods retain their official status but are carried only
by reference. Any laboratory which uses a surplus method and
wishes the text reprinted in the next edition must so notify
AOAC.
Editorial Conventions
(30) For sake of simplicity, abbreviations CI and I instead
of Cl 2 and I 2 are used for chlorine and iodine. Similar abbre-
viations have been used in other cases (O, N, H). The same
abbreviation may also be used for the ion where no ambiguity
will result.
(31) Reagents and apparatus referenced with only a letter,
e.g., (c), will be found in the reagent or apparatus section of
that method.
(32) To conserve space, most of the articles and some prep-
ositions have been eliminated.
Manufacturers and Suppliers
(33) Names and addresses of manufacturers and suppliers,
and trade names of frequently mentioned materials, are fur-
nished below solely as a matter of identification and conve-
nience, without implication of approval, endorsement, or cer-
tification. The same products available from other suppliers or
other brands from other sources may serve equally well if proper
tests indicate their use is satisfactory. These firms when men-
tioned in a method are given by name only (without ad-
dresses).
Ace Glass Inc., 1430 Northwest Blvd, Vineland, NJ 08360
Aldrich Chemical Co., Inc., 940 W St. Paul Ave, Milwaukee,
WI 53233
Alltech Associates, Inc., 2051 Waukegon Rd, Deerfield, IL
60015
American Cyanamid Co., Agricultural Div., 1 Cyanamid Plaza,
Wayne, NJ 07470
(ASBC) American Society of Brewing Chemists, 3340 Pilot Knob
Rd, St. Paul, MN 55121
(ATCC) American Type Culture Collection, 12301 Parklawn
Dr, Rockville, MD 20852
Analabs Inc., 140 Water St, Norwalk, CT 06854
Analtech Inc., 75 Blue Hen Dr, PO Box 7558, Newark, DE
19714
Applied Science, 2051 Waukegan Rd, Deerfield, IL 60015
J.T. Baker, Inc., 222 Red School Ln, Phillipsburg, NJ 08865
BBL Microbiology Systems, Div. of Becton, Dickinson & Co. ,
PO Box 243, Cockeysville, MD 21030
Beckman Instruments, Inc., 2500 Harbor Blvd, PO Box 3100,
Fullerton, CA 92634
Bio-Rad Laboratories, 1414 Harbour Way South, Richmond,
CA 94804
Brinkmann Instruments, Inc., Cantiague Rd, Westbury, NY
11590
Bur dick & Jackson Laboratories, Inc., Div. of Baxter Health-
care Corp., 1953 S Harvey St, Muskegon, Ml 49442
Burrell Corp., 2223 Fifth Ave, Pittsburgh, PA 15219
Calbiochem Corp., 10933 N Torrey Pines Rd, LaJolla, CA
92037
Carborundum Co., PO Box 337, Niagara Falls, NY 14302
(CGW) Corning Glass Works, Laboratory Products Dept,
Corning, NY 14830
Curtin Matheson Scientific, Inc., 9999 Veterans Memorial Dr,
PO Box 1546, Houston, TX 77038
Difco Laboratories, PO Box 331058, Detroit, MI 48232-7058
Dohrmann, Div. of Xertex Corp., 3240 Scott Blvd, Santa Clara,
CA 95050
Dow Chemical Co., Sample Coordinator, 9001 Bldg, PO Box
1706, Midland, MI 48647-1706
Dow Corning Corp., PO Box 999, Midland, MI 48686-0997
EJ. du Pont de Nemours & Co., Inc., Instrument Products
Div., Concord Plaza, 1007 Market St, Wilmington, DE
19898
Eastman Kodak Co., Eastman Organic Chemicals, 343 State
St, Rochester, NY 14650
Elanco Products Co., Div. of Eli Lilly & Co., Elanco Ana-
lytical Laboratories, Lilly Corporate Center, Indianapolis,
IN 46285
EM Sciences, A Div. of EM Industries, 480 Democrat Rd,
Gibbstown, NJ 08027
Fischer & Porter Co., Lab Crest Scientific, E County Line
Rd, Warminster, PA 18974
Fisher Scientific Co., 1 Reagent Ln, Fair Lawn, NJ 07410
Floridin Co., 3 Penn Center, Pittsburgh, PA 15235
Foss Food Technology Corp., 10355 W 70th St, Eden Prairie,
MN 55344
Gelman Scientific Inc., 600 S Wagner Rd, Ann Arbor, MI
48106
Gist-Brocades USA, PO Box 241068, Charlotte, NC 28224-
1068
Hamilton Co., PO Box 17500, Reno, NV 89510
Hess & Clark Laboratories, Div. of Rhodia, Inc., 7th & Or-
ange Sts, Ashland, OH 44805
Hewlett-Packard Co., Avondale Div., Rte 41, PO Box 900,
Avondale, PA 19311-0900
Hewlett-Packard Co., Mail Stop 20B3, 3000 Hanover St, Palo
Alto, CA 94304
Hoffmann-La Roche, Inc., 340 Kingsland St, Nutley, NJ 07110
ICI Americas, Inc., Western Research Center, 1200 S 47th St,
PO Box 4023, Richmond, CA 94804-0023
ICN Pharmaceuticals, Inc., Life Sciences Group, 26201 Miles
Rd, Cleveland, OH 44128
(IEC) International Equipment Co., Div. of Damon, 300 Sec-
ond Ave, Needham Heights, MA 02194
Kimble Glass Inc., Crystal Ave, Vineland, NJ 08360
Kontes Glass Co., Spruce St, Vineland, NJ 08360
Kopp Glass Co., 2108 Palmer St, Swissvale, PA 15218
Labconco Corp., 88 1 1 Prospect Ave, Kansas City, MO 64132
Lurex Scientific, 1298 North West Blvd, PO Box 2420, Vine-
land, NJ 08360
Mallinckrodt Chemicals Works, Science Products Div., 675
McDonnell Blvd, PO Box 5840, St. Louis, MO 63134
Manville Filtration & Minerals, PO Box 519, Lompoc, CA
93438
Matheson Scientific, Inc., see Curtin Matheson Scientific, Inc.
E. Merck, Frankfurter Str 250, Postfach 4119, D6100 Darm-
stadt, West Germany
Merck & Co., Inc., Chemical Div., PO Box 2000, Rahway,
NJ 07065
Mettler Instrument Corp., PO Box 71, Hightstown, NJ 08520
Millipore Corp., Ashby Rd, Bedford MA 01730
Mobay Corp., Agricultural Chemicals Div., Hawthorne Rd,
PO Box 4913, Kansas City, MO 64120-0013
Monsanto Chemical Co., 800 N Lindberg Blvd, St. Louis,
MO 63167
(NBS) National Bureau of Standards, see NIST
(NF) National Formulary, see USP
New York Laboratory Supply Co., 510 Hempstead Tnpk, West
Hempstead, NY 11552
(NIST) National Institute of Standards and Technology, Gaith-
ersburg, MD 20899
Orion Research Inc., 529 Main St, Boston, MA 02149
Perkin-Elmer Corp., 761 Main Ave, Mail Station 256, Nor-
walk, CT 06859-0256
Pierce Chemical Co., PO Box 117, Rockford, IL 61105
Rheodyne Inc., PO Box 996, Cotati, CA 94928
Rohm & Haas Co., Independence Mall West, Philadelphia,
PA 19105
Salsbury Laboratories, Charles City, I A 50616
Sargent-Welch Scientific Co., 7300 N Linder Ave, PO Box
1026, Skokie, IL 60077
(S&S) Sleicher & Schuell, Inc., 10 Optical Ave, Keene, NH
03431
(SEPCO) Scientific Equipment Products Co., Div. of JAG In-
dustries, Inc., 2201 Aisquith St, Baltimore, MD 21218
Scientific Products Inc., Div. of Baxter Healthcare Corp., 1430
Waukegan Rd, McGaw Park, IL 60085-6787
Searle Analytic, Inc., 2000 Nuclear Dr, Des Plaines, IL 60018
Shell Chemical Co., 1 Shell Plaza, Houston, TX 77002
Sigma Chemical Co., PO Box 14508, St. Louis, MO 63178
G. Frederick Smith Chemical Co., PO Box 23214, Columbus,
OH 43223
Supelco, Inc., Supelco Park, Bellefonte, PA 16823-0048
Technicon Instruments Corp., 511 Benedict Ave, Tarry town,
NY 10591
Thomas Scientific, 99 High Hill Rd, 1-295, PO Box 99, Swe-
desboro, NJ 08085
Tracor Instruments, Austin, Inc., 6500 Tracor Ln, Bldg 27-7,
Austin, TX 78726-2100
UVP, Inc., 5100 Walnut Grove Ave, PO Box 1501, San Ga-
briel, CA 91778-1501
Union Carbide Corp., Old Ridgebury Rd, Danbury, CT 06817
Uniroyal Chemical, Elm St, Naugatuck, CT 06770
The Upjohn Co., 7000 Portage Rd, Kalamazoo, MI 49001
(USP) United States Pharmacopeia!. Convention, Inc., 12601
Twinbrook Pkwy, Rockville, MD 20852
Varian Instrument Group, 505 Julie Rivers Rd, Su gad and, TX
77478
VWR Scientific, PO Box 7900, San Francisco, CA 94120
Waters Associates, Inc., 34 Maple St, Milford, MA 01757
Whatman, Inc., 9 Bridewell PI, Clifton, NJ 07014
Abbreviations
(34) The following abbreviations, many of which conform
with those of Chemical Abstracts, are used. In general, prin-
ciple governing use of periods after abbreviations is that period
is used where final letter of abbreviation is not the same as
final letter of word it represents. Periods are not used with
units, except inch(es) and gallon(s). Hour(s), second(s), inch(es),
and foot(feet) appear as hr or h, sec or s, " or in., and ' or ft,
because of new abbreviations adopted in recent methods.
Abbreviation Word
a absorptivity(ies)
A absorbance(s) thruout (not restricted to for-
mulas); not absorption. A' is used for std; Ao
for blank; 3 digit subscript numerals usually
denote wavelengths in nm
AA atomic absorption
Ac CH 3 CO- (acetyl, not acetate)
ACS American Chemical Society
addn addition
addnl additional
ale. alcoholic (not alcohol)
alk. alkaline (not alkali)
alky alkalinity
amp ampere(s)
amt amount
anal. analytical(ly)
anhyd. anhydrous
AOCS American Oil Chemists' Society
APHA American Public Health Association
app. apparatus
approx. approximate(Iy)
aq. aqueous
ASTM American Society for Testing and Minerals
arm. atmosphere, atmospheric
av. average (except as verb)
Be. degree Baume
bp boiling point
Bu butyl
C degrees Celsius (Centigrade)
ca about, approximately
calc. calculate
calcd calculated
calcg calculating
calcn calculation
Cat. No. Catalog Number
centrf. centrifuge
centrfd centrifuged
centrfg centrifuging
Chap. Chapter
chem. chemical (ly)
chromatgc chromatographic
chromatgd chrornatographed
chromatgy chromatography
Ci curie(s)
CI Colour Index
CIPAC Collaborative International Pesticides Analyti-
cal Council
cm centimeter(s)
compd compound
com. commercial(ly)
cone. concentrate (as verb or noun)
coned concentrated
concg concentrating
Abbreviation
Word
concn
concentration
const
constant
contg
containing
cP
centipoise
cpm
counts per minute
cryst.
crystalline (not crystallize)
crystd
crystallized
crystg
crystallizing
crystn
crystallization
cu in.
cubic inch(es)
dc
direct current
det.
determine
detd
determined
detg
determining
detn
determination
diam.
diameter
diat. earth
diatomaceous earth
dil.
dilute
dild
diluted
dilg
diluting
diln
dilution
distd
distilled
distg
distilling
distn
distillation
DMF
N , N-di methy If orm amide
DMSO
dimethyl sulfoxide
EDTA
ethylenedinitrilotetraacetic acid (or -tetraace-
tate)
e.g.
for example
elec.
electric(al)
equiv.
equivalent
est.
estimate
estd
estimated
estg
estimating
estn
estimation
Et
ethyl
EtOH
ethanol (the chemical entity C 2 H 5 OH)
evap.
evaporate
evapd
evaporated
evapg
evaporating
evapn
evaporation
ext
extract
extd
extracted
extg
extracting
extn
extraction
F
degrees Fahrenheit (°C - (5/9) X (°F - 32))
FAO
Food and Agriculture Organization
Fig.
Figure (illustration)
fl oz
fluid ounce(s) (29.57 mL)
fp
freezing point
ft
foot (30.48 cm)
g
gram(s)
g
gravity (in centrfg)
gal.
gallon(s) (3.785 L)
GC
gas chromatography
gr-
grain(s)
g-s
glass- stoppered
HCHO
formaldehyde
HOAc
acetic acid (not HAc)
h or hr
hour(s)
ht
height
id
inner diameter (or dimension)
in.
inch(es) (2.54 cm)
inorg.
inorganic
insol.
insoluble
XIX
Abbreviation
Word
Abbreviation
Word
IR
infrared
pptd
precipitated
ISO
Internationa] Organization for Standardization
PPtg
precipitating
JAOAC
Journal of the Association of Official Analyt-
pptn
precipitation
ical Chemists (after 1965)
Pr
propyl
Journal of the Association of Official Agricul-
prep.
prepare
tural Chemists (before 1966)
prepd
prepared
kg
kilogram(s)
prepg
preparing
L
liter(s)
prepn
preparation
LC
liquid chromatography
psi
pounds per square inch (absolute)
lb
pound(s) (453.6 g)
psig
pounds per square inch gage (atmospheric
liq.
liquid
pressure = 0)
in
meter(s); milli — as prefix
Pt
pint(s) (473 mL)
m
molal
QAC
quaternary ammonium compound
M
molar (as applied to concn), not molal
qt
quart(s) (946 mL)
ma
milliampere (cf amp)
qual.
qualitative(ly)
mag.
magnetic(ally)
quant.
quantitative(ly)
manuf.
manufacturer
®
Trademark name — (Registered)
max.
maximum
Rr
distance spot moved/distance solv. moved
mech .
mechanical(ly)
(TLC)
Me
methyl
r-b
round-bottom (flask)
MeOH
methyl alcohol
ref.
reference
mg
milligram(s)
resp.
respectively
min
minute(s)
rpm
revolutions per minute
min.
minimum
sat.
saturate
mixt.
mixture
satd
saturated
ml
milliliters)
satg
saturating
mm
millimeter(s)
satn
saturation
mp
melting point
-SCN
thiocyanate
mp.
millimicron (10~ 6 mm); use nanometer (nm)
SDF
special denatured formula (applied to alcohol)
(1(T 9 m)
s or sec
second(s)
mv
millivolt
sep.
separate (ly)
MW
molecular weight
sepd
separated
TV
normal (as applied to concn); in equations,
sepg
separating
normality of titrating reagent
sepn
separation
N
Newton (10 5 dynes)
sol.
soluble
n
refractive index
soln
solution
neg.
negative
solv.
solvent
neut.
neutral
sp gr
specific gravity (apparent density)
neutze
neutralize
spectrophtr
spectrophotometer
neutzd
neutralized
spectrophtric
spectrophotometric(ally)
neutzg
neutralizing
sq
square
neutzn
neutralization
SRM
Standard Reference Material of National Insti-
NF
National Formulary
tute of Standards and Technology
NFPA
National Food Processors Association
std
standard
HIST
National Institute of Standards and Technology
std dev.
standard deviation
ng
nanogram (10~ 9 g)
stdzd
standardized
nm
nanometer (10~ 9 m); formerly mp,
stdze
standardize
No.
number
stdzg
standardizing
-OAc
acetate (cf Ac)
stdzn
standardization
-OCN
cyanate
T
transmittance
od
outer diameter (or dimension)
tech.
technical
org.
organic
temp.
temperature
oxidn
oxidation
titr.
titrate
oz
ounce(s) (28.35 g)
titrd
titrated
p
pico (10~ 12 ) as prefix
titrg
titrating
Pa
Pascal (1 Newton/m 2 ; 9.87 X 10" 6 atm.; 7.5
titrn
titration
X 10" 3 mm Hg (torr); 1.45 X 1(T 4 psi)
TLC
thin layer chromatography
par.
paragraph(s)
U
unit
pet ether
petroleum ether
USDA
United States Department of Agriculture
phthln
phenolphthalein
USP
United States Pharmacopeia
pos.
positive
uv
ultraviolet
powd
powdered (as adjective)
v
volt(s)
ppb
parts per billion (1/10 9 )
v/v
both components measured by vol.
ppm
parts per million (1/10 6 )
vac.
vacuum
ppt
precipitate
vol.
XX
volume; also volumetric when used with flask
Abbreviation
Word
Abbreviation
w/w
both components measured by wt
1
WHO
World Health Organization
%
wt
weight
>
M-
micron (0.001 mm); use micrometer (jim) (10~ 6
m)
<
M£
microgram(s) (10 6 g)
^
jjlL
microliter(s) (10~ 6 L)
(am
micrometer(s) (10~ 6 m); formerly jjl
>
A
difference (e.g., &A - (A - A'))
'
foot (feet) (T = 30.48 cm)
I
"
inch(es) (1" - 2.54 cm)
J
Word
per
percent (parts per 100); percentage
more than; greater than; above; exceeds (use
with numbers only)
less than; under; below (use with numbers only)
not more than; not greater than; equal to or less
than
not less than; equal to or greater than; equal to
or more than; at least
standard taper
standard spherical joint
Collaborative Study Procedures of the Association
of Official Analytical Chemists
The Association of Official Analytical Chemists (AOAC) is
a unique, nonprofit scientific organization whose primary pur-
pose is to serve the needs of academia, government regulatory
and research agencies, and industry for analytical methods for
compliance, quality control, and research purposes. The goal
of the Association is to provide methods which will perform
with the necessary accuracy and precision under usual labo-
ratory conditions (/). Since its formation in 1884, AOAC has
provided a mechanism to select methods of analysis from pub-
lished literature or develop new methods, collaboratively test
them through interlaboratory studies, approve them, and pub-
lish the approved methods for a wide variety of materials re-
lating to foods, drugs, cosmetics, pesticides, feeds, fertilizers,
forensic science, and products affecting the public health and
welfare. Its membership is composed of scientists from gov-
ernment, academia, and industry laboratories in many coun-
tries who work within AOAC's established procedures as re-
searchers, methods collaborators, and committee members.
AOAC has more than 100 years of experience in utilizing
the collaborative study as a means of determining the reli-
ability of analytical methods for general purposes and, espe-
cially, for regulatory purposes. In fact, AOAC's major con-
tribution to analytical science has been to bring the collaborative
study technique for the validation of analytical methods to a
high degree of perfection . In such a study, laboratories analyze
identical sample sets which cover the range of applicability of
a method previously selected as being useful and practical.
The purpose of the study is to establish the characteristics of
the method with respect to accuracy, precision, sensitivity,
range, specificity, limit of detection, .limit of reliable mea-
surement, selectivity, practicality, and similar attributes, as re-
quired.
ORGANIZATION AND PROCEDURES FOR AOAC
COLLABORATIVE STUDIES
The collaborative study is organized and directed by an an-
alyst designated as the Associate Referee for the specific sub-
ject under investigation. Currently, some 700 Associate Ref-
erees appointed by the Association are responsible for as many
topics. An Associate Referee is selected for his or her knowl-
edge, interest, and experience in the subject matter field. The
Associate Referee operates under the scientific guidance and
support of a General Referee, who is in turn responsible for a
product area. The Associate Referee reviews the literature and
selects one or two of the appropriate analytical methods avail-
able, modifying them as needed. Alternatively, he or she may
develop or adapt a method used in his or her laboratory for
the analyte and matrix under study, testing it thoroughly before
designing a collaborative study. The General Referee is kept
informed of such preliminary studies.
The samples analyzed in a collaborative study are normally
prepared and distributed to the participants by the Associate
Referee. The Association follows the "Guidelines for Collab-
orative Study Procedure to Validate Characteristics of a Method
of Analysis," as accepted by IUPAC and adopted by AOAC
(see p. 673) for the number of participating laboratories and
number of materials.
Laboratories with at least some experience in the general
subject matter are selected as collaborators. Because the ob-
jective of the study is to evaluate the method, as contrasted to
evaluating the analyst (2), all analysts are instructed to follow
the method exactly as written even though they may not concur
with the Associate Referee's selection among possible alter-
natives. The content of the analyte in the samples is unknown
to the participants.
All individual results obtained by the collaborators are re-
ported to the Associate Referee, who compiles and evaluates
them. Since statistical treatment of the data is considered es-
sential in a rigorous evaluation of the method for accuracy,
precision, sensitivity, and specificity, it is now required for all
studies. The Association considers this of such importance that
it provides statistical assistance in all cases where it is other-
wise unavailable to the Associate Referee. A statistical manual
(3) is also provided.
The Associate Referee makes the initial judgment on the
performance of the method. If he or she recommends ap-
proval, it passes to the General Referee, the appropriate Meth-
ods Committee, and then to the chairman of the Official Meth-
ods Board. If all parties recommend approval, the method
receives interim official first action approval. The method is
then presented at the Association's annual business meeting
for vote for adoption as official by the membership,
Approved methods and supporting data are published in the
Journal of the Association of Official Analytical Chemists. They
are subject to scrutiny and general testing by other analysts for
2 years before final adoption. They may be modified and re-
studied collaboratively as needed, should feedback from gen-
eral use reveal flaws in the method or in its written set of
directions. Approved methods are included in the Associa-
tion's Official Methods of Analysis, the compendium of all
adopted methods, which is updated every 5 years.
The preceding summary of AOAC's modus operandi rec-
ognizes the need for healthy skepticism toward results ob-
tained by analytical methods which have not undergone such
rigorous scrutiny and interlaboratory testing of their accuracy,
precision, specificity, and practicality.
SELECTION OF METHODS FOR STUDY
A certain degree of variability is associated with all mea-
surements. Much of the research on analytical chemistry is an
attempt to minimize that variability. But there are many dif-
ferent types of variability in analytical work. We often find
that when we attempt to minimize one kind, we must neces-
sarily permit expansion in another kind. In practical analytical
XXII
chemistry, the problem often comes down to which variability
is to be minimized.
Some examples of this point may be helpful. In atomic weight
determination, everything — especially practicality — is sacri-
ficed for accuracy. A high degree of accuracy and practicality
is required in the assay of precious metals, but the fire assay
used is generally applicable to little else besides metals and
minerals. In clinical chemistry, within-laboratory precision
(repeatability) is critical, and often is of greater interest to clin-
ical laboratories than absolute accuracy or agreement with the
values of other laboratories (reproducibility). In drug analysis,
a high degree of accuracy is required in the therapeutic range
because the analytical values determining the identity, strength,
quality, and purity of pharmaceutical preparations, as laid down
in pharmacopoeial specifications, are directly related to clin-
ical value. With poly nuclear hydrocarbons, specificity is im-
portant, since some of these compounds are carcinogenic while
others are not. In applying the famous Delaney clause of the
United States Federal Food, Drug, and Cosmetic Act, all at-
tributes of the analytical methods are secondary to the detec-
tion of extremely small concentrations (detectability), or to ex-
hibiting a high degree of response for small changes in
concentration (sensitivity) .
There is a very special case involving accuracy, where the
"true value" is determined by the method of analysis. Many
legal specifications and standards for food and agricultural
products define ill-defined components such as moisture, fat,
protein, and crude fiber in terms of reference methods. There-
fore, the precision of these methods becomes the limiting fac-
tor for their performance. In fact, most analyses involved in
commercial transactions require primarily that the buyer and
seller agree on the same value (analytically and economically),
regardless of where it stands on an absolute scale.
The point of these examples is that although methods of
analysis are characterized by a number of attributes — accu-
racy, precision, specificity, sensitivity, detectability, depend-
ability, and practicality — no method is so flawless that all these
qualities can be maximized simultaneously. For any particular
analysis, the analyst must determine, on the basis of the pur-
pose of the analysis, which attributes are essential and which
may be compromised.
Unfortunately, the literature is replete with examples indi-
cating that an individual analyst, and especially the originator
of a method of analysis, is not an unbiased judge of the relative
merits of the methods of analysis which he or she develops
and uses. In our experience, the collaborative study provides
impartial data on the suitability of the method. The data, in
many cases, speak for themselves.
The collaborative study, or ring test or round robin test, as
it is called in other organizations, provides the basic infor-
mation on the performance of analytical methods. The extent
of the information will depend on the number of samples pro-
vided, the number of analyses performed, and the number of
laboratories participating. The data should be unbiased be-
cause the composition of the samples is known only to the
administrator of the study. Some of the requirements of the
study and their relationship to the characteristics and attributes
of the method are as follows:
(1) Accuracy. Samples must be of defined composition (by
spiking, by formulation, or by analytical consensus).
(2) Specificity. Samples should contain related analytes.
(3) Sensitivity. Samples should differ from each other or from
negative samples by a known amount.
(4) Applicability. Samples should include the concentration
range and matrix components of interest.
(5) Blanks. Samples should include different matrices with
"none" of the component of interest.
(6) Precision. Instructions should request replicate analyses
by the same or different analysts in the same laboratory, pref-
erably on different days. By far a better procedure is to include
"blind" (unknown to the analyst) replicate samples in the se-
ries.
(7) Practicality. Instructions should request information as
to the actual and elapsed time required for the analyses; the
availability of reagents, equipment, and standards; and any
necessary substitutions. When practice samples are included,
the number of analyses required to achieve the stated recovery
and repeatability should be reported.
PROCEDURAL DETAILS OF COLLABORATIVE STUDY
As numerous beginners in this field have discovered, much
preliminary work must be done before sending out samples:
(1) The method must be chosen and demonstrated to apply
to the matrices and concentrations of interest.
(2) The critical variables in the method should have been
determined and the need for their control must be emphasized
[a ruggedness test (4) is useful for this purpose].
(3) The method should be written in detail by the Associate
Referee and tested by an analyst not previously connected with
its development.
(4) Unusual standards, reagents, and equipment must be
available from usual commercial sources of supply, or suffi-
cient quantities must be prepared or obtained to furnish to the
participants.
(5) The samples must be identical and homogeneous so that
the analytical sample error is only a negligible fraction of the
expected analytical error.
(6) A sufficient number of samples must be prepared to cover
typical matrices and the concentration range of interest (tol-
erance, maximum or minimum specifications, likely levels of
occurrence, etc.).
(7) A minimum of 8 laboratories and sufficient samples must
be included to provide a minimum of 40 data points. Addi-
tional laboratories and samples are recommended.
(8) Samples must be stable and capable of surviving the ri-
gors of commercial transportation.
(9) Reserve samples should be prepared and preserved to
replace lost samples and to permit reanalysis of samples con-
sidered as outliers to attempt to discover the cause of abnormal
results.
(10) The instructions must be clear. They should be re-
viewed by someone not connected with the study to uncover
potential misunderstandings and ambiguities.
(11) If the analyte is subject to change (e.g., bacterial lev-
els, nitroglycerin tablets), provision must be made for all par-
ticipants to begin the analysis at the same time.
(12) Practice samples of a known and declared composition
should be furnished with instructions not to analyze the un-
knowns until a specified degree of recovery and repeatability
(or other attribute) has been achieved.
(13) Provision should be made when necessary for submis-
sion of standard curves, tracings of recorder charts, or pho-
tographs of thinlayer plates in order to assist in determining
possible causes of error.
OTHER TYPES OF SNTERLABORATORY STUDIES
This type of collaborative study, which is designed to de-
termine the characteristics of a method, must be carefully dis-
tinguished from other types of inter] aboratory studies which
by design or through ignorance provide other kinds of infor-
mation. The most important types of other studies are:
(1) Those studies which require the collaborators to inves-
tigate the variability of parts of methods or applicability to
different types of samples. (An interl aboratory study is usually
an inefficient way of obtaining this type of information.)
(2) Those studies which permit an analyst to use any method
desired. Such studies invariably produce such a wide scatter
of results that the data are of little value for evaluation of meth-
ods. They may be useful in selecting a method from a number
of apparently equivalent methods, provided the purpose is em-
phasized beforehand and the participants provide a description
of the method used in order to permit a correlation of the de-
tails of the methods with apparent biases and variabilities.
(3) Those studies which are used for quality control pur-
poses, whose participants are not permitted sufficient time to
gain familiarity with the method, or who permit deviations to
enter into the performance of the analyses on the grounds that
the deviation is obviously an improvement which could not
possibly affect the results of the analysis, or who claim to have
a superior method.
The following definitions were agreed on as part of the
guidelines for collaboration between AOAC and the Collab-
orative International Pesticides Analytical Council Ltd. (CIPAC)
(5).
Collaborative study. An analytical study involving a number
of laboratories analyzing the same sample(s) by the same
method(s) for the purpose of validating the performance of the
method(s).
Preliminary interlaboratory study. An analytical study in
which two or more laboratories evaluate a method to determine
if it is ready for a collaborative study.
Laboratory performance check. The analysis of very care-
fully prepared and homogeneous samples, normally of known
active ingredient content, to establish or verify the perfor-
mance of a laboratory or analyst.
SUMMARY
The collaborative study is an experiment designed to eval-
uate the performance of a method of analysis through the anal-
ysis of a number of identical samples by a number of different
laboratories. With proper design, it provides an unbiased eval-
uation of the performance of a method in the hands of those
analysts who will use it. A collaborative study must be distin-
guished from those studies designed to choose a method or to
determine laboratory or analyst performance.
REFERENCES
(i) Handbook for AOAC Members, AOAC, Arlington, YA
(1989).
(2) H. Egan, "Methods of Analysis; An Analysis of Meth-
ods," J. Assoc. Off. Anal. Chem., 60, 260-267 (1977).
(3) W. J. Youden and E. H. Steiner, "Statistical Manual of
the AOAC: Statistical Techniques for Collaborative Tests.
Planning and Analysis of Results of Collaborative Tests,"
AOAC, Arlington, VA (1975).
(4) W. J. Youden, "The Collaborative Test," J. Assoc. Off.
Agric. Chem., 46, 55-62 (1963).
(5) "Guidelines for Collaboration Between the Association of
Official Analytical Chemists (AOAC) and the Collabora-
tive International Pesticides Analytical Council Ltd. (CI-
PAC)," J. Assoc. Off. Anal. Chem., 57, 447-449 (1974).
BIBLIOGRAPHY
D. Banes, "The Collaborative Study as a Scientific Concept,"
7. Assoc. Off. Anal. Chem., 52, 203-206 (1969).
W. Horwitz, "Problems of Sampling and Analytical Meth-
ods," J. Assoc. Off. Anal. Chem., 59, 1197-1203 (1976).
Reprinted with permission from: Analytical Chemistry (March 1978) 50, 337A-340A, with minor
updating revisions. Published 1978 American Chemical Society
1. Agricultural Lsrr laterials
Frank J. Johnson, Associate Chapter Editor
National Fertilizer Development Center, Tennessee Valley Authority
924.01 Sampling of Liming Materials
Procedure
(Caution: See safety note on calcium oxide.)
Take sample representative of lot or shipment. Avoid dis-
proportionate amt of surface or any modified or damaged zone.
(a) Burnt or lump lime, in bulk. — Collect composite sample
of ^10 shovelfuls/car, with proportionate amts from smaller
lots, taking each shovelful from different part of lot or ship-
ment. Immediately crush to pass 5 cm (2") diam. circular
opening, mix thoroly and rapidly, reduce composite to ca 2
kg (5 lb) sample by riffling or quartering, and place in labeled,
dry, air-tight container.
(b) Hydrated lime and ground burnt lime, in bags. — Select
10 bags from different parts of each lot or shipment of <20
tons and 1 addnl bag for each addnl 5 tons. Use sampling tube
to withdraw top to bottom core from each bag selected. Com-
bine cores, mix thoroly and rapidly, reduce composite to ca 1
kg (2 lb) by riffling or quartering, and place in dry, air-tight
container.
(c) Ground limestone and ground marl, in bags. — Proceed
as in (b).
(d) Ground limestone, ground burnt lime, ground marl, and
slag, in bulk. — Use slotted sampling tube to withdraw samples
to full sampler depth from 10 points in lot or shipment. Pro-
ceed as in (b), beginning "Combine cores, ..."
Refs.: JAOAC 7, 252(1924); 48, 95(1965).
CAS-1317-65-3 (limestone)
924.02 Mechanical Analysis of
Liming Materials
Procedure
(Caution: See safety note on calcium oxide.)
If entire sample is not to be dried, obtain lesser portions by
riffling or quartering. Dry at 1 10° to const wt and cool to room
temp.
Obtain 90-150 g dry sample by riffling or quartering. Break
any agglomerates formed during drying by rolling dry sample
with hard rubber roller on hard rubber mat, wet sieving, or by
equally effective means that does not result in crushing the
limestone.
Wet sieving. — Place 100 g sample on No. 200 sieve and
wash with moderate stream of tap H 2 at max. gage pressure
of 0.28 kg/sq cm (4 Jb/sq in.) until H 2 passing sieve is clear,
with care to avoid loss of sample by splashing. Dry material
remaining on sieve at 105° and transfer to No. 100 sieve in
series with No. 200 sieve of same diam. and depth. Shake 8
min in mech. shaker. (If wet sieving is used to break agglom-
erates, do wet sieving on sieve having smallest opening to be
used in final testing. After drying, transfer to sieves to be used
in final testing. If only 1 sieve is to be used, do not transfer.)
Quant, transfer weighed sample to 8" diam. std sieve or set of
sieves (e.g., Nos. 10, 20, 40, 60, 80, and 100 or other ap-
propriate combination).
Sieve by lateral and vertical motion accompanied by jarring
action. Continue >5 min or until addnl 3 min of sieving time
fails to change results of any sieve fraction by 0.5% of total
sample wt. Do not overload any sieve when assaying closely
sized materials.
Det. wt of each sieve fraction and report as % of total sam-
ple wt.
Refs.: JAOAC 7, 252(1924); 55, 539(1972); 48, 95(1965); 52,
322(1969).
924.03
Liming Materials
Preparation of Sample
Procedure
Reduce dried sample, 924.02, to amt sufficient for analysis
and grind >225 g (0.5 lb) reduced sample in mortar, ball mill,
or other mech. app. to pass No. 60 sieve. Mix thoroly, and
store in air-tight container.
Refs.: JAOAC 7, 252(1924); 48, 95(1965).
955.01 Neutralizing Value
for Liming Materials
Final Action
(Uncorrected for sulfide content)
A. Reagents
(a) Sodium hydroxide std soln.
0.25/V. Prep, and stdze as
-0.5/V. Stdze against (a),
in 936.16
(b) Hydrochloric acid std soln
using phthln.
B. Indicator Titrimetric Method
Place 0.5 g burnt or hydrated lime (1 g ground limestone or
ground marl), prepd as in 924.03, in 250 mL erlenmeyer; add
50 mL HC1 std soln and boil gently 5 min. Cool, and titr.
excess acid with NaOH std soln, using phthln. For burnt and
hydrated lime, report as % CaO; for limestone and marl, report
as % CaC0 3 equivalence.
% CaC0 3 equivalence of sample
- 2.5 x (mL HC1 - mL NaOH/2)
% CaO equivalence = 2.8 X (mL HCl - mL NaOH/2)
C. Potentiometric Titration Method
(Applicable to liming materials contg large amt of Fe ' 2 or col-
oring matter, but not to silicate materials)
Proceed as in 955.01B thru "Cool, ..." Transfer to 250
mL beaker and insert glass and calomel electrodes of pH me-
ter, buret contg 0.25N NaOH, and mech. stirrer. Stir at mod-
erate speed to avoid splash. Deliver NaOH rapidly to pH 5,
then dropwise until soln attains pH 7 and remains const 1 min
while stirring. (If end point is passed, add, from 1 mL Mohr
pipet, just enough 0.5iV HC1 to bring pH to <7, and back-titr.
slowly to pH 7.) Add mL of excess acid, if used, to initial 50
Agricultural Liming Materials
AOAC Official Methods of Analysis (1990)
mL in calcg. Report as % CaC0 3 or CaO equivalence as in
955.01B
Ref.: JAOAC 38, 240(1955).
D. Approximate Proportions of Calcium and Magnesium in
Magnesic Limestone
Slightly acidify titrd soln, 955.01B or C, transfer to 250 mL
vol. flask, and dil. to vol. Det. Ca in 50 mL aliquot as in
927.02, beginning ". . . dil. to ca 100 mL . . ." Subtract its
CaC0 3 equivalence from total CaC0 3 equivalence, 955. 01B
or C, and assign difference as CaC0 3 equivalence of the Mg
content of the limestone.
CAS-7440-70-2 (calcium)
CAS-131 7-65-3 (limestone)
CAS-7439-95-4 (magnesium)
928.01 Caustic Vafue for Liming Materials
Titrimetric Method
Final Action 1965
A. Apparatus (Figure 928.01)
Use 500 mL Pyrex erlenmeyer, A, and fritted glass filter
(Corning Glass Works No. 39535, 30F), F. Connect filter to
siphon tube B with thick- wall rubber tubing. Use receiving
flasks M and TV calibrated to deliver 50 and 100 mL, resp. S
is suction flask.
B. Determination
Transfer portion of sample, 924.03, to weighing bottle and
det. wt bottle and contents in atm. of min. moisture and C0 2
content. With polished, narrow-point spatula calibrated to hold
ca 1.5 g, withdraw sample to be used and det. exact wt by
difference. Insert sample directly into dry flask, A, fitted with
tight rubber stopper.
Prep, sucrose soln immediately before use by placing 25 g
granulated sucrose in measuring flask calibrated to deliver 500
mL. Dissolve sucrose with cold C0 2 -free H 2 and dil. to vol.
Holding both erlenmeyer contg sample and flask contg sucrose
soln in slightly inclined position, insert neck of sucrose soln
flask short distance into erlenmeyer, and carefully transfer su-
crose soln with synchronized rotary motion of both flasks to
prevent granulation of lime. Stopper erlenmeyer securely, ag-
itate, and add, if desired, some clean dry beads. Completely
dissolve uncoated caustic lime by six 1 min agitations at 2 or
3 min intervals. Invert flask to trap any solid particles between
stopper and neck and crush by carefully twisting stopper. Let
stand 15 min and filter as follows:
Connect filter cone F with siphon B and close stopcock D.
Connect receiving flasks, apply suction, and quickly connect
erlenmeyer A contg lime soln with stopper E. Open stopcock
C and filter 25-50 mL soln. Close C and open D to release
suction. Remove M and replace with similar dry flask. Close
D, open C, and continue filtration until both M and TV are filled
at least to marks. To disconnect system, close stopcock C, and
gently press down outlet of flask M and then outlet of flask
N, to remove any excess liq. above marks. Let intermediate
connection empty, open stopcock D, and remove M and N.
Titr. first 50 mL, or pilot aliquot, of filtered soln with 0.5/V
HO, using phthln. To covered 200 mL beaker add twice vol.
0.57V acid required for this titrn, add second (100 mL) aliquot
of filtered soln to this acid and phthln, and complete titrn,
Calc. caustic value of sample: X - 1V/W
where X = % active CaO; V = mL 0.5VV acid used/ 100 mL
lime soln; W = g sample
Refs.: Ind. Eng. Chem. 20, 312(1928). JAOAC 11, 152(1928);
12, 146(1929).
CAS- 1305-78-8 (calcium oxide)
FIG. 928.01— Apparatus for automatic filtration and measurement of lime solutions
AOAC Official Methods of Analysis (1 990)
Slags
955.02 Carbon Dioxide in Liming Materials
Knorr Alkalimeter Method
Final Action 1965
A. Apparatus and Reagents
Knorr alkalimeter with C0 2 absorption train. — Fill guard
tube of alkalimeter with Ascarite. Connect upper end of con-
denser to absorption train consisting of 5 or 6 U-shape, g-s
drying tubes (or equiv.) joined in series. Fill first tube with
H 2 S0 4 and second with Ag 2 S0 4 -H 2 S04 soln (10 g Ag 2 S0 4 in
100 ml H 2 S0 4 ) to remove acidic gases other than C0 2 . Fill
third tube with Mg(C10 4 ) 2 to absorb H 2 0. Fill inlet 2 / 3 of fourth
and succeeding tubes with Ascarite to absorb C0 2 , and outlet
V,3 of each tube with Mg(C10 4 ) 2 . Connect last tube in train with
aspirating bottle or suction source.
Condition app. daily before use, and also when freshly filled
tube is placed in train, by aspirating air at rate of 2-3 bubbles/
sec thru dry alkalimeter assembly and absorption train until
C0 2 absorption tubes attain const wt (usually 20-30 min). Tare
against similarly packed tubes. Use std procedure for wiping
tubes with dry, lint-free cloth before each weighing.
B. Determination
Transfer 3 g burnt or hydrated lime or 0.5-1.0 g limestone
or marl, prepd as in 924.03, to dry alkalimeter flask. Mo-
mentarily open stopcocks of first 2 C0 2 absorption tubes to
air to equalize pressure, weigh tubes Sep., and place in posi-
tion in train. With assembled alkalimeter connected to ab-
sorption train, adjust rate of aspiration of air thru system to ca
2 bubbles /sec. Close funnel stopcock, remove alkalimeter guard
tube, fill funnel with 50 mL HC1 (1 +4), and replace guard
tube. Open funnel stopcock and let acid run slowly into flask,
taking care that evolution of gas is so gradual as not to ma-
terially increase flow thru tubes. After all acid is added, agitate
alkalimeter assembly to ensure complete dispersion of sample
in acid soln. Continue aspiration, gradually heat contents of
flask to bp, and boil 2-3 min after H 2 begins to condense.
Discontinue heating, and continue aspiration 15-20 min or un-
til app. cools. Remove, equalize internal and external pres-
sure, and re weigh absorption tubes.
Increase in wt = wt C0 2 . (Material increase in wt of second
tube usually indicates exhaustion of first tube, but may result
from too rapid evolution of C0 2 in relation to aspiration rate.)
Report % CaC0 3 .
Ref.: JAOAC 38, 413(1955).
CAS- 124-38-9 (carbon dioxide)
CALCIUM SILICATE SLAGS
944.01 Neutralizing Value for
Calcium Silicate Slags
Titrimetric Method
Final Action 1965
(Uncorrected for sulfide content)
(a) Blast furnace slag. — Transfer 0.5 g sample, ground to
pass No. 80 sieve, to 250 mL erlenmeyer. Wash down with
small portions H 2 and add 35 mL 0.57V HC1 while swirling.
Heat to gentle boil over burner, agitating suspension contin-
uously until bulk of sample dissolves. Boil 5 min and cool to
room temp.; then dil. with C0 2 -free H 2 to ca 150 mL and
add 1 mL 30% H 2 2 and 5 drops bromocresol green. (Dissolve
0.1 g tetrabromo-m-cresolsulfonphthalein in 1.5 mL 0.1 NaOH,
and dil. to 100 mL with H 2 0.) Back-titr. with 0.5N NaOH,
adding first 15 mL rapidly and titrg drop wise thereafter, vig-
orously agitating contents of stoppered flask after each addn,
until indicator tint matches or slightly exceeds that of pH 5.2
phthalate buffer soln, 941. 17C, of like vol. and indicator concn,
after 2-3 sec agitation.
(b) Rock phosphate reduction furnace slag. — Transfer 0.5
g sample to 250 mL beaker. Wash down with small portions
H 2 and add, stirring continuously, 50 mL HO Ac (1 + 4).
Heat to bp and boil 5 min, stirring frequently. Evap. to dryness
on steam bath. Add 20 mL of the HO Ac, dil. to 150 mL, and
heat to bp; add NH 4 OH (1 + 1) to distinct yellow of Me red.
Digest ca 10 min on hot plate. Filter by gravity thru 9 cm
paper, catching filtrate in 100 X 50 mm lipped Pyrex crystg
dish; wash beaker 3 times and paper 5 addnl times with neut.
0.5N NH 4 OAc. Evap. filtrate on hot plate. Adjust heat so bub-
bles breaking thru viscous surface film are released gently to
avoid spattering. (To expedite dehydration, repeat treatments
with 25 mL hot H 2 and evapn 2 or 3 times.) Continue heating
residue on hot plate until no HOAc odor remains. Heat addnl
10 min at full heat of hot plate; then ignite 10 min at 550°.
Cool, wet residue with 15 mL H 2 0, place watch glass over
dish, and add 25 mL 0.5N HC1 thru lip of dish. Heat 5 min
over burner at gentle simmer. Rinse watch glass, filter sus-
pended matter on 9 cm paper, catching filtrate in 250 mL er-
lenmeyer, and wash dish and filter 3 times with hot H 2 0. Titr.
excess acid with 0.5N NaOH to distinct yellow of Me red.
Net acid used x 5 = neutzg value of slag in terms of %
CaC0 3 equivalence.
Refs.: JAOAC 27, 74, 532(1944); 28, 310(1945); 31, 71(1948).
CAS-471-34-1 (calcium carbonate)
948.01 Sulfide Sulfur in
Calcium Silicate Slags
Titrimetric Method
Final Action 1965
(Note: CdS0 4 is toxic; see also safety note on toxic dusts.)
A. Reagents
(a) Zinc dust. — Low in Pb.
(b) Absorbent soln.— Dissolve 20 g CdS0 4 .2 2 /3H 2 in H 2
and dil. to 1 L. Adjust to pH 5.6 potentiometrically or col-
orimetrically. If colorimetrically, match sep. 50 mL aliquot to
buffer of same pH, 941. 17C.
(c) Sodium hydroxide std soln. — 0.17V. Prep, and stdze as
in 936.16.
(d) Std acid.— AN HC1. Stdze against std alkali, (c), us-
ing Me red.
(e) Methyl red indicator. — Dissolve 0.2 g Me red in 100
mL alcohol.
B. Apparatus
Fit 250 mL erlenmeyer with 2-hole No. 5.5 stopper. Insert
thru stopper 60 mL separator with stem drawn out to 2 mm
and bent upward at tip, adjusting separator so stem is 6 mm
from bottom of flask. Also insert thru stopper 6 mm glass out-
let tube. Connect with amber rubber tubing to inlet of 25 x
150 mm tube half filled with H 2 and heated to near bp before
and during detn. Connect in series 2 addnl tubes of same size,
each contg 25 mL absorbent soln and held in 600 mL beaker
filled with cold H 2 0.
C. Determination
Fill absorbent tubes with absorbent soln and heat H 2 tube
to gentle boiling. Weigh I g slag, ground to pass No. 80 sieve,
Agricultural Liming Materials
AOAC Official Methods of Analysis (1990)
into evolution flask, add 1 g Zn dust, and wash down sides
with 5-10 mL H 2 0; mix with flat-end rod and connect flask
to app. Add 50 mL HC1 (1 + 4) to separator and let acid flow
into reaction flask while swirling contents. If necessary, apply
pressure to transfer acid and close stopcock while a little of
the acid is still above it. Heat to bp; then regulate to maintain
active but not too vigorous boiling for 10 min. Swirl flask
frequently after adding acid and for first 5 min of boiling. To
disconnect, hold inlet in first absorbent tube firmly with one
hand and quickly pull off rubber tubing with other hand with-
out pinching.
Filter CdS suspension by gravity thru 9 cm paper into 250
mL erlenmeyer and wash with H 2 to vol. of 100 mL. Add
4 drops Me red indicator and agitate vigorously while titrg
slowly with 0.1N NaOH to exact tint of ref. soln (50 mL ab-
sorbent soln dild to 100 mL, with identical indicator concn,
in 250 mL erlenmeyer). If end point is passed so that Cd(OH) 2
ppts, add 1-2 mL O.lvV HO, let stand until ppt disappears,
and complete titrn dropwise, agitating vigorously.
% CaC0 3 equivalence of sulfide S in sample
- net mL 0.17VNaOH/2
g Sulfide S/detn - mL 0.LV NaOH X 0.0016
% Sulfides- g sulfides x 100
Refs.: J AOAC 31, 715(1948); 32, 73(1949).
CAS-7704-34-9 (sulfur)
GRAVIMETRIC ELEMENTAL ANALYSES
963.01 Elemental Analysis of
Liming Materials
Preparation of Sample Solution
First Action 1963
Final Action 1965
(Caution: See safety notes on wet oxidation, nitric acid,
and perchloric acid.)
Prep, samples as in 924.03, preferably in agate mortar. Grind
silicates to pass No. 100 sieve, and dry all samples at 105°.
Weigh 2 g limestone or 0.5 g silicate. If sample contains
org. matter, transfer to Pt crucible and place in cold furnace.
Raise temp, gradually to 1000° and hold 15 min. Transfer sam-
ple to 400 mL beaker and, if ignited, moisten cautiously with
H 2 0. Add 10 mL HN0 3 and evap. on hot plate at low heat
until mixt. becomes pasty. Cool, and add 10 mL H 2 and 20
mL 60% HC10 4 . Boil to heavy fumes of HCIO4, cover, and
fume slowly until soln is colorless or slightly yellow (5-10
min). Do not evap. to dryness. Cool to <100° and add 50 mL
H 2 0. Filter thru Whatman 41H or finer paper into 250 mL vol.
flask. Wash thoroly with hot H 2 to remove all traces of HCIO4.
Reserve filtrate and washings for prepn of Sample Solns X and
Y, 963.02
963.02 Silica in Liming Materials
Gravimetric and Titrimetric Methods
First Action 1963
Final Action 1965
(See also 965.07.)
(Caution: See safety notes on hydrofluoric acid and
perchloric acid.)
Transfer paper with Si0 2 to uncovered Pt crucible and heat
gently with low flame until paper chars without flame. Par-
tially cover crucible and cautiously burn C. Finally cover com-
pletely and heat with blast lamp or in furnace at 1150-1200°.
Cool in desiccator and weigh. Repeat to const wt (W). Treat
with ca 1 mL H 2 0, 2 drops H 2 S0 4 (1 + 1), and 10 mL HF.
Cautiously evap. to dryness in hood. Heat 2 min at 1050—
1100°, cool in desiccator, and weigh (B).
W — B ~ g Si0 2 in sample
g Si0 2 X 0.4674 = g Si
(a) Sample Soln X.— (0.008 g limestone or 0.002 g silicate/
mL.) Fuse residue from Si detn with 0.5 g Na 2 C0 3 by heating
covered crucible 10 min over Meker burner. Cool, fill crucible
2 / 3 full with H 2 0, and add 2 mL 60% HC10 4 dropwise, with
stirring. Warm if necessary to dissolve melt. Add to filtrate
and washings reserved for prepn of Sample Soln X in 963.01.
Dil. to 250 mL with H 2 0.
(b) Sample Soln Y.— (0.00016 g limestone or 0.00004 g sil-
icate/mL.) Dil. 10 mL Sample Soln X to 500 mL with H 2 0.
Refs.: JAOAC 46, 603(1963); 47, 1019(1964).
CAS-763 1-86-9 (silicon dioxide)
917.01 Aluminum, Iron, Phosphorus,
and Titanium Oxides in Liming Materials
Gravimetric Method
Final Action 1965
(Alternatively, Al, Fe, Mn, P, and Ti may be detd colori-
metrically as in 965.01, 965.02, 965.03, 965.04, 965.05, and
965.06.)
To 125 mL aliquot Soln X from 963.02(a), add 10 mL HC1
and few drops Me red indicator; heat to gentle boil and add
NH4OH (1 + 1) until ppt forms and indicator just changes to
distinct yellow. Boil <2 min and filter rapidly. Wash ppt 6-
8 times with hot 2% NH 4 N0 3 soln. Return ppt and filter to
original beaker, add 10 mL HC1, and macerate filter with po-
liceman. Dil. with H 2 0, heat to dissolve ppt, dil. to ca 200
mL, and reppt as above. Wash thoroly with the hot NH4NO3
soln until CI -free. Combine first and second filtrates and save
for Ca and Mg detns.
Place ppt in Pt crucible and dry. Ignite gently to oxidize C,
heat to bright red ca 10 min, cool in desiccator, and weigh in
covered crucible as Fe 2 3 + A1 2 3 + P 2 5 + Ti0 2 .
Refs.: U.S. Geol. Survey Bull. 700, p. 106. Ind. Eng. Chem.
9, 1114(1917). JAOAC 48, 95(1965).
CAS- 1344-28-1 (aluminum oxide)
CAS- 1309-37-1 (ferric oxide)
CAS- 13 14-56-3 (phosphorus pentoxide)
CAS- 13463-67-7 (titanium dioxide)
917.02 Calcium in Liming Materials
Gravimetric and Titrimetric Methods
Final Action 1965
Cone, combined filtrates and washings from 917.01 to ca
50 mL; make slightly aik. with NH 4 OH (1 + 1); while still hot,
add satd (NH a ) 2 C 2 O a soln dropwise as long as any ppt forms,
and then enough excess to convert Mg salts also to oxalate.
Heat to bp, let stand ^3 hr, decant clear soln thru filter, pour
15-20 mL hot H 2 on ppt, and again decant clear soln thru
filter. Dissolve any ppt remaining on filter by washing with
hot HC1 (1+9) into original beaker, wash 6 times with hot
H 2 0, and then reppt at bp by adding NH 4 OH and a little satd
AOAC Official Methods of Analysis (1990)
Elemental Analysis
(NH4) 2 C 2 04 soln. Let stand as before, filter thru same filter,
and wash with hot H 2 G until Cl-free, Reserve filtrates and
washings from both pptns for detn of Mg, 919. GIB.
Complete detn by one of following methods and report as
% CaO:
(a) Ignite ppt in crucible, either over S-free blast lamp, or
in elec. furnace at 950°, to const wt, cool in desiccator, and
weigh as CaO.
(b) Incinerate filter over low flame, mix ignited ppt with
finely pulverized and dried mixt. of equal parts of (NH 4 ) 2 S0 4
and NH 4 Cl, and drive off excess sulfate by carefully heating
upper portion of crucible. Complete ignition, cool in desic-
cator, and weigh as CaS0 4 .
(c) Perforate apex of cone; wash CaC 2 4 ppt into beaker
used for pptn; then wash filter with hot H 2 S0 4 (1 +4), and titr.
at 85-90° with 0.17V KMn0 4 .
Refs.: U.S. Geol. Survey Bull. 700, p. 106. Ind. Eng. Chem.
9, 1114(1917).
CAS- 1305-78-8 (calcium oxide)
919.01 Magnesium in Liming Materials
Gravimetric Method
Final Action 1965
A. Reagent
Phosphate soln. — Dissolve 100 g (NH 4 ) 2 HP0 4 in hot H 2 0,
dil. to 1 L, and add 5 mL CHC1 3 .
B. Determination
To combined filtrates and washings, 917.02, add 2 mL \M
citric acid, 100 mL NH 4 OH, and 50 mL alcohol. Then add 25
mL of the phosphate soln, with const stirring, and let stand
12-24 hr. Filter, wash twice with NH 4 OH (1 +9), and dissolve
ppt in HN0 3 (1+4), washing soln into original beaker to vol.
of 100-150 mL. Add 1/10 vol. NH 4 OH and 2 drops of the
phosphate soln. Stir vigorously and let stand ^3 hr. Filter thru
gooch, wash with NH 4 OH (1+9), moisten filter with satd soln
of NHqNOs made slightly ammoniacal, ignite, and weigh as
Mg 2 P 2 7 . Report as % MgO, Correct wt Mg 2 P 2 O v for co-pptd
Mn 2 P 2 7 by detg Mn as in 973.55 A.
Ref.: Washington, "Chemical Analysis of Rocks," 3rd Ed.,
1919, p. .181.
CAS- 1309-48-4 (magnesium oxide)
CHELOMETRIC ELEMENTAL ANALYSES
962.01 Calcium and Magnesium
in Liming Materials
EDTA Titrimetric Methods
First Action 1962
Final Action 1965
(Not applicable to samples with high phosphate content or contg
<2% Mg)
(Caution: See safety note on cyanides.)
A. Reagents
(a) Buffer soln.— pH 10. Dissolve 67.5 g NH 4 C1 in 200 mL
H 2 0, add 570 mL NH 4 OH, and dil. to 1 L.
(b) Potassium hydroxide-potassium cyanide soln. — Dis-
solve 280 g KOH and 66 g KCN in 1 L H 2 0.
(c) Potassium cyanide soln. — 2%. Dissolve 2 g KCN in 100
mL H 2 0.
(d) Eriochrome black T indicator soln. — Dissolve 0.2 g in-
dicator (HOC 10 H 6 N:NC 10 H4(OH)(NO 2 )SO3Na) (Eastman Ko-
dak P6361, or equiv.) in 50 mL MeOH contg 2 g NH 2 OH.HCl.
Store <1 month.
(e) Magnesium std solns. — 0.25 and 1.00 ing/mL. Dis-
solve 0.25 and LOO g Mg turnings in HC1 (1 + 10) and dil.
each to 1 L with double distd H 2 0.
(f ) Calcium std soln. — 1 mg/mL. Dissolve 2.4973 g CaC0 3 ,
primary std grade, previously dried 2 hr at 285°, in HC1 (1 + 10).
Dil. to 1 L with double distd H 2 0.
(g) Calcein indicator. — Grind together 1 g indicator (2',7'-
bis[[bis(carboxymethyl)amino]methyl]-fluorescein, sodium
derivative, sodium salt), 10 g charcoal (Norite A is satisfac-
tory), and 100 g KC1. (Indicator is described in Anal. Chem.
28, 882 (1956), and is available from Eastman Kodak.)
(h) Disodium dihydrogen EDTA std solns. — (1) 0.4%. —
Dissolve 4 g Na 2 H 2 EDTA in 1 L H 2 0. Stdze against std Ca
and Mg solns. (2) 0.1%. — Prep, as in (J), using 1 g
Na 2 H 2 EDTA, and stdze against 0.25 mg/mL Mg std soln.
B. Standardization
(a) For calcium. — Pipet 10 mL std Ca soln into 300 mL
erlenmeyer and add 10 mL H 2 0. Add 10 mL KOH -KCN soln
and ca 35 mg calcein indicator. Using mag. stirrer and arti-
ficial light, titr. with 0.4% EDTA std soln to disappearance
of all green. Titr. >3 aliquots and use av. to calc. titer Ca soln
= 10/mL EDTA soln.
(b) For magnesium. — Pipet 10 mL 0.25 and 1.00 mg/mL
Mg std solns into 300 mL erlenmeyers and add 100 mL H 2 0.
Add 5 mL pH 10 buffer, 2 mL 2% KCN soln, and 10 drops
eriochrome black T indicator. Using mag. stirrer and artificial
light, titr. with 0.1. and 0.4% EDTA std solns, resp., until
color changes permanently from wine red to pure blue. Titr.
>3 aliquots and use av. to calc. titer Mg soln = 2.5/mL EDTA
soln, or 10/mL EDTA soln, resp.
C. Determination
Dry sample at 1 10° to const wt and cool to room temp. Grind
to pass No. 60 or 80 sieve and mix thoroly. Accurately weigh
ca 0.5 g into 250 mL beaker, add 20 mL HC1 (1 + 1), and evap.
to dryness on hot plate. Dissolve residue in 5 mL HC1 (1 + 10),
dil. to ca 100 mL with H 2 0, and digest over low flame 1 hr.
Cool, transfer to 200 mL vol. flask, dil. to vol., mix, and let
settle or filter.
(a) For calcium. — Pipet 10 mL aliquot into 300 mL erlen-
meyer and titr. as in 962.01B(a), observing end point thru soln
and away from light. % Ca = (Titer EDTA std soln for Ca)
X mL EDTA std soln x 2/g sample.
(b) For magnesium. — (For agricultural limestones contg >4%
Mg.) For Ca + Mg, pipet 10 mL aliquot into 300 mL erlen-
meyer and titr. with 0.4% EDTA soln as in 962.01B(b).
% Mg = (Titer EDTA std soln for Mg) x [(mL EDTA std
soln in Ca + Mg titrn) - (mL EDTA std soln in Ca titrn)] X
2/g sample.
(c) For magnesium. — (For agricultural limestones contg 2-
4% Mg.) Pipet 10 mL aliquot (0.5-1.0 mg Mg) into 300 mL
erlenmeyer and add exact vol. of 0.4% EDTA soln required
for Ca detn. Titr. with 0.1% EDTA soln as in 962.01B(b).
% Mg = (Titer EDTA std soln for Mg) x mL EDTA std
0.1% soln x 2/g sample.
Refs.: JAOAC 45, 1(1962); 46, 611(1963); 48, 95(1965); 50,
190(1967).
CAS-7440-70-2 (calcium)
CAS-7439-95-4 (magnesium)
Agricultural Liming Materials
AOAC Official Methods of Analysis (1990)
COLORIMETRIC ELEMENTAL ANALYSES
965.01 Elemental Analysis
of Liming Materials
Preparation of Sample Solution
First Action 1965
Final Action 1975
(Carry reagent blanks thru detn with stds and samples. Treat
aliquots of blank soln (corresponding to aliquot sizes of sample
solns taken for analysis) as in Determination for appropriate
element and correct values for samples accordingly.)
Det. A.l, Fe, Mn, P, and Ti in solns prepd by HC10 4 diges-
tion, 963.01 and 963.02, or NaOH fusion, 965.01. Det. Si
only in soln prepd by NaOH fusion.
{Caution: See safety notes on perchloric acid, NaOH, and
KOH.)
Prep, samples as in 924.03, preferably in agate mortar. Grind
samples to pass No. 100 sieve and dry at 105°.
(a) Sample Soln X. — (0.005 g limestone or 0.002 g silicate/
mL.) Place 0.5 g limestone or 0.2 g silicate in 75 ml Ni cru-
cible. If sample contains org. matter, place uncovered crucible
in cold furnace, raise temp, gradually to 900°, and hold 15
min. Remove crucible from furnace and let cool. Mix 0.3 g
KN0 3 with sample and add 1.5 g NaOH pellets. Cover cru-
cible with Ni cover and heat 5 min at dull redness over gas
flame. (Do not fuse in furnace.) Remove from flame and swirl
melt around sides. Cool, add ca 50 mL H 2 0, and warm to
disintegrate fused cake. Transfer to 150 mL beaker contg 15
mL 5N HCIO4 (1(60%)+1). Scrub crucible and lid with po-
liceman, and wash any residue into beaker. Transfer to 100
mL vol. flask and dil. to vol. (Sample Soln X). (This soln is
acidic and is normally clear and free of insol . matter. Occa-
sionally particles of oxidized Ni from crucible appear. When
this occurs, let particles settle before taking aliquots.)
(b) Sample Soln y.— (0.00015 g limestone or 0.00004 g sil-
icate/mL.) Dil. 15 mL limestone Sample Soln X or 10 mL
silicate Sample Soln X to 500 mL with H 2 0.
Ref.: JAOAC 47, 1019(1964).
965.02 Aluminum in Liming Materials
Colorimetric Method
First Action 1965
Final Action 1975
A. Reagents
(a) Aluminum std solns. — (1) Stock soln. — 100 |xg Al/mL.
To 0.1000 g pure Al metal in 30 mL beaker, add 6 mL HC1
U + l).
Cover with watch glass and warm gently until Al completely
dissolves. Dil. to 1 L with H 2 0. (2) Working soln. — 4 |ULg Al/
mL. Dil. 20 mL stock soln to 500 mL.
(b) Aluminon soln. — Dissolve sep. in H 2 0: 0.5 g NH 4 au-
rintricarboxylate in 100 mL; 10 g acacia (gum arable) in 200
mL; and 100 g NH 4 OAc in 400 mL. Filter acacia soln. Add
56 mL HC1 to NH 4 OAc soln and adjust pH to 4.5 with HC1
or NH4OH. Combine 3 solns and dil. to 1 L with H 2 0.
(c) Antifoam soln. — Disperse 0.03 g silicone defoamer (Dow
Corning Corp. Antifoam A) in 100 mL H 2 0.
(d) Thiogly colic acid soln.— b\\. 1 mL HSCH 2 COOH to 100
mL with H 2 0.
B. Preparation of Standard Curve
Transfer aliquots of std soln contg 0, 4, 20, 40, 60, and 80
fxg Al to 100 mL vol. flasks and proceed as in detn. Prep, std
curve by plotting %T against jxg Al on semilog paper.
C. Determination
Use Sample SolnX for limestones contg <0.2% or silicates
contg <0.8% Al and adjust pH of aliquot to 4.5 with NH 4 0H.
For materials contg greater conens of Al , use Sample Soln Y
and omit pH adjustment.
Transfer aliquot (<20 mL contg <80 fig Al) of Sample Soln
X or Y to 100 mL vol. flask. Dil. to 20 mL with H 2 0. Add 2
mL thioglycolic acid soln, 0.5 mL antifoam soln, and 10 mL
aluminon soln. Place flask in boiling H 2 20 min (250 mL
beaker contg 125 mL H 2 holds 100 mL vol. flask conve-
niently). Remove flask from H 2 and let cool ca 30 min. Dil.
to 100 mL with H 2 0. Use |mg Al soln, 965. 02B, to set 100%
T at 525 nm. Read %T for sample soln and det. |mg Al from
std curve. Calc. % Al in sample.
Ref.: JAOAC 47, 1019(1964).
CAS-7429-90-5 (aluminum)
965.03 Iron in Liming Materials
Cotorimetric Method
First Action 1965
Final Action 1975
A. Reagents
(a) Iron std solns. — (7) Stock soln. — 100 |xg Fe/mL. Dis-
solve 0.1000 g pure Fe metal in 5 mL 2iV HC1 and dil. to 1
L with H 2 0. (2) Working soln. — 5 |xg Fe/mL. Dil. 25 mL
stock soln to 500 mL.
(b) 2 ,4 ,6-Tripyridyl-s-triazine (TPTZ) soln. — Dissolve 0.500
g TPTZ in few drops HC1 and dil. to 1 L with H 2 0.
(c) Hydroxy lamine hydrochloride soln. — Dissolve 50 g
NH 2 OH.HCl in H 2 0. Add 10 mL TPTZ soln and 0.5 g
NaC10 4 .H 2 0, and dil. to 500 mL with H 2 0. Transfer to sep-
arator, add 25 mL nitrobenzene, and shake several min. Let
phases sep. and discard lower nitrobenzene phase contg Fe.
Repeat extn 2 or 3 times.
(d) Acetate buffer soln. — Dissolve 164 g anhyd. NaOAc in
H 2 0. Add 115 mL HO Ac, 10 mL NH 2 OH.HCl soln, 0.05 g
TPTZ, and 1 g NaC10 4 .H 2 0, and dil. to 1 L with H 2 0. Trans-
fer to separator, add 25 mL nitrobenzene, and shake several
min. Let phases sep. and discard lower nitrobenzene phase.
Repeat extn 3 or 4 times.
B. Preparation of Standard Curve
Treat aliquots of std soln contg 0, 5, 50, and 100 |mg Fe as
in detn. Prep, std curve by plotting %T against jxg Fe on semi-
log paper.
C. Determination
Use Sample Soln X (<5 mL) for limestones contg <0.05%
or silicates contg <0.2% Fe and Sample Soln Y for materials
contg greater conens of Fe.
Transfer aliquot (< 1 00 \ig Fe) of Sample Soln X or Y to 1 00
mL vol. flask. Add 3 mL NH 2 OH.HCl soln and 10 mL TPTZ
soln. Add NH4OH dropwise until Fe derivative remains violet
on mixing. Add 10 mL buffer soln and dil. to 100 mL. Use
\xg Fe soln, 965.03B, to set 100% T at 593 nm. Read %T
for sample soln and det. |xg Fe from std curve. Calc. % Fe in
sample.
Ref.: JAOAC 47, 1019(1964).
CAS-7439-89-6 (iron)
AOAC Official Methods of Analysis (1990)
Elemental Analysis
965.04 Manganese in Liming Materials
Colorimetric Method
First Action 1965
Final Action 1975
A. Reagents
(a) Manganese std soln. — 50 \Lg Mn/mL. Dissolve 0.0500
g pure Mn metal in 20 mL 0.5 N H 2 S0 4 and dil. to 1 L with
H 2 0.
(b) Acid mixture.— Add 800 mL HN0 3 and 200 mL H 3 P0 4
to H 2 and dil. to 2 L.
B. Preparation of Standard Curve
Treat aliquots of std soln contg 0, 50, 100, 300, and 500
jxg Mn as in detn. Prep, std curve by plotting %T against jxg
Mn on semilog paper.
C. Determination
Transfer aliquot (<500 jig Mn) of Sample Soln X to 150
mL beaker. Add 25 mL acid mixt. and 0.3 g KI0 4 . Bring to
bp and keep near boiling temp. 10 min after color develops.
Let cool, transfer to 50 mL vol. flask, dil. to vol., and mix.
Use jxg Mn soln, 965. 04B, to set 100% T at 525 nm. Read
%T for sample soln and det. jxg Mn from std curve. Calc. %
Mn in sample.
Ret.: JAOAC 47, 1019(1964).
CAS-7439-96-5 (manganese)
965.05 Phosphorus in Liming Materials
Colorimetric Method
First Action 1965
Final Action 1975
(Do not clean glassware with detergents contg P.)
A. Reagents
(a) Phosphorus std solns . — ( 1 ) Stock soln . — 1 00 |JLg P/mL .
Dissolve 0.4393 g KH 2 P0 4 in H 2 and dil. to 1 L. (2) Working
soln. — 5 |mg P/mL. Dil. 25 mL stock soln to 500 mL.
(b) Ammonium molybdate soln. — Dissolve 20 g (NH 4 ) 6 *
Mo 7 24 .4H 2 in 500 mL H 2 0. Add 285 mL H 2 S0 4 , cool, and
dil. to 1 L with H 2 0.
(c) Hydrazine sulfate soln. — Dissolve 2 g N 2 H 4 .H 2 S0 4 in
H 2 and dil. to 1 L.
B. Preparation of Standard Curve
Treat aliquots of std soln contg 0, 5, 50, and 75 (xg P as in
detn. Prep, std curve by plotting %T against (mg P on semilog
paper.
C. Determination
Transfer aliquot (<15 mL contg <75 jxg P) of Sample Soln
X to 100 mL vol. flask. Add 5 mL NH 4 molybdate soln and
mix. Add 5 mL N 2 H 4 .H 2 S0 4 soln, dil. to 70 mL with H 2 0,
and mix. Place flask in boiling H 2 9 min. Remove, cool rap-
idly, and dil. to vol. Use \Lg P~soln, 965.05B, to set 100%
T at 827 nm. Read %T for sample soln and det. \xg P from
std curve. Calc. % P in sample.
Ref.: JAOAC 47, 1019(1964).
CAS-7723-14-0 (phosphorus)
965.06 Titanium in Liming Materials
Colorimetric Method
First Action 1965
Final Action 1975
A. Reagents
(a) Titanium std solns. — (/) Stock soln. — 1 00 |xg Ti/mL.
Place 0.1668 g Ti0 2 and 2 g K 2 S 2 7 in Pt crucible. Heat cov-
ered crucible gently at first and then at dull red ca 15 min.
Dissolve melt in 50 mL H 2 S0 4 (1 + 1) and dil. to 1 L with
H 2 0. (2) Working soln.— 5 \xg Ti/mL. Dil. 25 mL stock soln
to 500 mL.
(b) Acetate buffer soln. — pH 4.7. Dissolve 41 g anhyd.
NaOAc in LLO, add 30 mL HOAc, and dil. to 1 L.
(c) Disodium-1 ,2-dihydroxybenzene-3 ,5 -disulfonate {Tiron)
soln. — Dissolve 4 g Tiron in H 2 and dil. to 100 mL.
B. Preparation of Standard Curve
Treat aliquots of std soln contg 0, 5, 50, and 75 |xg Ti as
in detn, but do not add dithionite to stds. Prep, std curve by
plotting %T against ^g Ti on semilog paper.
C. Determination
Transfer aliquot (<75 u.g Ti) of Sample Soln X to 50 mL
beaker. Dil. to c a 25 mL with H 2 0. Add 5 mL Tiron soln and
then NH 4 OH (1 +9) dropwise until soln is neut. to Congo Red
paper. (Tiron soln must be added before pH is adjusted.)
Transfer to 50 mL vol. flask, add 5 mL buffer soln, dil. to
vol. with H 2 0, and mix thoroly. Add 25 mg dithionite (Na 2 -
S 2 4 ) and dissolve with min. agitation (to avoid reappearance
of blue). Use |xg Ti soln, 965.06B, to set 100% T at 410
nm. Read %T for sample soln within 15 min after adding di-
thionite. Det. jxg Ti from std curve. Calc. % Ti in sample.
Ref.: JAOAC 47, 1019(1964).
CAS-7440-32-6 (titanium)
965.07 Silicon in Liming Materials
Colorimetric Method
First Action 1965
Final Action 1975
(Clean all glassware with HCl (1 + 1).)
A. Reagents
(a) Silicon std soln.— 20 |xg Si/mL. Place 0.0428 g pure
Si0 2 in 75 mL Ni crucible and treat as in 965.01(a), but dil.
with H 2 to 1 L instead of 100 mL.
(b) Tartaric acid soln. — Dissolve 50 g tartaric acid in H 2
and dil. to 500 mL. Store in plastic bottle.
(c) Ammonium molybdate soln. — Dissolve 7.5 g (NH 4 ) 6 -
Mo 7 24 .4H 2 in 75 mL H 2 0, add 10 mL H 2 S0 4 (1 + 1), and
dil. to 100 mL with H 2 0. Store in plastic bottle.
(d) Reducing soln. — Dissolve 0.7 g Na 2 S0 3 in 10 mL H 2 0.
Add 0.15 g l-amino-2-naphthol-4-sulfonic acid and stir until
dissolved. Dissolve 9 g NaHS0 3 in 90 mL H 2 0, add to first
soln, and mix. Store in plastic bottle.
B. Preparation of Standard Curve
Treat aliquots of std soln contg 0, 20, 100, and 200 |mg Si
as in detn. Prep, std curve by plotting %T against \xg Si on
semilog paper.
8 Agricultural Liming Materials AOAC Official Methods of Analysis (1990)
C. Determination and let stand ^30 min. Use \xg Si soln, 965.07B S to set
Transfer 10 mL Sample Soln Y to 100 mL voh flask (use 100% T at 650 nm. Read %T for sample soln and det. \xg Si
Sample Soln X for limestones contg <0.2% Si) and add 1 mL from std curve. Calc. % Si in sample.
NH 4 molybdate soln with swirling. Mix well, and let stand 10 d ^ . iaOAC 47 1019(1964^
min. Add 4 mL tartaric acid soln with swirling, and mix well.
Add 1 mL reducing soln with swirling, dil. to vol., mix well, CAS-7440-21-3 (silicon)
2, Fertilizers
Frank J. Johnson, Associate Chapter Editor
National Fertilizer Development Center, Tennessee Valley Authority
929.01 Sampling of Solid Fertilizers
Finai Action 1974
(a) Bagged fertilizers. — Use slotted single or double tube
trier with solid cone tip, constructed of stainless steel or brass.
(Do not use unplated brass for samples on which micronu-
trients are to be detd.) Trier length, exclusive of handle, should
be approx. length of filled bag to be sampled, but >25"; length
of slot >23"; width of slot ^0.5"; and id >: 5 / 8 ".
Take sample as follows: Lay bag horizontally and remove
core diagonally from end to end. From lots of >10 bags, take
core from each of 10 bags. When necessary to sample lots of
<10 bags, take 10 cores but at least 1 core from each bag
present. For small packages (<10 lb), take 1 entire package
as sample.
(b) Bulk fertilizers, including railroad car-size lots.- — Use
trier of design represented in Table 929.01.
Draw 10 vertical cores distributed in std concentric sampling
pattern (Fig. 929.01A) of such design that each core represents
approx. equal fractions of lot.
Bulk shipments may be sampled at time of loading or un-
loading by passing sampling cup, Fig. 929. 0IB (mouth di-
mensions: width 3 / 4 ", length 16" or as long as max. diam. of
stream), thru entire stream of material as it drops from belt or
chute. Make sampling such as to assure >10 equal-timed-spaced
passes thruout transfer operation. Stream samples are not ap-
plicable unless uniform continuous flow of fertilizer is main-
tained for >3 min while lot is being sampled.
(c) Preparation of sample .—Place composite sample in air-
tight container and deliver entire sample to laboratory. Reduce
composite sample in laboratory, using riffle.
Refs.: JAOAC 12, 97(1929); 33, 424(1950); 38, 108,541
(1955); 50, 190,382(1967); 51, 859(1968); 55, 709
(1972).
969.01 Sampling of Liquid Fertilizers
Final Action
(In absence of free ammonia)
(a) Clear so Ins.- — (Mixed liqs and N solns.) Secure sample
directly from mixing vat, storage tank, or delivery tank after
thoro mixing. Take sample from surface or thru direct tap.
Flush direct tap, or delivery line and faucet, and collect sample
in glass or polyethylene container. Alternatively, lower sample
container into well mixed material thru port in top of tank and
let fill. Seal container tightly.
(b) Fluid fertilizers with suspended material. — (Salt sus-
pensions and slurries.) Agitate material in storage until thoroly
mixed (15 min usually adequate) before taking sample. Sam-
ple directly as in (a), or use 500 mL Missouri or Indiana sam-
pling bottle, Fig. 969.01. Lower sampling bottle from top
opening to bottom of tank and raise slowly while filling. Transfer
to sample bottle and seal tightly.
Alternatively, secure sample from tap on recirculation line
after agitating and recirculating simultaneously until thoroly
mixed. Draw sample while recirculating. If recirculation line
is attached to manifold delivery line, allowing cross-contam-
ination, pump ca 30 cm (1') or 2000 L (500 gal.) into tem-
porary storage tank, then sample from recirculation line as above
or from delivery line. Transfer to sample bottle and seal tightly.
Ref.: JAOAC 52, 592(1969).
959.01 Sampling of Ammoniacal Solutions
First Action 1959
Final Action 1960
A. Apparatus
(a) Container. — Polyethylene reagent-form bottle with but-
tress-type cap, 1 L (1 qt) capacity.
(b) Sample flow control apparatus .—Construct from fol-
lowing fittings: IV2 x X W reducing bushing; l / 4 " tee; V/ nipple
12-18" long (length not critical); two 7/ stainless steel, blunt-
nose needle valves with hose connections (Hoke No. 3712M4Y;
Hoke Inc., 1 Tenakill Pk, Cresskill, NJ 07626). All fittings
except valves can be either Al or stainless steel. (See Fig.
959.01.)
Attach valves directly to tee, which is then attached to re-
ducing bushing thru nipple. To both valves attach 1 / 4 " id Tygon
tubing (Hoke No. 62065 hose connection), 12" length to sam-
ple valve and sufficient length to vent valve to reach disposal
area or container. To free end of sample tubing attach 3" length
of ] /V' glass or stainless steel tubing inserted thru No. 4 rubber
stopper. To exit end of metal tube attach addnl 6" length of
Tygon tubing. Make certain all connections are tight. App. can
be attached directly to tank cars, but requires addnl coupling,
which varies with installation, to attach to storage tanks. IV2"
"quick coupler" (Ever-Tite Coupling Co., 254 W 54th St, New
York, NY 10019) suffices in most cases.
Ref.: JAOAC 42, 500 (1959).
B. Sampling
Prep, sample bottle in laboratory by adding ca 500 mL H 2 0,
replacing cap, and weighing accurately (±0.1 g). Attach sam-
pling app. to car or tank and, with sample valve closed, flush
line thru vent valve. Partially collapse sample bottle, insert
sample tube with stopper, and seal tightly. With sample tube
dipping below surface of H 2 in bottle, throttle vent valve to
maintain small flow of soln and partially open sample valve,
collecting ca 100 mL sample. (Bottle should not expand to full
size during this time.) Close sample valve, remove sample tube,
partially collapse bottle, and cap tightly. Reweigh (±0.1 g)
and calc. wt sample. Cool to 20°, transfer to 1 or 2 L vol.
flask, dil. to vol. with H 2 0, mix thoroly, and take aliquots for
analysis.
959.02 Sampling of Anhydrous Ammonia
First Action 1959
Final Action 1960
(Caution: Use extreme care in handling anhyd. NH 3 . Suitable
gas mask and rubber gloves are required. See safety note on
ammonia. )
10
Fertilizers
AOAC Official Methods of Analysis (1990)
Table 929.01
Trier Specifications
Trier
Length, in.
od, in.
id, in.
Compartments
No. Size, in.
Missouri
552 Grain*
Missouri "D" b
59
63
49
17«
1 : Vs
1%
7s
17s
1
8
11
1
3
37 a
43
Triers available from:
a Seedburo Equipment Co., 1022 W Jackson Blvd, Chicago, IL 60607.
"Boyt Tool & Die Co., 917 Maple St, West Des Moines, IA 50265.
A. Sampling
Use sample tube of thermal shock-resistant glass calibrated
to contain 100 mL and graduated in 0.05 mL subdivisions up
to 0.5 mL. (Dupont special oil centrf. tube or ASTM long-
form oil tube is satisfactory.) Flush line and fill tube to 100
mL mark with sample in such manner that condensing mois-
ture will not enter sample tube. (Skirt attached to end of sam-
ple line will drain moisture away.)
B. Water and Nitrogen
Immediately close sample tube with tight-fitting rubber stop-
per into which is inserted tight-fitting piece of 6 mm id glass
tubing 5™ 8 cm long, bent at its exit from outer end of stopper
to let gases escape but to exclude entrance of moisture or mois-
ture-laden air. Place in H 2 bath at approx. air temp, and let
NH 3 evap. When temp, of sample tube is ca that of bath, re-
move tube, wipe outer surface, and det. vol. of residue.
% H 2 in sample = mL residue x C
where C = 0.74, 0.70, or 0.66 for pressures in original con-
tainers of 100, 150, or 200 psi, resp.
% N - (100 - % H 2 0) x 0.8224
Ref.: JAOAC 42, 500(1959).
CAS-7727-37-9 (nitrogen)
7
<^ 4 ^***
8
[3
©
5 )
10
^. 6 ^
9
FIG. 929.01 A— Sampling pattern
OPEN
MOUTH
s Air escape hole 1/8"
-Fluid intake tube: 1/4" for solutions
3/8" for suspensions
and slurries
Nylon cord
500 mL polyethylene
bottle
700 g of lead wts
(may be placed
inside or
attached
outside of
bottle)
MISSOURI
Air escape hole 3 mm
Fluid intake tube 7 mm
Stainless steel jacket
INDIANA
FIG. 969.01 — Missouri and Indiana weighted restricted-fill fluid
fertilizer sampling bottles designed to fill while being lowered
(and raised) in storage tanks
929.02 Preparation of Fertilizer Sample
Final Action
Reduce gross sample to amt sufficient for analysis or grind
>225 g (0.5 lb) of reduced sample without previous sieving.
NEEDLE VALVE - BLUNT
NEEDLE
FIG. 929.01 B— Sampling cup
COUPLER
FIG. 959.01— Sampling apparatus for ammoniacal solutions,
including "quick coupler" for attaching to storage tanks
AOAC Official Methods of Analysis (1990)
Water
11
For fertilizer materials and moist fertilizer mixts, grind to pass
sieve with 1 mm circular openings, or No. 20 sieve; for dry
mixts that tend to segregate, grind to pass No. 40 sieve. Grind
as rapidly as possible to avoid loss or gain of moisture during
operation. Mix thoroly and store in tightly stoppered bottles.
Refs.: JAOAC 12, 98(1929); 24, 253(1941).
917.03* Bone, Tankage,
and Basic Slag Fertilizers
Mechanical Analysis
Final Action
Surplus 1970
See 2.008, 11th ed.
957.01 Phosphate Rock Fertilizers
Mechanical Analysis
Final Action
A. Apparatus
(a) Water pressure control. — See Fig. 957.01. Connect
valve, A, std pressure gage, B, and aerator, C, with 3 /s" diam.
pipe.
(b) Sieves. — Nos. 100 and 200, bronze or stainless steel
cloth, checked against certified sieves. Sieves 8" diam. and 2"
in depth to sieve cloth are recommended for both wet and dry
sieving, but other sizes may be used if detd to be suitable un-
der conditions of method. (Other sieves in U.S. series may be
used, with precaution to ensure complete sepn of sample into
desired fractions.)
(c) Sieve shaker. — Ro-Tap (C-E Tyler, Inc., 3200 Besse-
mer City Rd, Hwy 274, PO Box 8900, Gastonia, NC 28053),
Syntron (FMC Corp., Material Handling Equipment Div., Ho-
mer City, PA 15748), or other suitable machine.
B. Reagent
Dispersing agent. — Dissolve 36 g Na hexametaphosphate
and 8 g Na 2 C0 3 in H 2 and dil. to I L.
C. Determination
(a) Ground phosphate rock. — Place 100 g sample on No.
200 sieve and wash with moderate stream of tap H 2 at max.
gage pressure of 0.28 kg/sq cm (4 Jb/sq in.) until H 2 passing
sieve is clear, with care to avoid loss of sample by splashing.
Dry material remaining on sieve at 105° and transfer to No.
100 sieve in series with No. 200 sieve of same diam. and depth.
Shake 8 min in mech. shaker. Det. % sample passing No. 100
sieve by subtracting wt of material retained on that sieve from
100. Det. % sample passing No. 200 sieve by subtracting sum
of wts of material retained on that sieve and on No. 100 sieve
from 100.
(b) Soft phosphate with colloidal clay. — Add 100 g sample
to rapidly stirred soln of 50 mL dispersing agent and 450 mL
tap H 2 0, with care to avoid contact of un wetted material with
shaft of stirrer and side of beaker. Stir 5 min after addn of
sample is completed. Transfer slurry to No. 200 sieve and pro-
ceed as in (a).
Ref.: JAOAC 40, 711(1957).
955.03* Ash (Acid-Insoluble) of Fertilizers
Final Action
Surplus 1970
See 2.015, 1 1th ed.
WATER
950.01 Water (Total) in Fertilizers
Final Action
(Not applicable to samples that yield volatile substances other
than H 2 at drying temp.)
Heat 2 g sample, 929.02, 5 hr in oven at 100±l o . In case
of NaN0 3 , (NH 4 ) 2 S0 4 , and K salts, heat to const wt at I30±l°.
Report % loss in wt as H 2 at temp. used.
Ref.: JAOAC 33, 260(1950).
O^
3/8" waterpipe
FIG. 957.01 — Apparatus for control of water pressure
965.08 Water (Free) in Fertilizers
Vacuum-Desiccation Methods
First Action 1965
Final Action 1974
(Caution: See safety note on magnesium perchlorate.)
A. Method I
Place 2 g prepd sample, 929.02, in tared weighing dish.
(Weigh extremely hygroscopic or damp materials by differ-
ence in covered dishes.) Dry sample at 25-30° (precise results
depend on as const a temp, as possible) in vac. desiccator over
anhyd. Mg(C10 4 ) 2 , P 2 5 , or BaO, under ^50 cm (20") or <55
cm (22") vac. (20-25 cm (8-10") absolute pressure) 16-18
hr. Reweigh, and report % loss in wt as free H 2 0.
B. Method //
(Not applicable to samples which yield volatile substances other
than H 2 0)
Weigh 2 g prepd sample, 929.02, into tared glass weighing
dish. Dry sample 2 hr± 10 min at 50± 1 .5° in oven under vac.
of 48-53 cm (19-21") (23-28 cm (9-1 1") absolute pressure).
(Temp, control within specified limits thruout oven chamber
12
Fertilizers
AOAC Official Methods of Analysis (1990)
is essential.) Maintain vac. by passing desiccated air thru
chamber. Coo] dried sample in desiccator and re weigh. Report
% loss in wt as free H 2 0.
Refs.: JAOAC 46, 582(1963); 47, 32, 1040(1964); 48, 98
(1965).
972.01 Water (Free) in Fertilizers
Alternative Extraction Method
First Action 1972
Final Action 1974
A. Principle
Free H 2 is extd with dioxane and detd by titrn with Karl
Fischer reagent.
B. Reagents
(Keep exposure of org. reagents to air at min.)
(a) Karl Fischer reagent. — Stabilized single soln (Fisher
Scientific Co., So-K-3, or equiv.) dild ca 1 + 1 with stabi-
lized diluent (Fisher, So-K-5, or equiv.), or soln equiv. to 2.5
mg H 2 0/mL. Stdze daily with ca 0.2 g Na tartrate. 2H 2 0. 1
mg Na tartrate. 2H 2 = 0.1566 mg H 2 0.
(b) Methanol. — Low in H 2 0.
C. Determination
Accurately weigh 2.5 g prepd sample, 929.02, into 125 mL
erlenmeyer, add 50.0 mL 1 ,4 -dioxane, stopper, mix by swirl-
ing, and let stand 15 min. Mix thoroly by swirling, and centrf.
in closed tube. {Caution: See safety note on centrifuges.)
Transfer 10 mL aliquot to titrn vessel contg pretitrd MeOH
and titr. with Karl Fischer reagent. (Discard contents of titrn
vessel after 3 titrns, replace with enough MeOH to cover elec-
trodes, and pretitr. before proceeding with next sample.) Det.
blank on 10 mL dioxane as above and subtract from sample
detns. Calc. and report as free H 2 0.
Refs.: JAOAC 52, 1127(1969); 55, 699(1972).
PHOSPHORUS
957.02 Phosphorus (Total) in Fertilizers
Preparation of Sample Solution
Final Action
A. Reagent
Magnesium nitrate soln. — Dissolve 950 g P-free
Mg(N0 3 ) 2 .6H 2 in H 2 and dil. to 1 L.
B. Preparation of Solution
{Caution: See safety notes on wet oxidation, nitric acid, per-
chloric acid, sulfuric acid, and oxidizers.)
Treat 1 g sample by (a), (b), (c), (d), or (e), as indicated.
Cool soln, transfer to 200 or 250 mL vol. flask, dil. to vol.,
mix, and filter thru dry filter.
(a) Materials containing small quantities of organic mat-
ter. —Dissolve in 30 mL HN0 3 and 3-5 mL HC1, and boil
until org. matter is destroyed (30 min for liqs and suspen-
sions).
*(b) Fertilizers containing much Fe or Al phosphate, and
basic slag.*— See 2.017, 10th ed.
(c) Organic material like cottonseed meal alone or in mix-
tures. — Evap. with 5 mL Mg(N0 3 ) 2 soln, 957. 02 A, ignite,
and dissolve in HC1.
*(d) Materials or mixtures containing large amounts of or-
ganic matter.* — See 2.017(d), 11th ed.
(e) All fertilizers . — Boil gently 30-45 min with 20-30 mL
HN0 3 in suitable flask (preferably Kjeldahl for samples contg
large amts of org. matter) to oxidize all easily oxidizable mat-
ter. Cool. Add 10-20 mL 70-72% HC10 4 . Boil very gently
until soln is colorless or nearly so and dense white fumes ap-
pear in flask. Do not boil to dryness at any time (Danger!).
(With samples contg large amts of org. matter, raise temp, to
fuming point, ca 170°, over period of ^1 hr.) Cool slightly,
add 50 mL H 2 0, and boil few min.
Ref.: JAOAC 40, 690(1957).
CAS-7723-14-0 (phosphorus)
958.01 Phosphorus (Total) in Fertilizers
Spectrophotometry Molybdovanadophosphate Method
Final Action
(Not applicable to materials yielding colored solns or solns
contg ions other than orthophosphate which form colored com-
plexes with molybdo vanadate. Not recommended for basic
slag.)
A. Apparatus
Photometer. — Spectrophtr with stray light filter and matched
1 cm cells. Analyst must det. suitability for use and conditions
for satisfactory performance. Means for dispelling heat from
light source is desirable.
B. Reagents
(a) Molybdovanadate reagent. — Dissolve 40 g NH 4 molyb-
date.4H 2 in 400 mL hot H 2 and cool. Dissolve 2 g NH 4
metavanadate in 250 mL hot H 2 0, cool, and add 450 mL 70%
HCIO4. {Caution: See safety notes on perchloric acid.) Grad-
ually add molybdate soln to vanadate soln with stirring, and
dil. to 2 L.
(b) Phosphate std soln.— Dry pure KH 2 P0 4 (52. 15% P 2 5 )
2 hr at 105°. Prep, solns contg 0.4-1.0 mg P 2 5 /mL in 0.1
mg increments by weighing 0.0767, 0.0959, 0.1151, 0.1342,
0.1534, 0.1726, and 0.1918 g KH 2 P0 4 and dilg each to 100
mL with H 2 0. Prep, fresh solns contg 0.4 and 0.7 mg P 2 5 /
mL weekly.
C. Preparation of Standard Curve
Pipet 5 mL aliquots of 7 std phosphate solns (2-5 mg P 2 5 /
aliquot) into 100 mL vol. flasks and add 45 mL H 2 0. Then,
within 5 min for entire series, add 20 mL molybdovanadate
reagent by buret or pipet, dil. to vol. and mix. Let stand 10
min.
Select 2 absorption cells (std and sample cells) and fill both
with 2 mg std. Set spectrophtr to 400 nm and adjust to zero
A with std cell. Sample cell must check zero A within 0.001
unit; otherwise read A for sample cell and correct subsequent
readings. (Choose cell showing pos. A against other as sample
cell so that this pos, A is always subtracted.) Using sample
cell, det. A of other stds with instrument adjusted to zero A
for 2 mg std. After each detn empty and refill cell contg 2 mg
std, and readjust zero to avoid error that might arise from temp.
changes. Plot A against concn in mg P 2 5 /mL std soln.
D. Preparation of Solution
Treat 1 g sample as in 957. 02B, preferably (e), when these
acids are suitable solv. (Soln should be free of N oxides and
NOC1.)
(a) For P 2 5 content <5%, dil. to 250 mL.
AOAC Official Methods of Analysis (1990)
Phosphorus
13
(b) For P 2 O s content >5%, dil. to such vol. that 5 or 10
mL aliquot contains 2-5 mg P 2 5 .
E. Determination
Pipet, into 100 mL vol. flasks, 5 mL aliquots of std phos-
phate solns contg 2 and 3.5 mg P 2 5 /aliquot, resp., and de-
velop color as in 958. 01C. Adjust instrument to zero A for 2
mg std, and det. A of 3.5 mg std. (It is essential that A of
latter std be practically identical with corresponding value on
std curve.)
(a) Samples containing up to 5% P 2 5 . — Pipet, into 100
mL vol. flask, 5 mL sample soln, 958.01D(a), and 5 mL std
phosphate soln contg 2 mg P 2 5 . Develop color and det. A
concurrently with and in same manner as for std phosphate
solns in preceding par., with instrument adjusted to zero A for
2 mg std. Read P 2 5 concn from std curve. With series of
sample solns, empty and refill cell contg 2 mg std after each
detn.
% P 2 5 in sample - 100
X [(mg P 2 O s from std curve - 2)/20]
(b) Samples containing more than 5% P 2 5 . — Pipet 5 or
10 mL sample soln, 958.01D(b), into 100 mL vol. flask.
Without adding std phosphate soln, proceed as in (a).
% P 2 O s in sample
= 100 x (mg P 2 5 from std curve/mg sample in aliquot)
Refs,: JAOAC 41, 517(1958); 42, 503(1959); 44, 133(1961).
CAS- 13 14-56-3 (phosphorus pentoxide)
962.02 Phosphorus (Total) in Fertilizers
Gravimetric Quinolinium Molybdophosphate Method
First Action 1962
Final Action 1965
A. Reagents
(Store solns in polyethylene bottles.)
(a) Citric-molybdic acid reagent. — Dissolve 54 g 100%
molybdic anhydride (Mo0 3 ) and 12 g NaOH with stirring in
400 mL hot H 2 0, and cool. Dissolve 60 g citric acid in mixt.
of 140 mL HC1 and 200 mL FLO, and cool. Gradually add
molybdic soln to citric acid soln with stirring. Cool, filter, and
dil. to 1 L. (Soln may be green or blue; color deepens on
exposure to light.) If necessary, add 0.5% KBr0 3 soln drop-
wise until green color pales. Store in dark.
(b) Quinoline soln. — Dissolve 50 mL synthetic quinoline,
with stirring, in mixt. of 60 mL HC1 and 300 mL H 2 0. Cool,
dil. to 1 L, and filter.
(c) Quimociac reagent. — Dissolve 70 g Na molybdate.
2H 2 in 150 mL H 2 0. Dissolve 60 g citric acid in mixt. of
85 mL HN0 3 and 150 mL H 2 0, and cool. Gradually add mo-
lybdate soln to citric acid-HN0 3 mixt. with stirring. Dissolve
5 mL synthetic quinoline in mixt. of 35 mL HN0 3 and 100
mL H 2 6. Gradually add this soln to molybdate-citric acid-HN0 3
soln, mix, and let stand 24 hr. Filter, add 280 mL acetone,
dil. to 1 L with H 2 0, and mix.
B. Preparation of Solution
Treat 1 g sample as in 957. 02B, dilg to 200 mL.
C. Determination
Pipet, into 500 mL erlenmeyer, aliquot contg <25 mg P 2 O s
and dil. to ca 100 mL with H 2 0. Continue by one of the fol-
lowing methods:
(a) Add 30 mL citric-molybdic acid reagent and boil gently
3 min. (Soln must be ppt-free at this time.) Remove from heat
and swirl carefully. Immediately add 10 mL quinoline soln
from buret with continuous swirling. (Add first 3-4 mL drop-
wise and remainder in steady stream.) Or:
(b) Add 50 mL quimociac reagent, cover with watch glass,
place on hot plate in well- ventilated hood, and boil 1 min.
After treatment by (a) or (b), cool to room temp., swirl
carefully 3-4 times during cooling, filter into gooch with glass
fiber filter paper previously dried at 250° and weighed, and
wash with five 25 mL portions of H 2 0. Dry crucible and con-
tents 30 min at 250°, cool in desiccator to room temp., and
weigh as (C9H 7 N) 3 H3[P04.12Mo0 3 ]. Subtract wt reagent blank.
Multiply by 0.03207 to obtain wt P 2 5 (or by 0.01400 for P).
Report as % P 2 5 (or % P).
Refs.: Z. Anal. Chem. 189, 243(1962). JAOAC 45, 40, 999
(1962); 46, 579(1963); 47, 420(1964).
CAS-7723-14-0 (phosphorus)
CAS- 13 14-56-3 (phosphorus pentoxide)
969.02 Phosphorus (Total) in Fertilizers
Alkalimetric Quinolinium Molybdophosphate Method
First Action 1969
Final Action 1975
A. Reagents
(a) Quimociac reagent. — See 962.02A(c).
(b) Sodium hydroxide std soln. — (1 mL — 1 mg P 2 5 .) Dil.
366.32 mL IN NaOH, 936.16, to 1 L with H 2 0.
(c) Nitric acid std soln. — Prep. HNO3 soln equiv. to concn
of (b) and stdze by titrg against (b), using phthln. (For greater
precision, use HNO3 soln corresponding to 1/5 concn of (b).)
(d) Citric acid. — \0% (w/v).
(e) Indicators. — (J) Thymol blue soln. — 0.1%. Add 2.2 mL
0AN NaOH to 0. 1 g thymol blue and dil . to 100 mL with 50%
alcohol. (2) Phenolphthalein. — 0.1%. Dissolve 0.1 g phthln
in 100 mL 50% alcohol. (3) Mixed indicator. — Mix 3 vols (/)
and 2 vols (2).
B. Preparation of Sample Solution
Treat 1 g sample as in 957.02B.
C. Determination
(a) Precipitation. — Transfer aliquot contg <30 mg P 2 5 and
^5 mL coned acid to 500 mL erlenmeyer, add 20 mL citric
acid soln, and adjust to ca 100 mL. Add 60 mL quimociac
reagent, immediately cover with watch glass, and place on me-
dium temp, hot plate. After soln comes to bp, move to cooler
portion of hot plate and boil gently 1 min. Let cool until flask
can be handled comfortably with bare hand.
(b) Filtration and washing. — Prep, pulped-paper pad ca 6
mm thick on perforated porcelain disk in funnel by adding >:2
approx. equal increments of H 2 suspension of pulped paper
and sucking dry with vac. between addns. Swirl flask, pour
contents onto filter, and wash flask with five ca 15 mL por-
tions H 2 0, adding washings to funnel. Immediately after fun-
nel has emptied, wash down sides with ca 15 mL H 2 to re-
move residual acetone, which causes excessively fast drying
and later lump formation if allowed to evap. Wash with 3 addnl
15 mL portions H 2 0, letting funnel empty between addns. Keep
drying of ppt to min. Using only jet of H 2 0, transfer ppt and
pad to pptn flask and break up pad with jet of H 2 0. Do not
smear ppt against funnel or flask.
(c) Titration. — Titr. with std NaOH soln and add 3-5 mL
excess. Add 1 mL mixed indicator and titr. with std HNO3
14
Fertilizers
AOAC Official Methods of Analysis (1990)
soln to grey-blue end point. If overtitrd (greenish-yellow), add
addnl excess std NaOH soln and titr. to grey-blue.
(d) Blank. — Det. blank on all reagents, adding known amt
(1-2 mg) of P 2 5 . Use 1 + 9 dilns of std NaOH and HN0 3
for titrn and subtract theoretical titer equiv. to P 2 5 added from
experimental titer. Calc. difference equiv. to 0.36637V NaOH
and subtract this blank from all sample detns.
Calc. and report as % P 2 5 .
Refs.: Z. Anal. Chem. 189, 243(1962). JAOAC 45, 40, 999
(1962); 49, 1201(1966); 52, 587(1969).
CAS- 13 14-56-3 (phosphorus pentoxide)
978.01 Phosphorus (Total) in Fertilizers
Automated Method
First Action 1978
Final Action 1980
A. Principle
Samples are extd for direct available P 2 5 or for total P2O5
detns. Destruction of coloring matter, hydrolysis of nonortho-
phosphates, and elimination of citrate effect are accomplished
by digestion with AN HC10 4 at 95°. Digested samples are re-
acted with molybdovanadate reagent, and A of resulting com-
plex is read in flowcell at 420 nm in range 0.15-0.35 mg
P 2 O s /mL.
B. Apparatus and Reagents
(Caution: See safety notes on perchloric acid.)
(a) Automatic analyzer.— Auto Analyzer with following
modules (Technicon Instruments Corp., or equiv.): Sampler II
or IV with 40/hr (4:1) cam; proportioning pump III; P 2 O s anal.
cartridge (with 2 heating baths, each contg 10.6 mL coil held
at 95±1°; or AAI type heating bath contg one 40' x 1.6 mm
id coil and holding constant temp, of 95±.l.°); A All single
channel colorimeter with 15 x 1.5 or 2.0 mm id flowcell and
420 nm interference filters; voltage stabilizer; and recorder.
Construct manifold as in flow diagram, Fig. 978.01.
(b) Molybdovanadate reagent. — Dissolve 16.5 g NH 4 mo-
lybdate.4H 2 in 400 mL hot H 2 0, and cool. Dissolve 0.6 g
NH 4 metavanadate in 250 mL hot H 2 0, cool, and add 60 mL
70% HCIO4. Gradually add molybdate soln to vanadate soln
with stirring. Add 2 mL wetting agent, (e), and dil. to 2 L.
(c) Perchloric acid.— AN. Add 342 mL 70% HCJO4 to 500
mL H 2 in 1 L vol. flask. Add 1 mL wetting agent, and dil.
to vol.
(d) Sampler wash soln. — Add 1 mL wetting agent to 1 L
H 2 0, and mix well.
(e) Wetting agent. — Ultrawet 60 L (Technicon No. T01-
0214), or equiv.
(f) Phosphorus std solns. — (J) Stock soln. — 10 mg P 2 5 /
mL. Dissolve 9.5880 g dried (2 hr at 105°) KH 2 P0 4 primary
std (52.15% P 2 O s ) in H 2 0, and dil. to 500 mL with H 2 0. (2)
Working solns.— -0.15, 0.19, 0.23, 0.27, 0.31, and 0.35 mg
P 2 5 /mL. Using 25 mL buret, accurately measure 7.5, 9.5,
11.5, 13.5, 15.5, and 17.5 mL stock soln into six 500 mL
vol. flasks. Dil. each to vol. with H 2 0, and mix. (3) Working
soln for samples <7% P 2 5 . — 2 mg P 2 5 /mL. Pipet 100 mL
stock soln into 500 mL vol. flask, dil. to vol. with H 2 0, and
mix.
C. Preparation of Samples
Prep, samples for direct available P 2 5 detn as in 960.03B(a).
Prep, samples for total P 2 5 detn as in 957.02B(a) or (e), and
dil. to 250 mL.
SAMPLER II
20T
MIXER'
INJECTION
FITTING
FLOW,ml/Min
\
0.32
AIR
95<t]
HEATING BATH
40'COIL
1.6mm ID.
0.60
HCIO4
0.23
SAMPLE
INJECTION
FITTING
0-32
AIR
1,00 MO-V REAGENT
0.23 DIG. SAMPLE
20 T
MIXER
MODIFIED AO'
WASTE *s-
0.60
Dl<
SAMPLER 11^-
WASTE^,
2.00
H20
0.80
FLOWCELL
COLORIMETER RECORDER
15 mm FLOWCELL
420 nm FILTERS
6 FT LENGTH 0.030 I.D.'
TRANSMISSION TUBING
WASTE
POSITIONED WITH CAPILLARY SIDEARM
ON BOTTOM
FIG. 978.01— -Flow diagram for automated analysis for phosphorus
AOAC Official Methods of Analysis (1990)
Phosphorus
15
D. Analytical System
(Technicon part numbers are given to aid in construction of
manifold; equiv. coils, fittings, etc., are satisfactory.)
Sample, air, and AN HC10 4 are combined thru injection fit-
ting (No. 116-0489) and mixed in 20T coil (No. 157-0248).
Stream proceeds to heating bath(s) before resample thru mod-
ified AO fitting. Resample, air, and mo lybdo vanadate reagent
are combined thru injection fitting (No. 116-0489). Mixing
and color development take place in two 20T coils (No. 157-
0248) before measurement at 420 nm. If only total P 2 5 sam-
ples are to be analyzed, heating bath can be removed and AN
HC10 4 soln replaced by sampler wash soln, (d). Heating bath(s)
and acid soln are necessary only when analyzing samples for
direct available P 2 5 or combination of direct available and
total P 2 O s detns.
If manifold is to be constructed following flow diagram, use
clear std pump tubes for all air and soln flows. All fittings,
coils, and glass transmission lines are AAII type and size. Use
1.6 mm glass transmission tubing for all connections after pump
to colorimeter. Construct modified AO fitting, following heat-
ing bath, by using AO fitting, N13 stainless steel nipple con-
nector, and 1.3 cm length of 0.035" id Tygon tubing. Insert
N 13 nipple approx. halfway into 0.035" Tygon tubing. Insert
tubing into side arm of AO fitting far enough so resample line
will not pump any air. Connect Dl fitting directly to waste
side of AO fitting; position Dl fitting with capillary side arm
on bottom. Attach 0.6 mL/min pump tube to top arm of Dl
fitting, and attach 1.8 m (6') of 0.030" id transmission tubing
to bottom arm. All air segments must pass thru 0.6 mL/min
tube, leaving continuous column of liq. in 1.8 m length of
tubing to provide for constant back pressure on heating bath
coil. Length of resample pump tube should be <2.5 cm from
shoulder at entrance end.
E. Start-Up
Start automatic system, place all lines in resp. solns, and let
equilibrate >30 min. Proceed as in 978. 01G.
F. Shut-Down
Pump water thru reagent lines >30 min. Do not remove
HCIO4 lines from reagent until 20 min after last sample is run.
G. Check and Calibration
After equilibration, set colorimeter to damp 1 position and
pump 0.15 mg P 2 5 /mL working std soln continuously thru
system. Adjust colorimeter baseline to read 10% of full scale.
Pump 0.35 mg P 2 5 /mL std and adjust std calibration to read
90% of full scale. Range of 0. 15-0.35 mg P 2 O s /mL will ex-
pand to read 10-90% of full scale. Check of bubble flow pat-
tern will give indication of performance of system. Perfect
bubble pattern is required to obtain optimum peak shapes. Check
for air bubble in flowcell if noisy conditions exist. To check
system carryover, place three 0.35 mg/mL stds, followed by
three 0. 15 mg/mL stds, thru system. If first 0.15 mg/mL std
following 0.35 mg/mL std is >1 chart division higher than
other 2, carryover is indicated. If carryover occurs, check en-
tire system for poor connections.
H. Determination
Pipet aliquot of sample soln (see Table 978.01) into 100 m'L
vol. flask, dil. to vol. with H 2 0, and mix by inversion 20
times. For sample contg <7% P 2 5 , pipet 10 ml working soln,
(f)(3), into flask before diln. Place 0.15-0.35 mg P 2 5 /mL
working std solns in tray in increasing order of concn, fol-
lowed by group of samples. Analyze lowest concn std in du-
Table 978.01 Standard Dilutions
% p 2 5
Expected
Aliquot |
mL)
Direct Available
Total
Factor
1-7
8-16
17-34
^35
50 + "spike"
no diln
50
25
25 + "spike"
50
25
15
1
0.5
2 for direct available
1 .667 for total
plicate, discarding first peak. Precede and follow each group
of samples with std ref. curve to correct for possible drift. If
drift between first and last set of stds is >2 chart divisions,
repeat sample analysis. Prep, std curve by averaging peak hts
of first and second set of stds. Plot av. peak ht of stds against
mg P 2 5 /mL contained in each std. Read mg P 2 5 /mL for
each sample from graph.
% P 2 5 = mg P 2 5 /mL from graph (-0.20, if spiked)
x F X 100
where F = factor from Table 978.01.
Refs.: JAOAC 61, 533(1978).
CAS- 13 14-56-3 (phosphorus pentoxide)
977.01 Phosphorus (Water-Soluble)
in Fertilizers
Preparation of Solution
Final Action
Place 1 g sample on 9 cm filter and wash with small portions
H 2 until filtrate measures ca 250 mL. Add H 2 in fine stream
directed around entire periphery of filter paper in circular path,
ensuring that H 2 and solids are thoroly mixed with each addn.
Let each portion pass thru filter before adding more and use
suction if washing would not otherwise be complete within 1
hr. If filtrate is turbid, add 1-2 mL HNO3, dil. to 250 mL,
and mix.
Ref.: JAOAC 60, 393,702(1977).
962.03 Phosphorus (Water-Soluble)
in Fertilizers
Gravimetric Quinolinium Molybdophosphate Method
Final Action
Pipet aliquot contg <25 mg P 2 5 into 500 mL erlenmeyer.
Dil., if necessary, to 50 mL, add 10 mL HN0 3 (3+1), and
boil gently 10 min. Cool, dil. to 100 mL, and proceed as in
962.02C(b).
962.04 Phosphorus (Water-Soluble)
in Fertilizers
Alkalimetric Quinolinium Molybdophosphate Method
Final Action 1974
Pipet aliquot contg <30 mg P 2 5 into 500 mL erlenmeyer.
Dil., if necessary, to 50 mL, add 10 mL HN0 3 (1 + 1), boil
gently 10 min, cool, and proceed as in 969.02C(a), beginning
"... add 20 mL citric acid soln . . ."
16
Fertilizers
AOAC Official Methods of Analysis (1990)
970.01 Phosphorus (Water-Soluble)
in Fertilizers
Spectrophotometry Molybdovanadophosphate Method
First Action 1970
Final Action 1974
Adjust concn according to 958.01D(a) or (b) and proceed
as in'958.01E.
963.03 Phosphorus (Citrate-Insoluble)
in Fertilizers
First Action 1963
Final Action 1964
A. Reagents
(a) Ammonium citrate soln. — Should have sp gr of 1.09 at
20° and pH of 7.0 as detd potentiometrically.
Dissolve 370 g cryst. citric acid in 1.5 L H 2 and nearly
neutze by adding 345 mL NH 4 OH (28-29% NH 3 ). If concn
of NH 3 is <28%, add correspondingly larger vol. and dissolve
citric acid in correspondingly smaller vol. H 2 0. Cool, and check
pH. Adjust with NH 4 OH (1+7) or citric acid soln to pH 7.
Dil. soln, if necessary, to sp gr of 1.09 at 20°. (Vol. will be
ca 2 L.) Keep in tightly stoppered bottles and check pH from
time to time. If pH has changed from 7.0, readjust.
(b) Other reagents and solns.—See 957. 02A, 958. 01B, or
962. 02 A.
B. Preparation of Extract
(a) Acidulated samples, mixed fertilizers, and materials
containing water-soluble compounds. — After removing H 2 0-
sol. P 2 5 , 977.01, transfer filter and residue, within 1 hr, to
200 or 250 mL flask contg 100 mL NH 4 citrate soln previously
heated to 65°. Close flask tightly with smooth rubber stopper,
shake vigorously until paper is reduced to pulp, and relieve
pressure by removing stopper momentarily. Continuously ag-
itate stoppered flask in const temp. app. at exactly 65°. (Action
of app. should be such that dispersion of sample in citrate soln
is continually maintained and entire inner surface of flask and
stopper is continually bathed with soln.)
Exactly 1 hr after adding filter and residue, remove flask
from app. and immediately filter by suction as rapidly as pos-
sible thru Whatman No. 5 paper, or equiv., using buchner or
ordinary funnel with Pt or other cone. Wash with H 2 at 65°
until vol. filtrate is ca 350 mL, allowing time for thoro drain-
ing before adding more H 2 0. If material yields cloudy filtrate,
wash with 5% NH 4 N0 3 soln. Prep, citrate-insol. residue for
analysis by one of following methods:
(7) Dry paper and contents, transfer to crucible, ignite until
all org. matter is destroyed, and digest with 10—15 mL HO
until all phosphate dissolves; or (2) treat wet filter and contents
as in 957.02B(a), (c), (d), or (e). Dil. soln to 250 mL, or other
suitable vol., mix well, and filter thru dry paper.
(b) N onacidulated samples. — Place 1 g sample (ground to
pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm
paper. Without previous washing with FLO, proceed as in (a).
If sample contains much org. matter (bone, fish, etc.), dis-
solve residue in sol. in NH 4 citrate as in 957.02B(c), (d), or
(e).
C. Determination
— Final Action 1974
(a) Gravimetric quinolinium molybdophosphate method. —
Treat I g sample as in 963.03B(a) or (b). Transfer aliquot of
citrate-insol. P^O^ contg <25 mg P^0 5 and proceed as in
962.02C.
(b) Spectrophotometry molybdovanadophosphate method. —
Treat 1 g sample as in 963.036(a) or (b). Adjust concn of
citrate-insol. P 2 5 soln as in 958.01D(a) or (b) and proceed
as in 958.01E.
(c) Alkalimetric quinolinium molybdophosphate method. —
Treat 1 g sample by 963.03B(a) or (b). Transfer aliquot of
citrate-insol. P 2 5 contg <5 mL coned acid to 500 mL erlen-
meyer. Add 20 mL 10% citric acid soln and dil. to 100 mL
with H 2 0. Continue as in 969.02C(a), beginning "Add 60 mL
quimociac reagent, ..."
Refs.: JAOAC 5, 443,460(1922); 6, 384(1923); 14, 182(1931);
19, 269(1936); 22, 254(1939); 42, 503, 512(1959); 52,
587(1969).
Z. Anal. Chem. 189, 243(1962). JAOAC 45, 40, 201,
999(1962); 46, 579(1963); 47, 420(1964).
CAS- 13 14-56-3 (phosphorus pentoxide)
960.01
Phosphorus (Citrate-Soluble)
in Fertilizers
Final Action 1960
Subtract sum of H 2 0-sol. and citrate-insol.
P 2 5 to obtain citrate- sol. P 2 5 .
P 2 5 from total
960.02 Phosphorus (Available) in Fertilizers
Indirect Method
Final Action 1960
Subtract citrate-insol. P 2 5 from total P 2 5 to obtain avail-
able P 2 5 .
960.03 Phosphorus (Available) in Fertilizers
Final Action
A. Reagents
(Caution: See safety notes on nitric acid, perchloric acid, and
sulfuric acid.)
(a) Nitric-perchloric acid mixture. — Add 300 mL 70%
HCI0 4 to 700 mL HNO3.
(b) Ternary acid mixture. — Add 20 mL H 2 S0 4 to 100 mL
HNO3, mix, and add 40 mL 70% HC10 4 .
(c) Modified molybdovanadate reagent. — Prep, as in
958.016(a)' except use 250 mL 70% HCIO4 instead of 450
mL.
3. Preparation of Solution
(a) Acidulated samples, mixed fertilizers, and materials
containing water-soluble compounds. — (J) Without filtration
of citrate digest. — Remove H 2 0-soJ. P 2 5 as in 977.01,
collecting filtrate in 500 mL vol. flask, but do not add HNO3
to filtrate. Treat H 2 0-insol. residue with NH 4 citrate soln as
in 963.03B(a). Exactly 1 hr after adding filter and residue,
remove flask from app. and transfer contents to flask contg
H 2 0-sol. fraction. Cool to room temp, immediately, dil. to
vol., mix thoroly, and let stand >2 hr before removing ali-
quot.
(2) With filtration of citrate digest.- — if desired, wash by
gravity into 500 mL Kohlrausch flask contg 5 mL HNO3 (1 + 1),
catching filtrate from insol. residue, 963.03B(a), in the Kohl-
rausch flask contg H 2 0-sol. fraction, and wash residue until
vol. soln in flask is ca 500 mL. Cool, dil. to 500 mL, and
AOAC Official Methods of Analysis (1990)
Nitrogen
17
(b) Nonacidulated samples. — Place 1 g sample (ground to
pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm
paper. Without previous washing with H 2 0, proceed as in (a)(7)
or (2). If (2) is used, wash residue until vol. soln is ca 350
ml. Cool, dil. to 500 mL, and mix.
Refs.: JAOAC 43, 478(1960); 44, 133, 232(1961); 46, 570
(1963); 60, 702(1977).
C. Alkalimetric Quinolinium Molybdophosphate Method
— Final Action 1974
Treat 1 g sample by appropriate modification of 960. 03B.
Transfer aliquot contg <30 mg P 2 5 and <10 mL NH 4 citrate
soln, 963.03A(a), to 500 mL erlenmeyer. DiL, if necessary,
to 50 mL, add 10 mL HN0 3 (1 + 1), and boil gently 10 min.
Cool, dil. to 100 mL, and continue as in 969.02C(a), begin-
ning "Add 60 mL quimociac reagent, ..."
Ref.: JAOAC 52, 587(1969).
D. Spectrophotometry Molybdovanadophosphate Method
—Final Action 1961
(Not applicable to materials yielding colored solns or solns
contg ions other than orthophosphate which form colored com-
plexes with molybdovanadate. Not recommended for basic
slag.)
Prep, std curve as in 958.01 C, using photometer, 958. 01 A.
Pipet, into 100 mL vol. flasks, 5 mL aliquots std phosphate
solns contg 2 and 3.5 mg P 2 5 /aliquot, 958.01B(b), resp., add
2 mL 70% HC10 4 , and develop color as in 958.01C. Adjust
instrument to zero A for 2 mg std and det. A of 3.5 mg std.
(A of latter must be practically identical with corresponding
value on std curve.)
Prep, sample as in 960. 03B.
(a) Samples containing up to 5% P 2 O s . — Pipet 10 mL sam-
ple soln into 125 mL erlenmeyer, and treat by one of following
methods (Caution: See safety notes on wet oxidation, nitric
acid, and perchloric acid):
(J) Add 5 mL 20% NaC10 3 soln and 10 mL HN0 3 -HC10 4
mixt., 960.03A(a). Boil gently until greenish-yellow color dis-
appears (ca 20 min), cool, and add 2 mL HC1. After vigorous
reaction subsides, evap. to fumes of HCIO4, and fume 2 min.
(2) Add 5 mL ternary acid mixt., 960.03A(b), swirl, boil
gently 15 min, and digest at 150-200° until clear white salt
or colorless soln remains. Evap. to white fumes and continue
heating 5 min.
Cool, add 15 mL H 2 0, and boil 5 min. Transfer to 100 mL
vol. flask, dil. to 50 mL, swirl, and cool to room temp. Add
5 mL std phosphate soln contg 2 mg P 2 5 and 20 mL modified
molybdovanadate soln, 960.03A(c). Dil. to 100 mL, and con-
tinue as in 958.01E.
(b) Samples containing more than 5% P20 5 . — Dil. soln to
such vol. that 5-10 mL aliquot contains 2-5 mg P?0 5 . Digest
as in (a)(7) or (2). Without adding std phosphate soln, con-
tinue as in (a).
Ref.: JAOAC 44, 233(1961).
E. Gravimetric Quinolinium Molybdophosphate Method
—First Action 1963
—Final Action 1964
(a) Solns containing no organic phosphorus. — Prep, sam-
ple as in 960.03B. Pipet, into 500 mL erlenmeyer, aliquot contg
<25 mg P 2 5 and <10 mL original NH 4 citrate soln. DiL, if
necessary, to ca 50 mL, add 10 mL H.NO3 (1 + 1), and boil
gently 10 min. Cool, dil. to 150 mL, and proceed as in
962.02C(a) or (b).
(b) Solns containing organic phosphorus . — (Caution: See
safety notes on wet oxidation, nitric acid, and perchloric acid.)
Select aliquot as in (a). Add 10 mL 20% NaC10 3 and 10 mL
HNO3-HCIO4 mixt., 960.03A(a). Boil vigorously until green-
ish-yellow color disappears (usually ca 30 min), cool, and add
2 mL HO. After vigorous reaction subsides, evap. to white
fumes, and continue heating 5 min. Cool, and proceed as in
962.02C(a) or (b).
Refs.: JAOAC 46, 570(1963); 47, 420(1964).
920.01
NITROGEN
Nitrates in Fertilizers
Detection Method
Final Action
Mix 5 g sample with 25 mL hot H 2 0, and filter. To 1 vol.
of this soln add 2 vols H 2 S0 4 , free from HN0 3 and oxides of
N, and let cool. Add few drops coned FeS0 4 soln in such
manner that fluids do not mix. If nitrates are present, junction
at first shows purple, afterwards brown, or if only minute amt
is present, reddish color. To another portion of soln add 1 mL
1% NaN0 3 soln and test as before to det. whether enough H 2 S0 4
was added in first test.
955.04 Nitrogen (Total) in Fertilizers
Kjeldah! Method
Final Action
(Provide adequate ventilation in laboratory and do not permit
accumulation of exposed Hg.)
A. Reagents
(a) Sulfuric acid.— -93-98% H 2 S0 4 , N-free.
(b) Mercuric oxide or metallic mercury. — HgO or Hg, re-
agent grade, N-free.
(c) Potassium sulfate (or anhydrous sodium sulfate). — Re-
agent grade, N-free.
(d) Salicylic acid. — Reagent grade, N-free.
(e) Sulfide or thiosulfate soln. — Dissolve 40 g com. K 2 S in
1 L H 2 0. (Soln of 40 g Na 2 S or 80 g Na 2 S 2 3 .5H 2 in 1 L
may be used.)
(f) Sodium hydroxide .—(Caution: See safety notes on so-
dium and potassium hydroxide.) Pellets or soln, nitrate-free.
For soln, dissolve ca 450 g solid NaOH in H 2 0, cool, and dil.
to 1 L. (Sp gr of soln should be >'L36.)
(g) Zinc granules. — Reagent grade,
(h) Zinc dust. — Impalpable powder.
(i) Methyl red indicator. — Dissolve 1 g Me red in 200 mL
alcohol.
(j) Hydrochloric or sulfuric acid std soln. — 0.5vV, or 0. \N
when amt of N is small. Prep, as in 936.15 or 890. 01 A.
(k) Sodium hydroxide std soln. — 0.17V (or other specified
concn). Prep, as in 936.16.
Stdze each std soln with primary std (see chapter on standard
solutions) and check one against the other. Test reagents be-
fore use by blank detn with 2 g sugar, which ensures partial
reduction of any nitrates present.
Caution: Use freshly opened H 2 S0 4 or add dry P 2 5 to avoid
hydrolysis of nitriles and cyanates. Ratio of salt to acid (wt:vol.)
should be ca 1:1 at end of digestion for proper temp, control.
Digestion may be incomplete at lower ratio; N may be lost at
higher ratio. Each g fat consumes 10 mL H 2 S0 4 , and each g
carbohydrate 4 mL H 2 S0 4 during digestion.
18
Fertilizers
AOAC Official Methods of Analysis (1990)
B. Apparatus
(a) For digestion. — Use Kjeldahl flasks of hard, moder-
ately thick, well-annealed glass with total capacity ca 500-800
mL. Conduct digestion over heating device adjusted to bring
250 mL H 2 at 25° to rolling boil in ca 5 min or other time
as specified in method. To test heaters, preheat 10 min if gas
or 30 min if elec. Add 3-4 boiling chips to prevent super-
heating.
(b) For distillation. — Use 500-800 mL Kjeldahl or other
suitable flask, fitted with rubber stopper thru which passes lower
end of efficient scrubber bulb or trap to prevent mech. car-
ryover of NaOH during distn. Connect upper end of bulb tube
to condenser tube by rubber tubing. Trap outlet of condenser
in such way as to ensure complete absorption of NH 3 distd
over into acid in receiver.
C. improved Kjeldahl Method for Nitrate-Free Samples
(Caution: See safety notes on sulfuric acid, sodium hydrox-
ides, and mercury.)
Place weighed sample (0.7-2.2 g) in digestion flask. Add
0.7 g HgO or 0.65 g metallic Hg, 15 g powd K 2 S0 4 or anhyd.
Na 2 S0 4 , and 25 mL H 2 S0 4 . If sample >2.2 g is used, increase
H 2 S0 4 by 10 mL for each g sample. Place flask in inclined
position and heat gently until frothing ceases (if necessary, add
small amt of paraffin to reduce frothing); boil briskly until soln
clears and then >30 min longer (2 hr for samples contg org.
material) .
Cool, add ca 200 mL H 2 0, cool <25°, add 25 mL of the
sulfide or thiosulfate soln, and mix to ppt Hg. Add few Zn
granules to prevent bumping, tilt flask, and add layer of NaOH
without agitation. (For each 10 mL H 2 S0 4 used, or its equiv.
in dild H 2 S0 4 , add 15 g solid NaOH or enough soln to make
contents strongly alk.) (Thiosulfate or sulfide soln may be mixed
with the NaOH soln before addn to flask.) Immediately con-
nect flask to distg bulb on condenser, and, with tip of con-
denser immersed in std acid and 5-7 drops indicator in re-
ceiver, rotate flask to mix contents thoroly; then heat until all
NH 3 has distd (>150 mL distillate). Remove receiver, wash
tip of condenser, and titr. excess std acid in distillate with std
NaOH soln. Correct for blank detn on reagents.
% N = [(mL std acid x normality acid) - (mL std NaOH
x normality NaOH)] x].4007/g sample
Ref: JAOAC 38, 56(1955).
D. Improved Kjeldahl Method for Nitrate-Containing Samples
(Not applicable to liqs or to materials with high C1:N0 3 ratio.
Caution: See safety notes on sulfuric acid and mercury.)
Place weighed sample (0.7-2.2 g) in digestion flask. Add
40 mL H 2 S0 4 contg 2 g salicylic acid. Shake until thoroly
mixed and Jet stand, with occasional shaking, >30 min; then
add (/) 5 g Na 2 S 2 3 .5H 2 or (2) 2 g Zn dust (as impalpable
powder, not granulated Zn or filings). Shake and let stand 5
min; then heat over low flame until frothing ceases. Turn off
heat, add 0.7 g HgO (or 0.65 g metallic Hg) and 15 g powd
K 2 S0 4 (or anhyd. Na 2 S0 4 ), and boil briskly until soln clears,
then >30 min longer (2 hr for samples contg org. material).
Proceed as in second par. of 955. 04C.
Ref.: JAOAC 51, 446(1968).
CAS-7727-37-9 (nitrogen)
970.02 Nitrogen (Tota!) in Fertilizers
Comprehensive Nitrogen Method
First Action 1970
Final Action 1975
(Applicable to all fertilizer samples.)
(Caution: See safety notes on sulfuric acid and mercury salts.)
A. Reagents
(a) Chromium metal. — 100 mesh, low N (Fisher Scientific
Co. No. C-318 or Sargent- Welch Scientific Co. No. C1 1432
is satisfactory).
(b) Alundum. — Boiling stones. 8-14 mesh (Thomas Sci-
entific No. 1590-D18, or equiv.).
(c) Dilute sulfuric acid. — Slowly add 625 mL H 2 S0 4 to 300
mL H 2 0. Dil. to ca 1 L and mix. After cooling, dil. to 1 L
with H 2 and mix. Avoid absorption of NH 3 from air during
prepn, particularly if stream of air is used for mixing.
(d) Sodium thiosulfate or potassium sulfide soln.—] 60 g
Na 2 S 2 3 .5H 2 0/L or 80 g K 2 S/L.
For other reagents, see 920. 02A.
B. Determination
Place 0.2-2.0 g sample contg <60 mg nitrate N in 500-
800 mL Kjeldahl flask and add 1.2 g Cr powder. Add 35 mL
H 2 or, with liqs, amt to make total vol. 35 mL. Let stand
10 min with occasional gentle swirling to dissolve all nitrate
salts. Add 7 mL HC1 and let stand >30 sec but <10 min.
Place flask on preheated burner with heat input set at 7.0-
7.5 min boil test, 920.02B(a). After heating 3.5 min, remove
from heat and let cool.
Add 22 g K 2 S0 4 , 1.0 g HgO, and few granules Alundum.
Add 40 mL dil. H 2 S0 4 , (c). (If adequate ventilation is avail-
able, 25 mL H 2 S0 4 may be added instead of dil. H 2 S0 4 . If
org. matter which consumes large amt of acid exceeds 1.0 g,
add addnl 1.0 mL H?S0 4 for each 0.1 g org. matter in excess
of 1.0 g.)
Place flask on burners set at 5 min boil test. (Pre-heated
burners reduce foaming with most samples. Reduce heat input
if foam fills > 2 / 3 of bulb of flask. Use variable heat input until
this phase is past.) Heat at 5 min boil test until dense white
fumes of H 2 S0 4 clear bulb of flask. Digestion is now complete
for samples contg ammoniacal, nitrate, and urea N. For other
samples, swirl flask gently and continue digestion 60 min more.
Proceed as in 955.04C, second par., substituting 970.02A(d)
for 920.02 A(e).
Refs.: JAOAC 53, 450(1970); 57, 10(1974); 68, 441(1978).
CAS-7727-37-9 (nitrogen)
978.02 Nitrogen (Total) in Fertilizers
Modified Comprehensive Nitrogen Method
First Action 1978
Final Action 1984
(Applicable to all fertilizer samples)
A. Reagents
See 920.02A(a), (c), (f), (i), (j), (k), 970.02A(a), (b), and
in addn:
Copper sulfate pentahydrate (or anhydrous copper sul-
fate). — Reagent grade, N-free.
AOAC Official Methods of Analysis (1990)
Nitrogen
19
B. Determination
{Caution: See safety notes on wet oxidation, sulfuric acid, and
sodium hydroxide.)
Proceed as in 970. 02B, par. I and 2, using 0.2-1.6 g sam-
ple. For samples contg orgs other than urea or urea-form, use
>0.5 g sample.
Add 15 g K 2 S0 4 or 12 g anhyd. Na 2 S0 4 , 0.4 g anhyd. CuS0 4
or 0.6 g CuS0 4 .5H 2 0, and ca 0.8 g Alundum granules. Add
37 inL H 2 S0 4 (1 + 1). (If adequate ventilation is available, 20
mL H 2 S0 4 may be added instead of H 2 S0 4 (1 + 1). If org. mat-
ter other than urea exceeds 1.0 g, add addnl 1.0 mL H 2 S0 4
for each 0.1 g fat or 0.2 g other org. matter in excess of 1 .0
g.)
Proceed as in 970. 02B, par. 4, substituting 75 min for 60
min in last sentence.
Cool flask until it can be handled without gloves, and add
ca 250 mL H 2 0. Swirl to dissolve contents, and cool <25°.
Add ca 0.8 g Alundum granules to minimize bumping, tilt
flask, and add layer of NaOH without agitation. (For each 10
ml H 2 S0 4 used, or its equiv. in H 2 S0 4 (1 + 1), add 15 g solid
NaOH or enough so In to make contents strongly alk.) Proceed
as in 955. 04C, par. 2, beginning "Immediately connect flask
to distg bulb ..."
Ref.: JAOAC 61, 299(1978).
CAS-7727-37-9 (nitrogen)
970.03 Nitrogen (Total) in Fertilizers
Raney Powder Method
First Action 1970
Final Action 1975
(Applicable to all fertilizer samples except "nitric phosphates"
contg nonsulfate S. Caution: See safety notes on sulfuric acid
and mercury salts.)
A. Reagents
(a) Raney catalyst powder No. 2813.-50% Ni, 50% Al
(W. R. Grace & Co., Davison Chemical Division, 10 E Bal-
timore St, PO Box 2117, Baltimore, MD 21203-2117). Cau-
tion: Raney catalyst powders react slowly in H 2 or moist air
to form alumina; avoid prolonged contact with air or moisture
during storage or use.
(b) Sulfuric acid-potassium sulfate soln. — Slowly add 200
mL H 2 S0 4 to 625 mL H 2 and mix. Without cooling, add
106.7 g K 2 S0 4 and continue stirring until all salt dissolves.
Dil. to ca 1 L and mix. Cool, dil. to 1 L with H 2 0, and mix.
Avoid absorption of NH 3 from air during prepn particularly if
stream of air is used for mixing.
For other reagents, see 920. 02 A.
B. Determination
Place 0.2-2.0 g sample contg <42 mg nitrate N in 500-
800 mL Kjeldahl flask (800 mL flask is preferred with samples
which foam considerably, especially orgs). Add 1.7 g Raney
catalyst powder, 3 drops tributyl citrate, and 150 mL H 2 S0 4 -
K 2 S0 4 soln. If org. matter exceeds 0.6 g, add addnl 2.5 mL
of this soln for each 0. 1 g org. matter in excess of 0.6 g.
Swirl to mix sample with acid and place flask on cold burner.
If burner has been in use, turn off completely >10 min before
placing flask on burner. After flask is on burner, set heat input
to 5 min boil test. When sample starts boiling, reduce heat to
pass 10 min boil test. After 10 min, raise flask to vertical po-
sition and add 0.7 g HgO and 15 g K 2 S0 4 . (Contents of Kel-
Pak No. 5 (Curtin Matheson Scientific, Inc.) without plastic
container may be used.) Replace flask in inclined position and
increase heat to 4-5 min boil test. (Reduce heat input if foam
fills >: 2 /3 of bulb of flask. Use variable heat input until this
phase is past.) Heat at 4-5 min boil test until dense white
fumes of H 2 S0 4 clear bulb of flask. Digestion is now complete
for samples contg only ammoniacal, nitrate, and urea N. For
other samples, swirl flask gently and continue digestion addnl
30 min.
Proceed as in 955. 04C, second par. If 800 mL Kjeldahl flasks
have been used, add 300 instead of 200 mL H 2 0.
Refs.: JAOAC 53, 450(1970); 57, 10(1974).
CAS-7727-37-9 (nitrogen)
920.03 Nitrogen (Ammoniacal)
in Fertilizers
Magnesium Oxide Method
Final Action
(Not applicable in presence of urea)
Place 0.7-3.5 g, according to NH 3 content of sample, in
distn flask with ca 200 mL H 2 and >:2 g carbonate-free MgO.
Connect flask to condenser by Kjeldahl connecting bulb, distil
100 mL liq. into measured amt std acid, 920.02A(j), and titr.
with std NaOH soln, 920.02A(k), using Me red, 920.02A(i).
920.04* Nitrogen (Ammoniacal)
in Fertilizers
Formaldehyde Titration Method
Surplus 1970
(Applicable to NH 4 N0 3 and (NH 4 ) 2 S0 4 )
See 2.058, 11th ed.
920.05* Nitrogen (Ammoniacal and Nitrate)
in Fertilizers
Ferrous Sulfate-Zinc-Soda Method
Final Action 1965
Surplus 1970
(Not applicable in presence of org. matter, Ca cyanamide, and
urea)
See 2.059, 11th ed.
892.01 Nitrogen (Ammoniacal and Nitrate)
in Fertilizers
Devarda Method
Final Action
(Not applicable in presence of org. matter, Ca cyanamide, and
urea)
Place 0.35 or 0.5 g sample in 600-700 mL flask and add
300 mL H 2 0, 3 g Devarda alloy (Cu 50, Al 45, Zn 5), and
20
Fertilizers
AOAC Official Methods of Analysis (1990)
5 mL NaOH soln (42% by wt), pouring latter down side of
flask so that it does not mix at once with contents. By means
of Davisson (J. ind. Eng. Chem. 11, 465(1919)) or other suit-
able scrubbing bulb that will prevent passing over of any spray,
connect with condenser, tip of which always extends beneath
surface of std acid in receiving flask. Mix contents of distg
flask by rotating. Heat slowly at first and then at rate to yield
250 mL distillate in 1 hr. Collect distillate in measured amt
std acid, 920.02A(j), and titr. with std NaOH soln, 920.02A(k),
using Me red, 920.02 A(i).
Refs.: Chem. Ztg. 16, 1952(1892). JAOAC 6, 391(1923); 15,
267(1932).
930.01 Nitrogen (Nitrate) in Fertilizers
Robertson Method
Final Action
(Applicable in presence of Ca cyan amide and urea. Caution:
See safety notes on sulfuric acid and mercury.)
(a) Det. total N as in 955.04D, 970.02B, or 970.03B.
(b) Det. H 2 Oinsol. N as in 945.01, but use 2.5 g sample.
Dil. filtrate to 250 mL.
(c) Place 50 mL portion filtrate in 500 mL Kjeldahl flask
and add 2 g FeS0 4 .H 2 and 20 mL H 2 S0 4 . (If total N is >5%,
use 5 g FeS0 4 .7H 2 0.) Digest over hot flame until all H 2 is
evapd and white fumes appear, and continue digestion at least
10 min to drive off nitrate N. If severe bumping occurs, add
10-15 glass beads. Add 0.65 g Hg, or 0.7 g HgO, and digest
until all org. matter is oxidized. Cool, dil., add the K 2 S soln,
and complete detn as in 955. 04C. Before distn, add pinch of
mixt. of Zn dust and granular "20-mesh" Zn to each flask to
prevent bumping.
Total N (a) - H 2 0-insol. N (b) = H 2 0-sol. N.
H 2 0-sol. N - N obtained in (c) - nitrate N.
Refs.: JAOAC 13, 208(1930); 15, 267(1932); 56, 392(1973).
930.02 Nitrogen (Nitrate) in Fertilizers
Jones Modification of Robertson Method
Final Action
(Applicable when H 2 0-sol. N need not be detd.)
{Caution: See safety notes on sulfuric acid and mercury.)
Weigh 0.5 g sample into Kjeldahl flask, add 50 mL H 2 0,
and rotate gently. Add 2 g FeS0 4 .7H 2 and rotate. Add 20
mL H 2 S0 4 . Digest over hot flame. When H 2 evaps and white
fumes appear, add 0.65 g Hg and proceed as in 955. 04C.
Total N — N thus found = nitrate N
Refs.: JAOAC 13, 208(1930); 15, 267(1932).
945.01 Nitrogen (Water-Insoluble)
in Fertilizers
Method I
Final Action
(See 955.058(a) and (b) for urea-formaldehyde or mixts contg
such compds.)
Place 1 or 1.4 g sample in 50 mL beaker, wet with alcohol,
add 20 mL H 2 0, and let stand 15 min, stirring occasionally.
Transfer supernate to 1 1 cm Whatman No. 2 paper in 60° long-
stem funnel 60 mm diam., and wash residue 4 or 5 times by
decanting with H 2 at room temp. (20-25°). Finally transfer
all residue to filter and complete washing until filtrate mea-
sures 250 mL. Det. N in residue as in 955. 04C.
970.04 Nitrogen (Water-Insoluble)
in Fertilizers
Method II
First Action 1974
10 mm id, 12 mm od, con-
A. Apparatus
Extraction tube. — Glass, 250 X
stricted to 3-4 mm at one end.
B. Determination
Weigh 3.0 g unground mixed sample and place in extn tube
contg small glass wool plug. Place addnl glass wool pad on
top of sample. Connect 250 or 500 mL separator to column
with 75 mm piece of rubber tubing. Close stopcock of sepa-
rator and add 250 mL deionized H 2 0. Open stopcock and let
quick rush of H 2 pass thru column. After initial rush of H 2 0,
close stopcock. Adjust flow thru stopcock to ca 2 mL/min.
Squeeze rubber connection to bring level of H 2 ca 25 mm
above column bed. System then operates as constant-head
feeder.
After H 2 wash is complete, disconnect column from rub-
ber tubing. Invert column over Kjeldahl flask and force con-
tents into flask with aid of pressure bulb. Wash traces of sam-
ple from tube into Kjeldahl flask and wash sample from walls
of digestion flask with min. H 2 0. Det. N in residue as in 970.02
or 970.03.
Refs.: JAOAC 53, 808(1970); 56, 853(1973).
930.03* Nitrogen Activity in Fertilizers
Removal of Water-Soluble Nitrogen
Final Action
Surplus 1967
(a) Mixed fertilizers. — See 2.058, 10th ed.
(b) Raw materials. — See 2.058, 10th ed.
935.01* Nitrogen (Water-Insoluble)
in Cyanamide
Final Action
Surplus 1970
920.06* Nitrogen Activity in Fertilizers
Water-Insoluble Organic Nitrogen
Soluble in Neutral Permanganate
Final Action
Surplus 1987
See 2.063, 11th ed.
See 2.059, 10th ed.
AOAC Official Methods of Analysis (1990)
Nitrogen
21
920.07* Nitrogen Activity in Fertilizers
Water-Insoluble Organic Nitrogen
Distilled from Alkaline Permanganate
Final Action
Surplus 1987
See 2.060-2.061, 10th cd.
955.05 Nitrogen Activity Index (Al)
of Urea-Formaldehyde Fertilizers
Final Action 1965
(Applicable to urea-formaldehyde compds and mixts contg such
compds)
A. Reagent
Phosphate buffer soln.—pH 7.5. Dissolve 14.3 g KH 2 P0 4
and 91.0 g K 2 HP0 4 in H 2 and dil. to 1 L. Dil. 100 mL of
this soln to 1 L.
B. Determination
(a) Crush sample (do not grind) to pass No. 20 sieve.
(b) Det. cold H 2 0-insol. N (WIN) as in 945.01, keeping
temp, at 25±2°. Stir at 5 min intervals during 15 min standing.
(c) Det. hot H 2 0-insol. N (HWIN) in phosphate buffer soln
as follows: Place accurately weighed sample contg 0.1200 g
WIN in 200 mL tall-form beaker. Add ca 0.5 g CaC0 3 to mixed
fertilizers contg urea-HCHO compds. From supply of boiling
buffer soln, add 100 mL from graduate to sample, stir, cover,
and immerse promptly in boiling H 2 bath so that liq. in beaker
is below H 2 level in bath. Maintain bath at 98-100°, checked
with thermometer, and stir at 10 min intervals. After exactly
30 min, remove beaker from bath and filter promptly thru 15
cm Whatman No. 12 fluted paper. If filtration takes >4 min,
discard detn. Repeat detn, stirring in 1 g Celite filter-aid just
before removing beaker from bath, and filter.
Wash insol. residue completely onto paper with boiling H 2
and continue washing until total vol. used is 100 mL. Com-
plete washing before filtrate becomes cloudy or its temp, drops
to <60°. Det. total N (HWIN) in wet paper and residue as in
955.04C, using 35 mL H 2 S0 4 when CaC0 3 has been added.
Activity index (Al) = (%WIN - %HWIN) x 100/% WIN
Refs: JAOAC 38, 436(1955); 44, 245(1961).
959.03 Urea in Fertilizers
Urease Method
First Action 1959
Final Action 1960
A. Reagent
Neutral urease soln. — Use fresh com. 1% urease soln, or
dissolve 1 g urease powder in 100 mL H 2 0, or shake 1 g jack
bean meal with 100 mL H 2 5 min. Transfer 10 mL soln to
250 mL erlenmeyer, dil. with 50 mL H 2 0, and add 4 drops
Me purple (available from Fisher Scientific Co.; No. So-I-9).
Titr. with 0.17V HO to reddish purple; then back-titr. to green
with 0. lA r NaOH. From difference in mL, calc. vol. 0. IN HC1
required to neutze remainder of soln (usually ca 2.5 mL/100
mL), add this amount of acid, and shake well.
Verify enzyme activity of urease source periodically. Dis-
card any source which does not produce soln capable of hy-
drolyzing 0.1 g urea/20 mL soln.
B. Determination
Weigh 1-10±0.01 g sample (<1.0 g urea) and transfer to
15 cm Whatman No. 12 fluted filter paper. Leach with ca 300
mL H 2 into 500 mL vol. flask. Add 75-100 mL satd Ba(OH) 2
soln to ppt phosphates. Let settle and test for complete pptn
with few drops satd Ba(OH) 2 soln. Add 20 mL 10% Na 2 C0 3
soln to ppt excess Ba and any sol. Ca salts. Let settle and test
for complete pptn. Dil. to vol., mix, and filter thru 15 cm
Whatman No. 12 fluted paper. Transfer 50 mL aliquot to 200
or 250 mL erlenmeyer and add 1-2 drops of Me purple. Acid-
ify with 2N HC1 and add 2-3 drops excess. Neutze soln with
0AN NaOH to first change in color of indicator. Add 20 mL
neutral urease soln, close flask with rubber stopper, and let
stand 1 hr at 20-25°. Cool flask in ice-H 2 slurry and titr. at
once with 0AN HC1 to full purple; then add ca 5 mL excess.
Record total vol. added. Back-titr. excess HC1 with 0AN NaOH
to neut. end point.
% Urea = [(mLO AN HC1 - mL 0. \N NaOH)
X 0.3003]/g sample
Refs.: Ind. Eng. Chem. Anal. Ed. 7, 259(1935). JAOAC 41,
637(1958); 42, 494(1959); 43, 123(1960).
CAS-57-13-6 (urea)
983.01 Urea and Methyleneureas
(Water-Soluble) in Fertilizers
Liquid Chromatographic Method
First Action 1983
Final Action 1984
A. Principle
Sample is ground to pass 40 mesh sieve, extd with H 2 0,
and filtered. Urea, methylenediurea (MDU), and dimethyl-
enetriurea (DMTU) are detd by liq. chromatgy using external
stds and refractive index detection.
B. Apparatus
(a) Liquid chromato graph. — With refractive index detector
and pump capable of delivering mobile phase at 2 mL/min at
pressures up to 2000 psig. Operating conditions: flow rate 1.0
mL/min (1500 psi); attenuator 8x; ambient temp.; injection
vol. 10 \xL. Sample injector with fixed sample loop preferred.
(b) Chromatographic column. — Partisil 5 ODS-3, 4.6 mm
id x 25 cm (Whatman, Inc.; other manufacturers' small par-
ticle reverse phase columns may be substituted with adjust-
ments in operating conditions).
(c) Strip chart recorder. — Range to match output of de-
tector.
C. Reagents
(a) Mobile phase. — LC grade H 2 0.
(b) Purified methylenediurea (MDU) and dime thy lenetri-
urea (DMTU).- — Ext 50 g N-only ureaformaldehyde (UF) fer-
tilizer with acetone 8 h on soxhlet extractor. Select UF fertil-
izer with high MDU /DMTU -to-urea ratio. Remove thimble
from extractor, let air-dry, and collect residue. Mix 30 g ace-
tone-washed residue in 300 mL H 2 and filter or centrf . Inject
100 mL supernate onto Waters Associates PrepPak 500 C-18
cartridge (5.7x30 cm) in preparative liq. chromatograph (Waters
Associates Inc. Prep-500, orequiv.) at ambient temp, and with
H 2 mobile phase at 150 mL/min. Collect top third of MDU
and DMTU peaks. Evap. collected fractions to dryness in hood,
using heat lamps. Dry using vac. over P 2 5 . Confirm identity
using anal. liq. chromatogy and elemental analysis: mp of pu-
22
Fertilizers
AOAC Official Methods of Analysis (1990)
rified material, detd in Pyrex, should be 205-207°d for MDU
and 231~232°d for DMTU.
(c) External std solns. — (A) Accurately weigh ca 1.0 g each
of urea (Baker Analyzed Reagent) and purified MDU, transfer
both weighed compds to same 100 mL vol. flask, and dil. to
vol. with H 2 0. (B) Accurately weigh 0.0125, 0.025, 0.050,
and 0.10 g purified DMTU into sep. 50 mL vol. flasks. (C)
Pipet 2, 5, 10, and 15 mL of mixed urea/MDU stds (A) into
the vol. flasks from ( B), resp. Dil. to ca 40 mL with H 2 and
warm as necessary to dissolve DMTU. Cool to room temp.
and dil. to vol. Approx. std contents - (7) 0.25 mg DMTU
+ 0.4 mg urea/MDU per mL; (2) 0.50 mg DMTU + 1.0 mg
urea/MDU per mL; (3) 1 .00 mg DMTU + 2.0 mg urea/MDU
per mL; (4) 2.00 mg DMTU + 3.0 mg urea/MDU per mL.
D. Preparation of Sample
Grind sample to pass 40 mesh sieve. Accurately weigh 2.000
g well mixed ground sample into 200 mL vol. flask. Add 150
mL distd or deionized H 2 0, place on wrist-action shaker 20
min, and dil. to vol. with H 2 0. Using glass fiber paper, filter
portion into 4 mL vial. Filter again thru 0.45 jim filter before
injection.
E. Determination and Calculations
Inject 10 |xL of each mixed std until peak hts agree ±2%.
Inject 10 [xL sample. Repeat stds after all samples have been
injected. Std peak hts should agree within 3% of initial std
peak hts. Average peak hts for each component and plot mg/
mL vs peak hts.
% Urea N = rag/mL (from graph) x 9.33/g sample
% MDU N = mg/mL (from graph) X 8.484/g sample
% DMTU N - mg/mL (from graph) x 8.236/g sample
Ref.: JAOAC 66, 769(1983).
CAS-57-13-6 (urea)
CAS- 1 3547- 1 7-6 (methy Jenediurea)
988.01 Triamino-s-Triazine
in Fertilizer Mixes
Liquid Chromatographic Method
First Action 1988
C. Reagents
(a) Sodium phosphate. — Anhyd., dibasic. Na 2 HP0 4 , re-
agent grade or equiv.
(b) Diethy lamine. — Reagent grade or equiv.
(c) Phosphoric acid. — Reagent grade or equiv.
(d) Water. — Deionized or distd.
(e) Buffer soln.— pH 4.0.
(f) Mobile phase. — Deionized H 2 contg 1% (w/v) anhyd.
Na 2 HP0 4 and 1 mL diethylamine/L. Adjust to pH 4 with H 3 P0 4 .
(g) Triamino-s-triazine std solns . — (I) Stock std so In . — 5 00
mg/L (ppm). Accurately weigh 50.0 mg triamino-s-triazine
ref. std (Melamine Chemicals, Inc., PO Box 748, Donaldson-
ville, LA 70346) into 100 mL vol. flask. Dissolve in and dil.
to vol. with deionized H a O. (2) Working std solns. — 50, 125,
and 250 mg/L. Pipet 10, 25, and 50 mL stock std soln into
sep. 100 mL vol. flasks and dil. to vol. with deionized H 2 0.
Use as calibration stds.
D. Preparation of Sample
Grind >225 g sample (triamino-s-triazine granules or dry-
mix blends with other fertilizers) to pass No. 40 sieve, mix
thoroly, and store in tightly stoppered bottle.
Accurately weigh 5-8 g well mixed, ground sample and
transfer to 2 L vol. flask. Dil. to vol. with deionized H 2 and
stir 2 h using stir bar and mag. stirrer. Filter portion for analy-
sis thru 1 (xm glass fiber filter. Pipet 1 mL filtrate into 100
mL vol. flask and dil. to vol. with deionized H 2 0.
E. Determination
Equilibrate column with mobile phase for 30-60 min. Inject
20 (xL std soln until peak hts agree ±2%. Inject 20 |ulL sample
with attenuation set to give largest possible on-scale peaks.
Reinject std after every 10th sample to verify calibration and
ensure accurate quantitation.
F. Calculations
Calc. amt triamino-s-triazine as follows:
Triamino-s-triazine, % - {PH/PH') X |C/(5 x W)] x 100
where PH and PH' - peak hts for sample and std, resp.;
C = concn of std, ppm; and W = sample wt, g.
Ref.: J AOAC 71, 611(1988).
CAS- 108-78-1 (l,3,5-triazine-2,4,6-triamine; melamine)
A. Principle
Ground sample is extd with H 2 and filtered. Triamino-s-
triazine is detd by liq. chromatgy using external std and UV
detection at 254 nm.
B. Apparatus
(a) Liquid chromatograph. — With UV detection at 254 nm.
Operating conditions: flow rate 1.0 mL/min (1200 psi); col-
umn temp, ambient; chart speed 0.5 cm/min; injection vol. 20
(xL; sample injector with fixed sample loop preferred. Pump
LC mobile phase thru column until system is equilibrated. Al-
low 10 min run time for each injection. Retention time for
triamino-s-triazine is 4-5 min. Re-equilibrate baseline before
each injection.
(b) LC column.— LiChrosorb RP-18, 25 cm x 4.5 mm. (Use
this type column; chemistry of triamino-s-triazine requires use
of polar solv. system.)
(c) Strip chart recorder. — To match output of detector.
(d) pH meter. — Sensitivity 0.01. Stdze with pH 4 buffer
soln.
(e) Filters. — 2.4 cm glass fiber (Whatman 934-H or equiv.).
960.04 Biuret in Fertilizers
Spectrophotometric Method
First Action 1960
Final Action 1980
(Applicable to urea only. Do not use for mixed fertilizers.)
4. Reagents
(a) Alkaline tartrate soln. — Dissolve 40 g NaOH in 500 mL
H 2 0, cool, add 50 g NaKC 4 H 4 6 .4f4 2 0, and dil. to 1 L. Let
stand 1 day before use.
(b) Copper sulfate soln. — Dissolve 15 g CuS0 4 .5H 2 in
CCVfree H 2 and dil. to 1 L.
(c) Biuret. — To recrystallize, weigh ca 10 g reagent grade
biuret, transfer to 2 L beaker, add 1 L absolute alcohol, and
dissolve. Cone, by gentle heating to ca 250 mL. Cool at 5°
and filter thru fritted glass funnel. Repeat crystn and dry final
product 1 hr at 105-1 10° in oven. Remove from oven, place
in desiccator, and cool to room temp.
(d) Biuret std soln. — 1 mg/mL. Dissolve 1 .0000 g recrystd
biuret in C0 2 -free H 2 and dil. to I L.
AOAC Official Methods of Analysis (1990)
Potassium
23
B. Preparation of Standard Curve
Transfer series of aliquots, 2-50 mL, of std biuret soln to
100 mL vol. flasks. Adjust vol. to ca 50 mL with C0 2 -free
H 2 0, add 1 drop Me red, and neutze with 0. IN H 2 S0 4 to pink
color. Add, with swirling, 20 mL alk. tartrate soln and then
20 mL CuS0 4 soln. Dil. to vol., shake 10 sec, and place in
H 2 bath 15 min at 30 ±5°. Also prep, reagent blank. Det. A
of each soln against blank at 555 nm (instrument with 500-
570 nm filter is also satisfactory) with 2-4 cm cell. Plot std
curve.
C. Determination
Continuously stir <10 g sample contg 30-125 mg biuret in
150 mL ca 50° H 2 30 min. Filter and wash into 250 mL vol.
flask, and dil. to vol. Transfer 50 mL aliquot to 100 mL vol.
flask and proceed as in 960. 04B.
Refs.: JAOAC 43, 499(1960); 57, 1360(1974); 59, 22(1976);
60, 323(1977); 62, 153, 330(1979); 63, 222(1980).
CAS-108-19-0 (biuret)
976.01 Biuret in Fertilizers
Atomic Absorption Spectrophotometric Method
First Action 1976
Final Action 1980
A. Apparatus and Reagents
(a) Atomic absorption spectrophotometer . — IL Model 353
(Instrumentation Laboratory, Inc., 113 Hartwell Ave, Lexing-
ton, MA 02173), or equiv., with Cu hollow cathode lamp.
(b) Copper sulfate soln. — Dissolve 15 g CuS0 4 .5H 2 in
H,0 and dil. to 1 L.
(c) Buffer soln.— pH 13.4. Dissolve 24.6 g KOH and 30 g
KC1 in H 2 and dil. to 1 L.
(d) Starch soln. — Treat 1 g sol. starch with 10 mL cold
H 2 0, triturate to thin paste, and pour gradually into 150 mL
boiling FLO contg 1 g oxalic acid. Boil until soln clears, cool,
and dil. to 200 mL. Prep, fresh weekly.
(e) Bromocresol purple indicator. — Dissolve 0.1 g bromo-
cresol purple in 19 mL 0AN NaOH and dil. to 250 mL with
H 2 0.
(f) Biuret.— See 960.04A(c).
(g) Biuret std soln. — .4 mg/mL. Dissolve 0.4000 g re-
cry std biuret in warm H 2 0, cool, transfer to 1 L flask, and
dil. to vol.
(h) Copper std solns. — Dil. aliquots of Cu stock soln,
965.09B(b), with H 2 to obtain >4 std solns within range of
detn, 1-4 |xg Cu/mL final soln.
B. Preparation of Standard Curve
Transfer aliquots of biuret std soln contg 0, 2, 4, 6, 8, 10,
and 12 mg biuret to sep. 100 mL vol. flasks, dil. to ca 30 mL
with H 2 0, and add 25 mL alcohol to each. While stirring with
mag. stirrer, add 2 mL starch soln, 10 mL CuS0 4 soln, and
20 mL buffer soln. Remove stirring bar, rinse, dil. to vol.,
mix thoroly, and let stand 10 min. With vac, filter ca 50 mL
thru dry 150 mL medium porosity fritted glass funnel into dry
flask. Transfer 25 mL aliquots of each filtrate to 250 mL vol.
flasks, acidify with 5 mL IN HCI, and dil. to vol. with H z O.
Proceed as in 965.09, using std solns, 976.01 A(h), to det.
complexed Cu in soln by AA spectrophotometry after adding
equiv. amts of alcohol, KOH soln, buffer soln, and \N HCI.
Take >3 readings of each soln. From mean value of Cu concn,
prepare std curve relating mg Cu found to mg biuret added.
Redet. daily.
C. Determination
(a) In urea. — Accurately weigh sample contg <10 mg biu-
ret, dissolve in H 2 0, transfer to 100 mL vol. flask, add 25 mL
alcohol, and proceed as in 976. 01B, beginning "While stirring
with mag. stirrer, ..." From Cu found, calc, biuret concn,
using std curve.
(b) In mixed fertilizers . — Transfer accurately weighed sam-
ple contg <40 mg biuret to 250 mL beaker and add 1 mL H 2
for each g of sample (5 g max.). Warm, add 65 mL alcohol
and 7 drops bromocresol purple, and adjust pH to first blue
color (pH 6-7) with 20% KOH. Place on hot plate, heat to
bp, cool, and, if pH has changed, make final adjustment to
first blue. Vac. -filter thru alcohol-washed paper pulp pad into
100 mL vol. flask. (If filtrate is not clear, improper pH ad-
justment has been made. Add HCI and readjust to pH 6-7.)
Wash pad and ppt with alcohol and dil. to vol. with alcohol.
Transfer 25 mL aliquot to 100 mL vol. flask, and proceed as
in 976. 01B, beginning "While stirring with mag stirrer, ..."
From Cu found, calc. biuret concn, using std curve and ap-
propriate diln factors. (Final aliquot can be varied to give Cu
concn between 1 and 4 |mg/mL.)
Refs.: JAOAC 59, 22(1976); 62, 153(1979); 63, 222(1980).
CAS-108-19-0 (biuret)
POTASSIUM
935.02* Potassium in Fertilizers
Lindo-Gladding Method
Final Action
Surplus 1970
See 2.076-2.078, llthed.
949.01* Potassium in Fertilizers
Wet-Digestion Method
Final Action
Surplus 1970
See 2.079-2.080, 11th ed.
945.02* Recovery of Platinum
Procedure
Final Action
Surplus 1970
See 2.081-2.083, 11th ed.
983.02 Potassium in Fertilizers
Flame Photometric Method
(Manual or Automated)
First Action 1983
Final Action 1985
(Caution: See safety notes on flame photometer.)
A. Method Parameters
Any flame photometer, manual or automated, capable of de-
tecting K, using Li as internal std, and meeting method per-
formance characteristics described below, is satisfactory. Sam-
24
Fertilizers
AOAC Official Methods of Analysis (1990)
pies are extd with ammonium oxalate soln or ammonium citrate
soln. Appropriate dilns of ext are mixed with LiN0 3 internal
std soln and aspirated or pumped into flame photometer. La 2 3
is added to LiN0 3 soln to eliminate the phosphate effect. Final
soln to be introduced to flame should have the following com-
position: (a) concn of K 2 in range such that std curve re-
sponse is linear over that range, (b) const amt of Li in range
5 to 40 ppm, (c) selected concn of La <1400 ppm, and (d)
0.2N HN0 3 . Exact concn of LiN0 3 and La 2 3 are optimized
for particular instrumentation as described in performance
specifications below. Ratio of K intensity at 768 nm to Li in-
tensity at 671 nm is detd, and compared with similar ratios
from std set of >6 stds, prepd from NBS or primary std KH 2 P0 4 .
Stds are arranged in ascending order and evenly distributed
thru chosen range.
&. Preparation of Sample
(a) Ammonium oxalate extraction. — Weigh 1 g sample into
500 mL vol. flask, add 50 mL 4% (NH 4 ) 2 C 2 4 and 125 mL
H 2 0, boil 30 min, and cool. Dil. to vol. with H 2 0, mix, and
filter or let stand until clear.
(b) Ammonium citrate extraction from direct available
phosphorus extract. — Prep, as in 960. 03B. (If solns must be
held overnight, add 3-4 drops of CHC1 3 .)
C. Performance Specifications
System performance criteria. — Detailed example of specific
instrumental system capable of meeting specified performance
criteria follows this performance section. It is necessary to ver-
ify that this or any other particular system meets all of the
following performance criteria before samples are analyzed.
Levels specified are to be considered min. acceptable levels.
Various criteria are written for automated instrument, but should
also apply to manual instrument systems.
(a) LiN0 3 concentration level. — Amt of LiN0 3 in final soln
aspirated into flame is adjusted partly for convenience of in-
strument parameters, but should be such that Li and K chan-
nels give roughly equal responses. This can be detd either by
displaying each channel's output sep., or by displaying ratio
of K to Li response and then interchanging Li and K filters
and displaying ratio again.
Using either procedure, sample midrange K + stds under
analysis conditions while varying concn of Li + until acceptable
concn of Li + is found.
(b) Noise. — Adjust detector output to 90% full scale with
high std sampled continuously. Noise must be <2% full scale
peak to peak. Note that some instruments, flow injection anal-
ysis systems, for example, are not designed to pump samples
continuously. In this case, substitute repeated sampling for
continuous sampling, and consider noise to be difference be-
tween adjacent peak maxima. (Optimum performance on ex-
ample instrument system described in this method is ca 1 / 2 %
peak to peak. To reduce noise on example system, stabilize
flame, stabilize pumping rate, stabilize back-pressure, change
pump tubes, clean manifold, and /or rework manifold to en-
sure adequate mixing. To det. min. noise limit of instrument,
collect system waste soln, connect short length of tubing di-
rectly to photometer aspirator, and aspirate waste soln directly
into flame.)
(c) Carryover. — Adjust detector output to give ca 10% and
90% full scale response for low and high stds, resp. Sample
3 high stds followed by 5 low stds on system under analysis
conditions. Carryover, defined as difference between first low
std and mean of other low stds, may not be >1% full scale.
(Optimum performance on example system is negligible car-
ryover. To reduce carryover on example system, clean man-
ifold and aspirator, check manifold connections for dead space,
redesign manifold shortening hydraulic system wherever pos-
sible, decrease sampling rate, and/or reduce std range.)
(d) Drift. —Adjust instrument to give detector response ca
50% full scale with middle std sampled continuously. Sample
middle std continuously for the time it would take to analyze
30 samples. For instruments not designed to sample continu-
ously, draw smooth line thru 30 middle std peaks. Drift may
not exceed 1% full scale per any 10 sample segment. (Opti-
mum performance on example system is zero drift. To reduce
drift on example system, stabilize room and soln temps, adjust
manifold to maintain const back-pressure, and /or stabilize
flame.) As long as drift does not exceed 1% per 10 peak level,
routine data may be further improved by inserting a middle std
periodically between groups of samples. This allows mathe-
matical peak ht correction, assuming linear drift.
(e) Precision.— -With instrument calibrated for 10% and 90%
full scale for low and high stds, resp., sample 30 middle stds
under analysis conditions. Range of instrument response may
not vary >2% full scale. (Optimum performance on example
system is 0.7% full scale. To improve precision on example
system, reduce noise, check sampler timing, and/or decrease
sampling rate.)
(f) Std curve. — Std curve consists of >6 different stds, evenly
distributed thru std concn range. Prep, solns from NIST or
primary std KH 2 P0 4 , dried 2 h at 105°. Include factor for ac-
tual purity of std material in calcns of std concn.
With instrument calibrated for ca 10% and 90% response
for low and high stds, resp., run stds in order of ascending
concn under analysis conditions. Response should be linear.
Mathematically perform first degree least squares fit to std curve
data. Alternatively, use calculator capable of least squares fits.
First order least squares fit may be performed as follows: As-
sume that points to be fitted are {X h Y } , (X 2 , Y 2 ), . . . (X n ,
Y„). Calc. means by:
X = -2Xj
n
1
-2/i
Slope of least square fitted line is given by:
bi
S(Xj - X)(Yi - Y) ax/d - nXY
(XX^-lMSXj) 2
%(X } - xf
Intercept for line is given by:
b = Y - bjl
Equation of resulting line is:
Y = b + bjX
Using derived equation and individual std responses, calc.
concn for each std. Compare calcd and known concns for each
std. Calcd value may not differ from known value by >±2%
in any one instance. Also, av. of absolute values of those %
differences may not be >1%. (Optimum performance on ex-
ample system is 0.75% and 0.37%, resp. To improve std curve
fit, optimize parameters (b) thru (e) above and /or reduce std
range.)
(g) Phosphate effect. — For example system, amt of La 2 3
in LiN0 3 reagent is sufficient to eliminate phosphate effect
(depression of instrument response to K by phosphate ion). If
other than example automated system is used, elimination of
phosphate effect must be verified. Using KN0 3 , prep. 200 mL
soln of K 2 with concn equal to twice that of highest std. Pipet
50 mL of that soln into each of two 100 mL vol. flasks. Dil.
one to vol. and mix. Add sufficient NH 4 H 2 P0 4 soln to the
other flask such that concn of P 2 5 will be as high as highest
concn of P 2 5 anticipated in any sample ext. Dil. to vol. and
mix. Sample 10 portions of each soln, alternating, under anal-
AOAC Official Methods of Analysis (1990)
Potassium
25
ysis conditions. Average 10 responses for each soln. Av. re-
sponses of the 2 solns must not differ from each other by >1%.
Select min. amt of La 2 3 which will eliminate phosphate ef-
fect. (Optimum performance of example system is <0.5%. To
improve performance, adjust amount of La 2 3 .)
(h) Overall performance of system. — Performance charac-
teristics mentioned above are worst case examples. A system
functioning marginally in many categories would probably fail
the following overall performance check.
Verify overall performance as follows: Ext and analyze once
each 20 different Magruder samples, or other similar perfor-
mance check samples previously detd by interlaboratory study.
Also ext and analyze 5 independent 1 g portions of NIST or
primary std KH 2 P0 4 . Randomize Magruder and KH 2 P0 4 sam-
ple order. Calc. % K 2 0. Av. bias of Magruder results, 2 (Ma-
gruder grand av. - calcd % K 2 O)/20, must be <±0.1. Av.
of absolute va]ue of differences must be <0.4. (Optimum val-
ues on example system are ca ±0.02 and —0.2, resp.)
For 5 analyses of KH 2 P0 4 , difference between mean of calcd
% K 2 and known % K 2 must not be >±0.2, and std de-
viation must not be >0.25. (Optimum values for example sys-
tem are ±0.1 and 0.15, resp.)
(i) Ongoing performance checks. — (/) Conduct daily per-
formance check by analyzing same performance check sample
at least once in every 60 regular samples, and at least once in
each run. (2) Repeat (h) above at least twice per year, and
whenever system has not been used for prolonged periods.
Example Automated instrument System
D. Apparatus
Automatic analyzer. — Auto Analyzer with following mod-
ules (available from Technicon Instruments Corp.): sampler II
or IV, pump III, flame photometer IV, and recorder. Com-
puter or calculator capable of least square fits is desirable.
E. Reagents
(a) Ammonium oxalate soln. — Dissolve 40 g (NH 4 ) 2 C 2 4 in
1 L H 2 0.
(b) Ammonium citrate soln. — Should have sp. gr. of 1.09
at 20° and pH of 7.0 as detd potentiometrically.
Dissolve 370 g cryst. citric acid in 1.5 L H 2 and nearly
neutze by adding 345 mL NH 4 OH (28-29% NH 3 ). If concn
of NH 3 is <28%, add correspondingly larger vol. and dissolve
citric acid in correspondingly smaller vol. H 2 0. Cool, and check
pH. Adjust with NH 4 OH (1+7) or citric acid soln to pH 7.
Dil. soln, if necessary, to sp. gr. of 1.09 at 20°. (Vol. will be
ca 2 L.) Keep in tightly stoppered bottles and check pH from
time to time. If pH has changed from 7.0, readjust.
(c) Lithium nitrate soln. — Dissolve 1 .642 g La 2 3 in 30 mL
HN0 3 , add 0.9935 g dried (2 h at 105°) LiN0 3 and 1 mL
Flaminox 1% soln (Fisher Scientific Co.), and dil. to 1 L with
H 2 0.
(d) Sampler wash and dilution water soln. — Dil. 1 mL
Flaminox 1% soln to 1 L with H 2 0.
(e) Potassium std solns.— (1) Stock std soln. — \ mg K 2 0/
mL. Dissolve 2.889 g dried (2 h at 105°) KH 2 P0 4 (NIST SRM
200) in H 2 0, and dil. to 1 L. (2) Working std solns. — 10, 20,
30, 40, 50, and 55 [ig K 2 0/mL. Accurately measure by buret
10, 20, and 30 mL stock std soln into 1 L vol. flasks, and 20,
25, and 27.5 mL into 500 mL vol. flasks. Add 0.2 g (NH 4 ) 2 C 2 4
per 500 mL final vol. if samples are prepd by ammonium ox-
alate extn, or add 12 mL ammonium citrate soln per 500 mL
final vol, if samples are prepd by ammonium citrate extn. Dil.
to vol. with H 2 and mix. (Add 3 mL CHC1 3 to preserve ci-
trate std solns for long periods.)
F. Analytical System
Assemble manifold as in Fig. 983.02. Use 1.6-2.0 mm id
glass transmission tubing for all reagent flow upstream from
Dl fitting. Use clear std pump tubes for air and soln stream
flow.
Air and H 2 are combined thru injection fitting (116-0492-
01). Hard thin-wall polyethylene tubing (ca 0.30 in. id) con-
nects air bar tubing to injection fitting. Sample is introduced
immediately downstream thru second injection fitting (194-
G0 12-01), designed to eliminate double peaks in recorder out-
put. Mixing of sample and H 2 occurs in double 10-turn coil
with insert (157-B089). LiN0 3 reagent is introduced thru in-
sert. Another 10-turn coil (157-0251) further mixes solns.
Portion of soln is aspirated to flame photometer thru A4 fit-
ting (116-0200-04). Hard, thin-wall polyethylene tubing (ca
0.045 in. id) connected to photometer is inserted and glued to
tee arm of A4 fitting. Remaining unaspirated soln is drawn
thru double 10-turn mixing coil (157-9248-01) and thru Dl
fitting (116-0203-01). Large diam. branch of Dl fitting leads
to pump and waste. Small diam. branch of Dl fitting is con-
0.23 ML /MIN
SAMPLE
c
D 0000 0000
A 1.00 ML /MIN
WATER
f °l
owo
B
0.32 ML/ MIN
AIR
0.80 ML/ MIN
UNO*
SAMPLER IV
7
~ .. 2.00 ML/ MIN
WATER
1 V w
TO WA
E 0000
~„ 0.80 ML/MIM
0000 G
F
TO 6'
OF .030
TY60N TUBING
-<r
(J)
1 TT 1
ywj
FLAME
PHOTOMET
ER
RECORDER
FIG. 983.02— Manifold for K 2 in fertilizers. A, injection fitting 116-0492-01; B, injection fitting 194-G012-01; C, double 10-turn
coil with insert 157-B089; D, 10-turn coil 157-0251; E, A4 fitting 116-0200-04; F, double 10-turn coil 157-0248-01; G, D1 fitting
116-0203-01
NORTHFAST WISCONSIN TFFhNsCAL C0U.E(it
LEARNING RESOURCE CENTER
GBEEM BAY, Wl 54307
26
Fertilizers
AOAC Official Methods of Analysis (1990)
nected to 6 ft (I . .83 m) of Tygon tubing (0.030 in. id) to waste.
Dl. fitting is oriented with small diam. branch low, so that only
soln, and no air, enters 0.030 in. tubing. This establishes const
back-pressure and therefore stable aspiration conditions at flame
photometer.
G. Startup and Shutdown Procedures
Start system and place reagent lines in proper solns. Let
equilibrate 30 min before beginning calibration. Adjust flame
photometer as follows: (/) damping control to damp 3 posi-
tion; (2) flame ht of main cone ca 4 cm; (3) atomizer adjust
control set to give atomization rate of ca 1.3-1.4 mL/min.
Rate of atomization is detd by subtracting rate of flow to waste
from rate of flow upstream from (disconnected) A4 fitting. Use
0.3 and 0.6 neut. density filters for Li and K detectors, resp.
Initially it may be necessary to manually fill system down-
stream from A4 fitting with H 2 0, making certain that 6 ft of
0.030 tubing is filled. System is shut down after pumping H 2
thru reagent lines 5:15 min.
H. Checkout and Calibration
After equilibration, pump 10 (xg K 2 0/mL std thru system
and adjust baseline control of photometer to read 10% full scale.
Pump 55 jmg K 2 0/mL std thru system and adjust std calibra-
tion control to read 90% full scale. If noisy conditions exist,
check for aspiration of air at A4 fitting, or check for air en-
tering lower arm of Dl fitting. If drift exists, check room and
solns for temp, stability. Std curves should be virtually linear.
/. Determination
Pipet aliquots of sample solns. Table 983.02, into 250 mL
vol. flask, dil. to vol. with H 2 0, and mix 15 times. For 10
mL aliquots of citrate extns, add 4 mL ammonium citrate so In
to aliquots before dilg to vol. Run samples in groups of 10.
Place 10 thru 55 fxg K 2 0/mL stds in order in sampler, pre-
ceded by extra 10 (xg/mL std. Place 30 |JLg/mL std after every
10th sample, to be used for drift correction. End series with
two 30 |xg/mL stds. Sample at rate of 40/h, 2:1 sample-to-
wash ratio.
J. Calculations
Correct sample peak hts for drift. Correct peak hts of first
10 samples as follows:
// c = // - |(O t - Z> )/14][L + 3]
where H c — corrected peak ht; H = uncorrected peak ht;
£>! - ht of first drift correction std; D = ht of 30 \xg std in
initial std sequence; and L - position No. of sample peak to
be corrected. Correct subsequent sample peak hts as follows:
# c = H - [D x - D ] - \{D y - D X )/11][P]
where D x = ht of drift std preceding sample to be corrected;
D y = ht of drift std following sample to be corrected; and
P = position No. of sample within group of 10.
Calc. least squares fitted curve of emission against K 2 concn.
Calc. (xg K 2 0/mL of corrected peak hts from equation:
% K 2 = (|xg K 2 0/mL X 12.5) /(aliquot X g sample)
Ref.: JAOAC 66, 1242(1983).
Table 983.02 K 2 Aliquots
K 2 Expected, %
Aliquot, mL
<2
2-6.49
6.50-19.99
>20
250 (no diln)
100
30
10
CAS-7440-09-7 (potassium)
CAS- 12 136-45-7 (potassium oxide)
955.06* Potassium in Fertilizers
Flame Photometric Method
Final Action
Surplus 1986
See 2.108-2.113, 14th ed.
971 .01 • Potassium in Fertilizers
Automated Flame Photometric Method
First Action 1971
Final Action 1973
Surplus 1986
See 2.114-2.118, 14th ed.
958.02 Potassium in Fertilizers
Volumetric Sodium Tetraphenylboron Method I
First Action 1958
Final Action 1960
(Caution: See safety notes on formaldehyde.)
A. Reagents
(a) Formaldehyde soln. —31%.
(b) Sodium hydroxide soln. — 20%. Dissolve 20 g NaOH in
100 mL H 2 0.
(c) Sodium tetraphenylboron (STPB) soln.— Approx. 1.2%.
Dissolve 12 g NaB(C 6 H 5 ) 4 in ca 800 mL H 2 0. Add 20-25 g
Al(OH) 3 , stir 5 min, and filter (Whatman No. 42 paper, or
equiv.) into 1 L vol. flask. Rinse beaker sparingly with H 2
and add to filter. Collect entire filtrate, add 2 mL 20% NaOH,
dil. to vol. with H 2 0, and mix. Let stand 48 hr and stdze.
Adjust so that 1 mL STPB = 1% K 2 0. Store at room temp.
(d) Benzalkonium chloride (BAC) soln, — Approx. 0.625%.
Dil. 38 mL 17% Zephiran chloride (Winthrop Laboratories;
also available at local pharmacies as benzalkonium chloride)
to 1 L with H 2 0, mix, and stdze. Cetyltri methyl ammonium
bromide may be substituted for Zephiran chloride. If other concn
is used, adjust vol.
(e) Clayton Yellow (Titan Yellow; Colour Index No.
19540).— 0.04%. Dissolve 40 mg in 100 mL H 2 0.
B. Standardization of Solutions
(a) BAC soln.— To 1.00 mL STPB soln in 125 mL erlen-
meyer, add 20-25 mL H 2 0, 1 mL 20% NaOH, 2.5 mL HCHO,
1.5 mL 4% (NH 4 ) 2 C 2 0^ and 6-8 drops indicator, (e). Titr. to
pink end point with BAC soln, using 10 mL semimicro buret.
Adjust BAC soln so that 2.00 mL - 1.00 mL STPB soln.
(b) Sodium tetraphenylboron soln. — Dissolve 2.500 g
KH 2 P0 4 in H 2 in 250 mL vol. flask, add 50 mL 4%
(NH 4 ) 2 C 2 4 soln, dil. to vol. with H 2 0, and mix. (It is not
necessary to bring to boil.) Transfer 15 mL aliquot (51.92 mg
K 2 0, 43.10 mg K) to 100 mL vol. flask; add 2 mL 20% NaOH,
5 mL HCHO, and 43 mL STPB reagent. Dil. to vol. with
H 2 0, mix thoroly, let stand 5—10 min, and pass thru dry filter.
Transfer 50 mL aliquot of filtrate to 125 mL erlenmeyer, add
6-8 drops indicator, (e), and titr. excess reagent with BAC
soln. Calc. titer as follows:
AOAC Official Methods of Analysis (1990)
Other Elements 27
F = 34.61/(43 mL - mL BAC) - % K 2 0/mL STPB reagent
Factor F applies to all fertilizers if 2.5 g sample is dild to 250
mL and 15 mL aliquot is taken for analysis. If results are to
be expressed as K rather than as K 2 0, substitute 28.73 for
34.61 in calcg F.
C. Determination
Place 2.5 g sample (1.25 g if K 2 >50%) in 250 mL vol.
flask, add 50 mL 4% (NH 4 ) 2 C 2 4 and 125 mL H 2 0, and boil
30 min. (If org. matter is present, add 2 g K-free C before
boiling.) Cool, dil. to vol. with H 2 0, mix, and pass thru dry
filter or let stand until clear. Transfer 15 mL aliquot sample
soln to 100 mL vol. flask and add 2 mL 20% NaOH and 5
mL HCHO. Add 1 mL std STPB soln for each 1% K 2 ex-
pected in sample plus addnl 8 mL excess to ensure complete
pptn. Dil. to vol. with H 2 0, mix thoroly, let stand 5-10 min,
and filter thru dry paper (Whatman No. 12 or equiv.). Transfer
50 mL filtrate to 125 mL erlenmeyer, add 6-8 drops indicator,
(e), and titr. excess reagent with std BAC soln.
% K 2 in sample = (mL STPB added - mL BAC) X F
where F = % K 2 0/mL STPB reagent. (Multiply by 2 if 1.25
g sample was used.)
Refs: Anal. Chem. 29, 1044(1957); 30, 1882(1958). JAOAC
41, 533(1958); 43, 472(1960).
CAS-7440-09-7 (potassium)
CAS- 13547- 17-6 (potassium oxide)
D. Determination
Transfer 25 mL aliquot of sample soln to 100 mL vol. flask.
(If org. matter is present, treat 100 mL portion with 1 g K-
free C and filter before transferring aliquot.) Add 8 mL HCHO
first and then 5 mL 20% NaOH soln, and wash down sides of
flask with H 2 0. Swirl and add 1 mL STPB for each 1.5 mg
K 2 expected in sample aliquot plus addnl 8 mL excess to
ensure complete pptn. Dil. to vol. with H 2 0, mix thoroly, let
stand 5-10 min, and pass thru dry filter (Whatman No. 12,
or equiv.). Transfer 50 mL aliquot filtrate to 125 mL erlen-
meyer, add 6-8 drops indicator, 958*02A(e), and titr. excess
reagent with std BAC soln.
% K 2 in sample - (mL STPB added - mL BAC) x F x 2
Calcn applies to all fertilizers if 1 g sample is dild to 500
mL and 25 mL aliquot is taken for analysis.
Ref: JAOAC 52, 566(1969).
CAS-7440-09-7 (potassium)
CAS- 13547- 17-6 (potassium oxide)
OTHER ELEMENTS
965.09 Nutrients (Minor) in Fertilizers
Atomic Absorption Spectrophotometric Method
First Action 1965
Final Action 1969
969.04 Potassium in Fertilizers
Volumetric Sodium Tetraphenylboron Method II
First Action 1969
Final Action 1970
(For use with sample prepd for available P detn)
(Caution: See safety notes on formaldehyde.)
A Reagents
See 958.02A(a), (b), (c), (d), and (e).
B. Standardization of Solutions
(a) BAC soln.— in 125 mL erlenmeyer, add 2.5 mL neut.
NH 4 citrate soln, 963.03A(a), 15-20 mL H 2 0, 4 mL HCHO,
and 2.5 mL 20% NaOH soln. Swirl; then add 4.00 mL STPB
soln and 6-8 drops indicator, 958.02A(e). Titr. to pink end
point with BAC soln, using 10 mL semimicro buret. Adjust
BAC soln so that 2.00 mL - 1.00 mL STPB soln.
(b) Sodium tetraphenylboron soln. — Dissolve 1.4447 g pri-
mary std KH 2 P0 4 in H 2 in 500 mL vol. flask, add 100 mL
neut. NH 4 citrate soln, 963.03A(a), dil. to vol. with H 2 0, and
mix. Transfer 25 mL aliquot (25.00 mg K 2 0, 20.75 mg K) to
100 mL vol. flask; add 8 mL HCHO and 5 mL 20% NaOH,
swirl, and add 25 mL STPB reagent. Dil. to vol. with H 2 0,
mix thoroly, let stand 5-10 min, and pass thru dry filter. Transfer
50 mL aliquot of filtrate to 125 mL erlenmeyer, add 6-8 drops
indicator, 958.02A(e), and titr. excess reagent with BAC soln.
Calc. titer as follows:
F = 25 mg K 2 0/(25 mL STPB - mL BAC)
= mg K 2 0/mL STPB reagent
if results are to be expressed as K rather than K 2 0, substitute
20.75 for 25 in calcg F.
C. Preparation of Sample
Prep, as in 960.03B.
(Caution: See safety notes on atomic absorption spectropho-
tometer.)
A. Apparatus and Reagent
(a) Atomic absorption spectrophotometer. — Several com.
models are available. Since each design is somewhat different,
with varying requirements of light source, burner flow rate,
and detector sensitivity, only general outline of operating pa-
rameters is given in Table 965.09. Operator must become fa-
miliar with settings and procedures adapted to his own app.
and use table only as guide to concn ranges and flame con-
ditions.
(b) Disodium EDTA soln.— 2.5%. Dissolve 25 g Na 2 H 2
EDTA in 1 L H 2 and adjust to pH 7.0 with 5N NaOH, using
pH meter.
B. Standard Solutions
(Do not use <2 mL pipets or <25 mL vol. flasks. Automatic
diln app. may be used. Prep, std solns in 0-20 \xg range fresh
daily.)
(a) Calcium solns. — (J) Stock soln. — 25 |xg Ca/mL. Dis-
solve 1.249 g CaC0 3 in min. amt 3/V HC1. Dil. to 1 L. Dil.
50 mL to 1 L. (2) Working std solns.— 0, 5, 10, 1.5, and 20
|xg Ca/mL contg 1% La. To 25 mL vol. flasks add 0, 5, 10,
15, and 20 mL Ca stock soln. Add 5 mL La stock soln and
dil. to 25 mL.
(b) Copper stock soln. — 1000 juig Cu/mL. Dissolve 1.000
g pure Cu metal in min. amt HN0 3 and add 5 mL HO. Evap.
almost to dryness and dil. to 1 L with 0. 1W HC1.
(c) Iron stock soln. — 1000 |xg Fe/mL. Dissolve 1.000 g pure
Fe wire in ca 30 mL 6N HC1 with boiling. DiJ. to 1 L.
(d) Lanthanum stock soln. — 50 g La/L. Dissolve 58.65 g
La 2 3 in 250 mL HC1, adding acid slowly. Dil. to 1 L.
(e) Magnesium stock soln. — 1000 |xg Mg/mL. Place 1.000
g pure Mg metal in 50 mL H 2 and slowly add 10 mL HCL
Dil. to 1 L.
28
Fertilizers
AOAC Official Methods of Analysis (1990)
(f ) Manganese stock soln. — 1000 |xg Mn/mL. Dissolve L582
g Mn0 2 in ca 30 mL 67V HO. Boil to remove CI and dil. to
1 L.
(g) Zinc stock soln, — 1000 {xg Zn/mL. Dissolve 1.000 g
pure Zn metal in ca 10 mL 67V HCL Dil. to 1 L.
(h) Other std solns. — Dil. aliquots of solns (b), (c), (e),
(f ), and (g) with 0.57V HC1 to make >4 std solns of each ele-
ment within range of detn.
C. Preparation of Sample Solutions
{Caution: See safety notes on wet oxidation, hydrofluoric acid,
and perchloric acid.)
(a) Inorganic materials and mixed fertilizers. — Dissolve 1 .00
g well ground sample in 10 mL HC1 in 150 mL beaker. Boil
and evap. soln nearly to dryness on hot plate. Do not bake
residue. Redissolve residue in 20 mL 27V HC1, boiling gently
if necessary. Filter thru fast paper into 100 mL vol. flask,
washing paper and residue thoroly with H 2 0. Measure ab-
sorption of soln directly, or dil. with 0.57V HC1 to obtain solns
within ranges of instrument. If Ca is to be detd, add enough
La stock soln to make final diln 1% La (i.e., 5 mL La to 25
mL flask, 20 mL to 100 mL flask, etc.).
(b) Fertilizers containing organic matter ( tankage y corn-
cobs, cottonseed hulls, etc.). — Place 1 .00 g sample in 150 mL
beaker (Pyrex, or equiv.). Char on hot plate and ignite 1 hr
at 500° with muffle door propped open to allow free access of
air. Break up cake with stirring rod and dissolve in 10 mL
HC1 as in (a).
(c) Fertilizers containing fritted trace elements. — Dissolve
<1.00 g well ground sample in 5 mL HC10 4 and 5 mL HF.
Boil and evap. to dense HC10 4 fumes. Dil. carefully with H 2 0,
filter, and proceed as in (a). Alternatively, dissolve sample in
10 mL HCI, 5 mL HF, and 10 mL MeOH. Evap. to dryness.
Add 5 mL HC1 and evap. Repeat HCI addn and evapn. Dis-
solve residue as in (a). (Normally Pt ware should be used;
Pyrex or other glassware may be used if Na, K, Ca, and Fe
are not to be detd.)
(d) For manganese . — (J ) Acid-soluble, for both Mn + ~ and
Mn +4 .—See (a), (b), and (c), and 972.02(b). (2) Acid-soluble,
for Mn' 2 only.— See 972.02(a), 940.02, and 941.02*. (3)
Water-soluble, for Mn +2 only. — See 972.03.
(e) For iron and zinc. — (7) Aqueous extraction. — Place 1.00
g sample in 250 mL beaker, add 75 mL H 2 0, and boil 30 min.
Filter into 100 mL vol. flask, washing paper with H 2 0. Dil.
to vol. and redil. if necessary. (2) Chelation extraction. — Place
1.00 g sample in 250 mL beaker, and add 5 cm (2") mag.
stirrer bar and 100 mL 2.5% EDTA soln. Stir exactly 5 min,
and filter thru Whatman No. 41 paper, or equiv. If filtrate is
cloudy, refilter immediately thru fine paper (Whatman No. 5,
or equiv.). Redil., if necessary, with 0.57V HCI.
D. Determination
(P interferes in Ca and may interfere in Mg detn with air-C 2 H 2
burners. Eliminate interference by adding La stock soln to std
and sample solns so that final dilns contain 1% La. P does not
interfere with Ca detn when N 2 0-C 2 H 2 flame is used.)
Set up instrument as in Table 965.09, or previously estab-
lished optimum settings for app. to be used. Less sensitive
secondary lines (Gatehouse, and Willis, Spectrochim. Acta 17,
710(1961)) may be used to reduce necessary diln, if desired.
Read ^4 std solns within anal, range before and after each
group of 6-12 samples. Flush burner with H 2 between sam-
ples, and re-establish absorption point each time. Prep, cal-
ibration curve from av. of each std before and after sample
group. Read concn of samples from plot of absorption against
fxg/mL.
Table 965.09 Operating Parameters
Wave-
Range,
Element
length, A
Flame
^g/mL
Remarks
Ca
4227
Rich Air-C 2 H 2
2-20
1% La, 1% HCI
4227
Rich N 2 OC 2 H 2
2-20
Requires special
burner
Cu
3247
Air-C 2 H 2
2-20
Fe
2483
Rich Air-C 2 H 2
2-20
Mg
2852
Rich Air-C 2 H 2
0.2-2
May need La
Mn
2795
Air-C 2 H 2
2-20
Zn
2138
Air-C 2 H 2
0.5-5
E. Calculations
% Element = (fxg/mL) x (F/sample wt) x 10~ 4
F — mL original diln X ml final diln/mL aliquot, if original
100 mL vol. is diJd.
Refs.: JAOAC 48, 406, 1100(1965); 50, 401(1967); 51,
847(1968); 58, 928(1975).
CAS-7440-70-2 (calcium)
CAS-7440-50-8 (copper)
CAS-7439-89-6 (iron)
CAS-7439-95-4 (magnesium)
CAS-7439-96-5 (manganese)
CAS-7440-66-6 (zinc)
949.02 Boron (Acid-Soluble) in Fertilizers
Titrimetric Method
Final Action
A. Apparatus
Use high sensitivity glass electrode pH meter for titrn. Use
assembly with burets, electrodes, and mech. stirrer, arranged
for convenient use with 250 mL beaker. Use ordinary 50 mL
burets for the 0.0257V NaOH and 0.027V HCL
B. Reagents
(a) Boric acid std soln. — Dissolve 1 g H 3 B0 3 in H 2 and
dil. to 1 L. 1 mL = 0.1748 mg B.
(b) Sodium hydroxide std soln. — C0 2 -free, ca 0.0257V. Stdze
as follows: Pipet 25 mL std H 3 B0 3 soln into 250 mL beaker,
add 3.0 g NaCl, acidify to Me red, dil. to 150 mL, boil to
expel C0 2 , cool, and titr. potentiometrically as in 949. 02C.
Det. blank by repeating titrn, substituting 25 mL H 2 for H3BO3
soln. Calc. B equivalence as follows:
mg B/mL = 4.369/[(mL NaOH soln) - (mL blank)]
Protect from atm. C0 2 by soda-lime tubes or other suitable
means.
(c) Methyl red indicator. — Dissolve 0. 1 g Me red in 50 mL
alcohol, dil. to 100 mL with H 2 0, and filter if necessary.
C. Determination
Weigh sample within 1 mg (1.0 g for up to 0.45% B, smaller
samples for above that content) and place in 250 mL beaker.
Add ca 50 mL H 2 and 3 mL HCI. Heat to bp and keep hot
until carbonates are decomposed. Keep soln hot but do not boil
during following phosphate removal:
Add 10% Pb(N0 3 ) 2 soln, usually 10 mL, or 1 mL for each
1.2% P 2 5 if P 2 5 content is known to be > 1 2% . Add NaHC0 3 ,
little at time, until soln approaches neutrality (often observed
by formation of white ppt in addn to insol . matter already pres-
ent). Add few drops Me red and continue adding NaHC0 3
gradually until just alk. to Me red (yellow or very slightly or-
AOAC Official Methods of Analysis (1990)
Other Elements
29
ange). Keep mixt. hot but not boiling (H 2 bath or steam bath
is best) 30 min, adding addnl small amts of NaHC0 3 if needed
to keep same indicator color. (If indicator is bleached by ni-
trate present, add more; if color is obscured by org. matter,
use external spot tests to follow neutzn.) After neutzn and
heating, 40-50 mL soln should remain.
Filter hot soln into 250 mL beaker and wash solids thoroly
with hot H 2 0. Acidify filtrate with few drops HO and boil
briefly to expel most of C0 2 . Neutze hot soln with 0.5N NaOH,
and reacidify with 0.5 A" HO, using 0.3-0.5 mL excess. DiJ.
to ca 150 mL and boil gently few min to expel remaining C0 2 .
Cool to room temp, in running H 2 0. Roughly neutze mixt.,
using C0 2 -free 0.5N NaOH, and place beaker in titrn assembly
with electrodes and stirrer immersed. Start stirrer and adjust
pH to exactly 6.30 by adding 0.0257V NaOH or 0.027V HC1 as
required. (When properly adjusted, pH should be steady; drift-
ing usually is due to incomplete removal of C0 2 .) When read-
ing of pH 6.30 is steady, read 0.025/V NaOH buret, add 20 g
mannitol or cryst. D-sorbitol, and titr. with 0.0257V NaOH to
pH 6.30. (Conveniently done with slidewire type instrument
by opening pH meter circuit when mannitol is added, leaving
scale setting at 6.30, closing circuit again when indicator color
shows that end point is being approached, and carefully adding
std NaOH soln until galvanometer needle returns to zero.
Somewhat slow approach to equilibrium, characteristic of glass
electrode, can be anticipated with practice so as not to overrun
end point.) When end point is reached, read buret again. Ob-
tain reagent blank by repeating detn with all reagents but with-
out sample.
% B = (mL NaOH soln in detn - mL blank)
x (mg B/mL NaOH soln)/(10 x g sample)
Refs.: JAOAC 32, 422(1949); 33, 132(1950); 36, 623(1953);
38, 407(1955).
CAS-7440-42-8 (boron)
949.03 Boron (Water-Soluble) in Fertilizers
Titrimetric Method
Final Action
(Not applicable in presence of >5% urea or urea-formaldehyde
resins)
Weigh 2.5 g sample into 250 mL beaker. Add 125 mL H 2 0,
boil gently ca 10 min, and filter hot thru Whatman No. 40
paper, or equiv., into 400 mL beaker. Wash solids well with
6 portions hot H 2 and dil. to >200 mL with H 2 0. Heat fil-
trate just to bp. Add 15 mL 10% BaCl 2 soln to ppt sulfates
and phosphates, and add powd Ba(OH) 2 , cautiously with stir-
ring, until just alk. to phthln, avoiding large excess. Boil in
open beaker >60 min to expel NH 3 . (Samples colored by org.
matter should be boiled longer.) If necessary, add H 2 to keep
vol. to >150 mL. Add and stir 1-2 teaspoonfuls Filter-Cel or
other inert filtering aid, and filter with suction thru packed
paper pads into 500 mL Pyrex erlenmeyer, Wash ppt 6 times
with hot boiled H 2 0. (Avoid too large wash vols which in-
crease vol. in flask to point of dangerous bumping in next step.)
Make filtrate just colorless to phthln with HO (1 + 5), add
Me red, and make just pink with the acid. Add 5 or 6 boiling
stones and stirring rod, cover with watch glass, and boil 5 min
to remove C0 2 . Cool in cold H 2 while covered. Wash cover
glass, stirrer, and sides of flask. Titr. to yellow of Me red with
std 0.05N NaOH, 936.16. Add 20 g D-mannitol and 1 mL or
more phthln, shake, and wash down sides of flask. Titr. to
pink end point. Det. blank in exactly same manner as sample.
1 m'L0.05iVNaOH
- 0.000540 g B or 0.00477 g Na 2 B 4 7 . 10H 2 O
Or, (Titer - blank) x factor
= lb Na 2 B 4 O 7 .10H 2 O/ton(f actor = 3.807 for 0.05/V NaOH).
Refs.: JAOAC 32, 422(1949); 33, 132(1950); 36, 623(1953);
38, 407(1955).
CAS-7440-42-8 (boron)
982.01 Boron (Acid- and Water-Soluble)
in Fertilizers
Spectrophotometric Method
First Action 1982
Final Action 1985
A. Apparatus and Reagents
(a) Spectrophotometer. — Beckman Model 24/25 (replace-
ment model DV-64), or equiv.
(b) Precision pipet. — 100 (jlL Sherwood Lancer (Monoject
Scientific, 200 Express St, Plainview, NY 11803), or equiv.
(c) Dispenser pipet. — Automatic (Repipet, Lab industries,
Inc., 620 Hearst Ave, Berkeley, CA 94710), or equiv., 5 mL
capacity.
(d) Boron std solns. — (J) Stock soln. — 100 jxg/mL. Dis-
solve 0.5716 g boric acid in H 2 and dil. to 1 L with H 2 0.
Mix well and transfer to plastic bottle. (2) Working solns. —
0, 5, 10, 15, 20, 25, 30, and 45 |ig/mL. Pipet 0, 5, 10, 15,
20, 25, 30, and 45 mL stock soln into sep. 100 mL vol. flasks,
dil. to vol. with 1% HO, mix well, and transfer to plastic
bottles. Solns are stable.
(e) Azomethine H color reagent. — Dissolve 0.9 g azome-
thine H (Pierce Chemical Co.) and 2.0 g ascorbic acid in 100
mL H 2 0. Store in refrigerator and discard after 14 days.
(f) Buffer-masking soln. — Dissolve 140 g ammonium ace-
tate, 10 g potassium acetate, 4 g nitrilotri acetic acid, disodium
salt 99+% (Aldrich Chemical Co., Inc., No. 10629-1), 10 g
(ethylenedinitrilo) tetraacetic acid, and 350 mL 10% acetic acid
(v/v) in H 2 and dil. to 1 L with H 2 0. Soln is stable.
(g) Color developing reagent. — Place 35 mL azomethine H
color reagent and 75 mL buffer- masking soln into 250 mL vol.
flask and dil. to vol. with H 2 0. Prep, fresh daily.
B. Preparation of Sample Solutions
(a) Acid-soluble boron. — Weigh 2.00 g sample into 100 mL
vol. flask, add 30 mL H 2 and 10 mL HO, stopper, and shake
15 min. Dil. to vol. with H 2 0, mix well, and filter immedi-
ately into plastic bottle. Dil. as necessary, so final soln for
color measurement falls within std curve.
(b) Water-soluble boron. — Weigh 2.00 g sample into 250
mL beaker, add 50 mL H 2 0, and boil ca 10 min. Filter hot
thru Whatman No. 40 paper, or equiv., into 100 mL vol. flask.
Wash ppt 6 times with hot, boiled H 2 until vol. in flask is
ca 95 mL. Cool, add 1.0 mL HO, dil. to vol. with H 2 0, and
mix. Transfer to plastic bottle immediately; dil. as necessary
so final soln for color measurement falls within std curve.
C. Determination
Pipet 100 |ulL aliquots of 0, 5, 10, 15, 20, 25, 30, and 45
jxg/mL std and 100 jjiL aliquots of sample solns into sep. 10
mL erlenmeyers. Add 5.0 mL color developing reagent by au-
tomatic pipet dispenser (5 mL pipet is suitable but slower) and
let stand 1 h at room temp. Transfer to 1 cm cell and read A
at 420 nm against H 2 0. Correct for reagent blank (0 mg/mL
std). Construct std curve by plotting A against |xg/mL stds and
read concns (jig/mL) of sample solns from std curve.
30
Fertilizers
AOAC Official Methods of Analysis (1990)
D. Calculation
Boron, % = (|mg/mL from std curve) X diln factor
x (100/g sample) x 1(T 6
Ref.: J AOAC 65, 234(1982).
CAS-7440-42-8 (boron)
945.03 Calcium (Acid-Soluble) in Fertilizers
Titrimetric Method I
Final Action
(Presence of other analytes pptd by oxalate, such as Ba and
Sr, will cause pos. bias in results.)
Weigh 2.5 g sample into 250 mL vol. flask, add 30 mL
HN0 3 and 10 mL HC1, and boil 30 min. Cool, dil. to vol.,
mix, and filter if necessary. Transfer 25 mL aliquot to beaker
and dil. to 100 mL. Add 2 drops bromophenol blue (grind 0.1
g bromophenol blue powder with 1.5 mL 0.1N NaOH and dil.
to 25 mL with H 2 0). Add NH 4 OH (1+4) until indicator changes
from yellow to green (not blue). If overrun, bring back with
HC1 (1 +4). (This gives pH of 3.5-4.0.) Dil. to 150 mL, bring
to bp, and add 30 mL satd hot (NH4) 2 C 2 04 soln slowly, stirring
constantly. If color changes from green to blue or yellow again,
adjust to green with HC1 (1+4). If yellow, adjust with NH 4 OH
to green. Digest on steam bath 1 hr, or let stand overnight,
and cool to room temp. Filter supernate thru quant, paper,
gooch, or fritted glass filter, and wash ppt thoroly with NH 4 OH
(1+50). Place paper or crucible with ppt in original beaker
and add mixt. of 125 mL H 2 and 5 mL H 2 S0 4 . Heat to >70°
and titr. with 0AN KMn0 4 until first slight pink appears. Cor-
rect for blank and calc. to Ca.
965.1 0* Calcium (Acid-Soluble)
in Fertilizers
Titrimetric Method II
Final Action
Surplus 1980
See 2.142, 14th ed.
945.04 Calcium (Acid-Soluble) in Fertilizers
Atomic Absorption Spectrometric Method
Final Action
See 965.09.
955.07*
Carbon (Carbonate)
in Fertilizers
Final Action
Surplus 1970
See 2.107-2.108, 11th ed.
928.02 Chlorine (Water-Soluble)
in Fertilizers
Final Action
A. Reagents
(a) Silver nitrate std soln. — Dissolve ca 5 g recrystd AgN0 3
in H 2 and dil. to 1 L. Stdze against pure, dry NaCl and adjust
so that 1 mL soln = 0.001 g CI.
(b) Potassium chromate indicator. — See 941.18B(b),
B. Determination
Place 2.5 g sample on 11 cm filter paper and wash with
successive portions boiling H 2 until washings total nearly 250
mL, collecting filtrate in 250 mL vol. flask. Cool, dil. to vol.
with H 2 0, and mix well. Pipet 50 mL into 150 mL beaker,
add 1 mL K 2 Cr0 4 indicator, and titr. with std AgN0 3 soln to
permanent red of Ag 2 Cr0 4 .
Refs.: JAOAC 11, 34,201(1928); 16, 69(1933).
CAS-7782-50-5 (chlorine)
965.11 Cobalt in Fertilizers
Cotorimetric Method
First Action 1965
Final Action 1969
{Caution: See safety notes on nitric acid and perchloric acid.)
A. Reagents
(Use H 2 free of interfering elements. Check by shaking 2
drops 0.01% dithizone in CC1 4 with 10 mL H 2 0. CC1 4 phase
should remain green.)
(a) Ternary acid mixture. — See 960.03A(b).
(b) Ammonium hydroxide. — Use fresh stock. (Reagent be-
comes contaminated with heavy metals on prolonged storage
in glass.)
(c) Isoamyl acetate. — Distd.
(d) 2-Nitroso-l-naphthol soln. —0.05%. Dissolve 0.05 g 2-
nitroso-1-naphthol in 8 drops 1W NaOH and 1 mL ILO. Add
50-60 mL H 2 and 6.5-7 mL NH 4 OH, and dil. to "lOO mL
with H 2 0. Divide into 2 ca equal parts and wash each part
twice in 100 mL centrf. tube with 20 mL isoamyl acetate. Shake
30 sec and centrf. after each addn. (It may be necessary to
remove part of aq . phase to ensure complete removal of for-
eign matter at interface.)
(e) Cobalt std so Ins. — (J) Stock soln, — 200 \xg Co/mL.
Dissolve 0.0808 g CoCl 2 .6H 2 in H 2 and dil. to 100 mL.
(2) Working soln. — 2 \xg Co/mL. Dil. I mL stock soln to 100
mL with H 2 0.
B, Determination
Slowly add 20 mL ternary acid mixt. to 2.00 g pulverized,
mixed fertilizer in 150 mL beaker. Cover with watch glass and
digest on steam bath overnight. Transfer to hot plate and heat
covered until dense white fumes appear. (At this point HNO3
will have been expelled. Take care not to lose significant amts
of H.CIO4.) Dil. sample contg undissolved residue with H 2 0,
transfer to 50 mL vol. flask, and dil. to vol. Transfer to 100
mL centrf. tube and centrf. 5 min at 2000 rpm. Transfer ali-
quot, contg 2-5 fxg Co, to 50 mL g-s centrf. tube. Add 10
mL 20% diammonium citrate soln and 2 drops phthln. Adjust
pH carefully to distinct pink with NH 4 OH (1 + 1) and add suc-
cessively 1 mL 10% Na 2 S 2 3 soln, 2 mL 2-nitroso-l-naphthol
soln, and 5 mL isoamyl acetate. (Only isoamyl acetate addn
requires high degree of precision.) Shake mixt. 5 min and let
sep. Centrf., if necessary. Draw off and discard aq. phase thru
glass capillary tube attached to vac. Wash isoamyl acetate phase
with two 5 mL portions \N NaOH and one 5 mL portion IN
HO. Shake 5 min after each addn, let layers sep., and draw
off and discard aq. phase. Centrf. 2 min at 1500 rpm and mea-
sure A or %T at 530 nm against isoamyl acetate. Det. Co from
calibration curve relating A or log %T to Co content of std
solns contg 0, 2, 4, and 5 (xg Co.
Refs: Anal. Chem. 30, 1153(1958). JAOAC 48, 412(1965).
CAS-7440-48-4 (cobalt)
AOAC Official Methods of Analysis (1990)
Other Elements
31
975.01 Copper in Fertilizers
Atomic Absorption Spectrometry Method
Final Action
See 965.09.
941.01* Copper in Fertilizers
Long Volumetric Method
Final Action
Surplus 1970
S*e 2.129-2.130, 11th ed.
942.01
Copper in Fertilizers
Short Volumetric Method
Final Action 1960
A. Reagents
(a) Sodium thiosulfate std soln. — 0.037V. Prep, daily by dilg
0.17V soln, 942.27. 1 mL 0.037V Na 2 S 2 3 = 1.906 mg Cu.
(b) Starch soln. — Mix ca 1 g sol. starch with enough cold
H 2 to make thin paste, add 100 mL boiling H 2 0, and boil
ca 1 min while stirring.
(c) Bromocresol green indicator. — Dissolve 0.1 g terra-
bromo-m-cresolsulfonphthalein in 1.5 mL 0.17V NaOH, and dil.
to 100 mL with H 2 0.
B. Determination
Place 2 g sample in 300 mL erlenmeyer and add 10 mL
HN0 3 and 5 mL H 2 S0 4 . Digest on hot plate to white fumes.
If soln darkens, owing to org. matter, cool slightly, add little
more HN0 3 , and digest again to white fumes, repeating op-
eration if necessary until org. matter appears to be destroyed.
Cool, add 50 mL H 2 0, boil ca 1 min, and cool to room temp.
Add bromocresol green, then NH 4 OH until indicator changes
to light green (pH 4). Cool again to room temp., and if in-
dicator changes back to more acid color, add NH 4 OH dropwise
until indicator becomes light green again, avoiding excess. Add
2 g NH^Fj (toxic; see safety notes on toxic dusts), mix well,
and let stand ca 5 min. Add 8-10 g KI, mix well, and titr.
with std Na 2 S 2 3 soln to light yellow. Add ca 1 mL starch
soln and continue titrg slowly until color is nearly same as just
before addn of the Kl and becomes no darker on standing 20
sec. Report as % Cu.
Ref.: JAOAC 25, 77,352(1942).
CAS-7440-50-8 (copper)
(d) Mercuric chloride saturated soln. — Shake HgCl 2 with
H 2 and let settle.
(e) Stannous chloride soln. — Dissolve 20 g Sn0 2 .2H 2 in
20 mL HC1, warming gently. Add 20 mL H 2 and dil. to 100
mL with HC1 (1 + 1). Keep warm until clear; then add few
granules Sn. Dispense from dropping bottle.
B. Preparation of Sample Solution
(a) Suitable for all fertilizers. — Treat 1 g as in 957.028(e),
using 15 mL HCIO4. Hold =>1 hr at ca 170° to remove HN0 3
completely. Dil. to 200 mL.
(b) Suitable for soluble salts and oxides. — Dissolve 1 g in
10 mL HC1, warming gently. Dil. to 200 mL.
C. Reduction
Heat aliquot of sample soln (100 mL and 50 mL, resp., for
samples contg <0.5 and 0.5-4.0% Fe) to bp. Add few drops
diphenylamine sulfonate soln; then SnCl 2 soln dropwise until
violet color is discharged and 2 drops excess. (Usually 1-6
drops are required. Larger amt may be used with samples contg
large amt of Fe.) If reduction does not occur, discard and pro-
ceed as follows with second aliquot:
Add few granules Zn, boil few min, and either filter off
excess Zn, washing with hot H 2 0, or let Zn dissolve. Heat to
bp and finish reduction with SnCl 2 and diphenylamine sulfo-
nate indicator as before. Add 10 mL HC1 (1 + 1). Adjust vol.
to 75-110 mL with H 2 0. Cool rapidly in cold H 2 0. Add 10
mL satd HgCl 2 soln, swirl gently, add 5 mL H 3 P0 4 , and titr.
immediately. (Small amt of HgCJ must ppt to ensure complete
reduction.)
D. Titration
Add I drop diphenylamine indicator by pipet (no more; ex-
cess will interfere with end point if amt of Fe is small). Titr.
with 0.0 17V K 2 Cr 2 7 soln. Since end point may be difficult to
see with very small amt Fe, approach end point slowly, al-
lowing few sec for color to develop. Titr. to permanent blue
(sometimes green with very small amt Fe). For samples contg
>4% Fe, use 0AN K 2 Cr 2 7 for titrn. 1 mL 0.17V K 2 Cr 2 7 =
0.00558 g Fe; 1 mL 6.017V = 0.000558 g Fe.
Ref.: JAOAC 50, 397(1967).
CAS-7439-89-6 (iron)
980.01 Iron in Fertilizers
Atomic Absorption Spectrophotometric Method
Final Action
See 965.09.
967.01 Iron in Fertilizers
Titrimetric Method
First Action 1967
Final Action 1969
(Note: Diphenylamine may be harmful. See safety notes on
mercury salts and toxic dusts.)
A. Reagents
(a) Diphenylamine soln. — Dissolve I g in 100 mL H 2 S0 4 .
(b) Diphenylamine sulfonate soln. — Dissolve 0.5 g in H 2
in 100 mL vol. flask and dil. to vol.
(c) Potassium dichromate std solns. — 0. IN and 0.017V. Prep.
0AN K 2 Cr 2 7 as in 949.13C. Prep. 0.017V soln by dilg 100
mL 0.17V soln to 1 L.
983.03 Iron (Chelated)
in Iron Chelate Concentrates
Atomic Absorption Spectrophotometric Method
First Action 1983
(Applicable to Fe ethylenediaminetetraacetate (EDTA), Fe hy-
droxy ethylethylenediaminetriacetate (HEDTA), Fe diethyl-
enetriaminepentaacetate (DTPA), Fe ethylenediaminedi-o-hy-
droxyphenylacetate (EDDHA), and Fe citrate. Not applicable
to mixed fertilizers, or to samples contg non-chelated metals
other than Fe.)
A. Principle
Sample is dissolved in H 2 0, and non-chelated Fe is pptd as
FeOH 3 at pH 8.5 and removed. Chelated Fe is detd by AAS,
using std solns contg Na 2 H 2 EDTA.
32
Fertilizers
AOAC Official Methods of Analysis (1990)
B. Apparatus and Reagents
(a) Sodium hydroxide soln. — 0.5N. Dissolve 20 g NaOH
in H 2 and dil. to 1 L.
(b) Disodium EDTA soln.— 0. 66%. Dissolve 0.73 g
Na 2 H 2 EDTA.2H 2 in H 2 0, and dil. to 100 mL.
(c) Iron std solns. — (/) Stock soln. — 1000 jxg Fe/mL. Dis-
solve 1 .000 g pure Fe wire in ca 30 mL 6N HC1 with boiling.
Dil. to 1 L. (2) Intermediate soln. — 100 fxg Fe/mL. Pipet 10
mL Fe stock soJn and 10 mL Na 2 H 2 EDTA soln into 100 mL
vol. flask and dil. to vol. with H 2 0. (3) Working solns. — Dil.
aliquots of intermediate soln with 0.5N HO to make >4 std
solns within range of detn (2-20 |xg Fe/mL).
(d) Atomic absorption spectrophotometer. — With air~C 2 H 2
flame. See 965.09A(a).
C. Determination
Weigh sample contg ca 40 mg Fe into 200 mL tall-form
beaker. Wet with 2-3 drops of alcohol and dissolve in 100
mL H 2 0. Add 4 drops of 30% H 2 2 , mix and adjust pH of
soln to 8.5 with 0.5N NaOH. If pH drifts above 8.8, discard
soln and repeat analysis. Transfer soln to 200 mL vol. flask,
dil. to vol. with H 2 Q, and mix. Filter soln thru quant, paper.
Pipet 10 mL filtrate into 200 mL vol. flask and dil. to vol.
with 0.5N HO. Measured of solns, using lean air-C 2 H 2 flame
as in 965. 09D and det. concn of Fe in sample (jxg Fe/mL)
from either calibration curve or digital concn readout. In same
manner, det. Fe blank on all reagents used.
% Chelated iron - (|xg Fe/mL in sample
— fig Fe/mL in blank) X 0.4/g sample
Refs.: J AOAC 66, 952(1983); 69, 280(1986).
984.01 Magnesium (Acid-Soluble)
in Fertilizers
Atomic Absorption Spectrophotometry Method
Final Action
See 965.09.
964.01
Magnesium (Acid-Soluble)
in Fertilizers
EDTA Titration Method
First Action 1964
Final Action 1965
(Applicable to samples contg <0.25% Mn or Zn)
A. Reagents
Use reagents 962.01A(a), (b), (c), (d), (f) (1 mL = 1 mg
Ca, equiv. to 0.6064 mg Mg), (g), (h) (stdzd as in 964.01B),
and in addn:
(a) Triethanolamine soln . — ( 1 + 1 ) .
(b) Potassium ferrocyanide soln. — Dissolve 4 g
K 4 Fe(CN) 6 .3H 2 in 100 mL H 2 0.
(c) Ferric ammonium sulfate soln. — Dissolve 136 g
FeNH 4 (S0 4 ) 2 .12H 2 in H 2 contg 5 mL H 2 S0 4 , and diL to 1
L. Filter if not clear.
B. Standardization
Pipet 10 mL Ca std soln into 300 mL erlenmeyer. Add 100
mL H 2 0, 10 mL KOH-KCN soln, 2 drops triethanolamine soln,
5 drops K 4 Fe(CN) 6 soln, and 15±1 mg caJcein indicator. Im-
mediately place flask on mag, or other mech. stirrer in front
of daylight fluorescent light and white background. While stir-
ring, titr. with EDTA soln to disappearance of all fluorescent
green and until soln remains pink. Titr. ^3 aliquots. From av.,
calc. Ca titer in mg/mL EDTA soln. Ca titer x 0.6064 = Mg
titer in mg/mL.
C. Preparation of Solution
(Caution: See safety notes on wet oxidation and perchloric
acid.)
(a) Organic materials. — Weigh 1 g sample into 250 mL
boiling flask or erlenmeyer. Add 5 mL HCl and 10 mL HN0 3 ,
and boil on hot plate or over low flame until easily oxidized
org. matter is destroyed (ca 15 min). Cool, add 5 mL 70-72%
HC10 4 , and heat to appearance of copious fumes and momen-
tary cessation of boiling, but not to dryness. Cool, and transfer
to 250 mL beaker with ca 100 mL H 2 0. Continue with pH
adjustment, as in 964. 01D.
(b) Inorganic materials and mixed fertilizers. — Weigh 1 g
sample into 250 mL beaker. Add 5 mL HCl and 10 mL HNO3.
Cover with watch glass and heat on asbestos mat on hot plate
nearly to dryness (ca 30 min). If soln remains colored from
org. residues, cool, add 5 mL HC10 4 (70-72%), and continue
heating to copious fumes and momentary cessation of boiling,
but not to dryness.
D. Determination
Cool prepd soln to room temp. Wash watch glass and inside
of beaker to ca 100 mL with H 2 0. Using pH meter with glass
electrode and mech. stirring, adjust to ca pH 3 with 30% KOH
soln and finally to pH 4.0 with 10% KOH soln. Add
FeNH 4 (S0 4 ) 2 soln, 5 mL for sample <7% P 2 5 , 10 mL for
sample 7-15% P 2 5 , 15 mL for sample 16-30% P 2 5 , and
proportionate amts for samples >30% P 2 5 . Adjust to pH 5.0
with KOH solns as above, or with HCl (1+4) if pH is >5.0.
Cool to room temp, and transfer to 250 mL vol. flask with
H 2 0. Dil. to vol. with H 2 and mix. Let stand until ppt settles.
Disturbing ppt as little as possible, filter enough soln for ali-
quots required for titrn thru dry 1 1 cm fluted paper, Whatman
No. 1 , or equiv.
Pipet two equal aliquots contg <15 mg Ca + JVlg (usually
25 mL) into two 300 mL erlenmeyers and dil. each to 100 mL
with H 2 0. To one (titrn J for Ca + Mg) add 5 mL pH 10
buffer soln, 2 mL KCN soln, 2 drops triethanolamine soln, 5
drops K 4 Fe(CN) 6 soln, and 8 drops eriochrome black T indi-
cator. Titr. immediately with EDTA soln, stirring and lighting
as in stdzn. Color changes are wine red, purple, dark blue, to
clear pure blue end point, becoming green if overtitrd.
To second aliquot (titrn 2 for Ca) add 10 mL KOH-KCN
soln, 2 drops triethanolamine soln, 5 drops K 4 Fe(CN) 6 soln,
and 15 ±1 mg calcein indicator. Titr. immediately with EDTA
soln as in stdzn.
(Titrn 1 - Titrn 2) x Mg titer EDTA
X 100/mg sample in aliquot = % Mg
Titrn 2 x Ca titer EDTA
x 10/mg sample in aliquot - % Ca
Ref.: JAOAC 47, 450(1964).
CAS-7439-95-4 (magnesium)
937.01 * Magnesium (Acid-Soluble)
in Fertilizers
Gravimetric Method
Final Action
Surplus 1970
See 2.123, 11th ed.
AOAC Official Methods of Analysis (1990)
Other Elements
33
940.01* Magnesium (Acid-Soluble)
in Fertilizers
Volumetric Method
Final Action
Surplus 1970
See 2.124, 11th ed.
937.02 Magnesium (Water-Soluble)
in Fertilizers
Final Action 1966
(a) In potassium-magnesium sulfate, magnesium sulfate, and
kieserite. — Weigh 1 g sample into 250 mL vol. flask, add 200
mL H 2 0, and boil 30 min. Cool, dil. to vol. with H 2 0, and
mix. If detn is to be conducted gravimetrically, 937.01*, or
volumetrically, 940.01A*, see 2.125, 11th ed.
(b) In other materials, including mixed fertilizers . — Weigh
1 g sample into 500 mL vol. flask, add 350 mL H 2 0, and boil
1 hr. Cool, dil. to vol., mix, and filter if necessary. If detn is
to be conducted gravimetrically, 937.01*, or volumetrically,
940.01A*, see 2.125, 11th ed.
(c) By EDTA method. — Transfer aliquot soln prepd as in
(a) or (b) to beaker and det. Mg as in 964. 01D, using HO or
KOH to adjust pH.
Refs.: JAOAC 20, 252(1937); 22, 270(1939); 23, 249(1940);
24, 268(1941); 25, 326(1942).
until brown fumes diminish; then boil 30 min. If org. matter
is not destroyed, cool, add 5 mL HN0 3 , and boil. Repeat pro-
cess until no org. matter remains, and boil until white fumes
appear. Cool slightly, and add 50 mL H 3 P0 4 (1+9). Boil few
min. Cool, dil. to 200 mL in vol. flask, mix, and let stand to
allow pptn of CaS0 4 .
Pipet 50 mL clear soln into beaker. Heat nearly to bp. With
stirring or swirling, add 0.3 g KIO A for each 15 mg Mn pres-
ent, and hold 30-60 min at 90-100°, or until color develop-
ment is complete. Cool, and dil. to measured vol. that will
provide satisfactory concn for colorimetric measurement by in-
strument chosen (usually <20 ppm Mn). Compare in color-
imeter against std KMn0 4 soln, or in spectrophtr at 530 nm.
Calc. to Mn.
Ref.: JAOAC 23, 249(1940).
CAS-7439-96-5 (manganese)
941.02* Manganese (Acid-Soluble)
in Fertilizers
Bismuthate Method
Final Action
Surplus 1970
(Applicable to Mn"' 2 only)
See 2.127-2.128, 11th ed.
972.02 Manganese (Acid-Soluble)
in Fertilizers
Atomic Absorption Spectrophotometric Method
First Action 1972
Final Action 1974
(a) Applicable to Mn +2 only. — Prep, sample soln as in
940.02, omitting the 50 mL H 3 P0 4 (1+9). Proceed as in
965.09D, using std solns prepd as in 965.09B(f) and (h), sub-
stituting 0.5N H 2 S0 4 for 0.5/V HC1 in 965.09B(h).
(b) Applicable to total Mn +2 and Mn A . — Prep, sample soln
as in 965. 09C. Proceed as in 965. 09D, using std solns prepd
as in 965.09(f) and (h).
Ref.: JAOAC 55, 695(1972).
940.02 Manganese (Acid-Soluble)
in Fertilizers
Colorimetric Method
Final Action
(Applicable to samples contg Mn +2 only and with <5% Mn)
A. Reagent
Potassium permanganate std soln. — 500 ppm Mn. Prep, and
stdze as in 940.35, except use 1.4383 g KMn0 4 and 0.12 g
Na oxalate. Transfer aliquot contg 20 mg Mn to beaker. Add
100 mL H 2 0, 15 mL H 3 P0 4 , and 0.3 g KI0 4 , and heat to bp.
Cool, and dil. to I L. Protect from light. Dil. this soln contg
20 ppm Mn with H 2 (previously boiled with 0.3 g K10 4 /L)
to make convenient working stds in range of concns to be com-
pared .
B. Determination
Place 1 g sample in 200 mL wide-neck vol. flask or 250
mL beaker. Add 10 mL H 2 S0 4 and 30 mL HN0 3 . Heat gently
972.03 Manganese (Water-Soluble)
in Fertilizers
Atomic Absorption Spectrophotometric Method
First Action 1972
Final Action 1974
(Applicable to Mn +2 only)
Place 1 g sample in 50 mL beaker, wet with alcohol, add
20 mL H 2 0, and let stand 15 min, stirring occasionally. Trans-
fer to 9 cm Whatman No. 5 paper, and wash with small por-
tions H 2 until filtrate measures ca 230 mL. Let each portion
pass thru paper before adding more. Add 3-4 mL H 2 S0 4 to
filtrate. Proceed as in 965. 09D, using std solns prepd as in
965.096(f) and (h), substituting 0.5N fLS0 4 for 0.5N HC1 in
965.09B(h).
Ref.: JAOAC 55, 695(1972).
974.01
Sodium in Fertilizers
Flame Photometric Method
First Action 1974
A. Reagents
See 955.06*, and in addn:
Sodium chloride. — Dry 2 hr at 105°.
B. Preparation of Solution
Prep, soln as in 955.06*, using 2.5 g sample (<4% Na) or
1.25 g (4-20% Na).
C. Preparation of Standard Curve
(a) Samples containing 1% or more sodium. — Proceed as
in 955.06*, using 1.271 1 g NaCl, range of diln 0-40 ppm Na,
intervals <5 ppm, and full scale for 40 ppm Na.
(b) Samples containing less than 1% sodium. — Proceed as
34
Fertilizers
AOAC Official Methods of Analysis (1990)
in 955.06*, using 1.2711 g NaCi, range of diln 0-10 ppm Na,
intervals 2 ppm, and full scale for 10 ppm Na.
D. Determination
Transfer 25 mL (<4% Na) or 10 mL (4-20% Na) sample
soln to 250 mL vol. flask, dil. to vol. with H 2 0, and mix (if
internal std instrument is used, add required amt LiN0 3 before
dilg to vol.). Atomize portions of sample several times to ob-
tain reliable av. readings for each soln. Det. ppm Na from std
curve (a) or (b). Calc. % Na as follows:
0-4%: ppm Na/10 = % Na
4-20%: ppm Na/2 = % Na
Refs.: JAOAC 55, 986(1972); 56, 859(1973); 57, 1402(1974).
CAS-7440-23-5 (sodium)
983.04 Sodium in Fertilizers
Atomic Absorption Spectrophotometry Method
First Action 1983
A. Reagents and Apparatus
(a) Ammonium oxalate soln. — Dissolve 40 g (NH 4 ) 2 C 2 04 in
1 L H 2 0.
(b) Sodium chloride .—Dry 2 h at 105°.
(c) Atomic absorption spectrophotometer . — Model AA6
(replacement model is Spectr-AA Series, Varian Instrument
Group), or equiv.
B. Preparation of Solution
Weigh 2.5 g (<4% Na) or 1.25 g (4-20% Na) sample into
250 mL vol. flask, add 125 mL H 2 and 50 mL (NH^QAt
soln, and boil 30 min. Cool, dil. to vol., mix, and pass thru
dry filter. For samples contg <1% Na, use this soln for detn.
For samples contg 1-20% Na, place 20 mL in 100 mL vol.
flask, dil. to vol. with H 2 0, and mix.
C. Preparation of Standard Curve
Dissolve 2.5421 g dried NaCl in H 2 and dil. to 1 L (1000
ppm Na). Prep, std solns to cover range 0-200 ppm at inter-
vals <20 ppm Na.
D. Determination
Set wavelength at 330.3 nm using air-C 2 H 2 flame. Aspirate
stds and samples. Plot curve from std values and det. Na con-
tent of sample solns from plot of A against ppm Na. Calc. %
Na as follows:
<1%: ppm Na X 25 /M = %Na
1-20%: ppm Na X 125/M = %Na
where M = wt of sample (mg) .
Ref.: JAOAC 66, 1234(1983).
CAS-7440-23-5 (sodium)
980.02 Sulfur in Fertilizers
Gravimetric Method
First Action 1980
Final Action 1985
(Caution; See safety notes on sodium hydroxide and perox-
ides.)
(a) Total sulfur (sulfate and elemental) in dry fertilizers. —
Accurately weigh sample contg 100-150 mg S into 400 mL
beaker, add 200 mL H 2 0, 15 mL HC1, heat to bp, and boil
gently ca 10 min. Filter thru gooch crucible contg glass fiber
paper and wash with hot H 2 0. Set washed crucible aside.
Quant, transfer filtrate back to beaker and bring nearly to
bp. Add slowly, with const stirring, slight excess (ca 15 mL)
10% BaCL soln. Digest on low temp, hot plate adjusted so
that soln does not boil, or on steam bath 1 hr, and let stand
at room temp, overnight. Filter thru gooch crucible contg glass
fiber paper previously dried at 250°, cooled, and weighed. Wash
with ten portions hot H 2 0, dry crucible and contents 1 hr at
250°, cool to room temp., and weigh.
% Sulfate S = g BaS0 4 x 0.1374 x 100/g sample
Wash insol. residue with five 10 mL portions acetone satd
with S, dry 1 hr at 100°, cool, and weigh. Wash residue with
three 5 mL portions CS 2 , drain, dry 1 hr at 100°, cool in des-
iccator, and weigh. Difference in wt = elemental S (S°).
Perform blank on S° portion of method by weighing 5.0 g
fertilizer sample contg no S° into 400 mL beaker and pro-
ceeding as above, beginning "add 200 mL H 2 0, .... Set
washed crucible aside." Omit sulfate S detn and continue, be-
ginning "Wash insol. residue with five. ..." Correct differ-
ence in wt (— elemental S) for blank found.
%S°
% Total S
gS x 100 /g sample
% Sulfate S + % S°
(If S° is <5%, use 5 g sample and repeat S° portion of detn.)
(b) Total sulfur (sulfate, sulfite, thiosuifate, and sulfide) in
liquid fertilizers. — Accurately weigh sample contg 100-150
mg S into 400 mL beaker. Add 50 mL H 2 0, 2 mL 50% NaOH,
and 2 mL 30% H 2 2 . Cover with watch glass and reflux 1 hr,
adding 1 mL increments H 2 2 (^5 mL total) as reaction sub-
sides. After 1 hr, wash watch glass and remove. Dil. to ca 175
mL with H 2 0, acidify with ca 10 mL MCI (1 + 1), and bring
to bp. Proceed as in (a), beginning "Add slowly, with constant
stirring, ..."
% Total S = g BaS0 4 x 0.1374 x 100/g sample
(c) Total sulfur in sulfur-coated urea and elemental sulfur
formulations. — Accurately weigh sample contg 200-300 mg
S into 125 mL g-s erlenmeyer, add ca 50 mL H 2 0, stopper,
and shake vigorously 30 sec. Filter quant, with suction thru
gooch crucible contg glass fiber paper and wash with H 2 0.
Proceed as in (a), beginning "Wash insol. residue with five
10 mL portions ..."
% S° = g S x 100/g sample
Refs.: JAOAC 63, 854(1980); 64, 420(1981).
CAS-7704-34-9 (sulfur)
942.02* Zinc in Fertilizers
Gravimetric Method
Final Action 1976
Surplus 1976
(For samples contg >0.1% Zn)
See 2.138, 12th ed.
942.03* Zinc in Fertilizers
Colorimetric Method
Final Action 1976
Surplus 1976
(For samples contg <4% Zn)
See 2.139, 12th ed.
AOAC Official Methods of Analysis (1990)
Other Elements 35
975.02 Zinc in Fertilizers
Atomic Absorption Spectrophotometric Method
Final Action 1976
See 965.09.
973.01 Zinc in Fertilizers
Zincon Ion Exchange Method
First Action 1973
Final Action 1976
(Clean all glassware with hot chromic acid or HN0 3 (1 + 1).
Rinse thoroly with H 2 0. Caution: See safety notes on chromic
and perchromic acids and nitric acid.)
A. Reagents
(a) Anion exchange resin. — 100-200 mesh, strong base,
polystyrene alkyl quaternary amine, 7% cross linkage.
(b) Zincon indicator. — Dissolve 0.12 g zincon (o-[[a~[(2~
hydroxy-5 -sulfophenyl)azo J benzyl idenejhydrazinoj benzoic acid,
Na salt) (J.T. Baker Inc., No. X690) in 5 mL 0.37V NaOH
and dil. to 100 mL with H 2 0. Prep, fresh weekly.
(c) Hydrochloric acid solns.—(l) 0.5N.— Dil. 20 mL HC1
to 500 mL with H 2 0. (2) 0.25/V.— Dil. 2 mL HC1 to 100 mL
with H 2 0. (3) 0.005N. —DiL 2.5 mL HCI to 6 L with H 2 0.
(d) Borate buffer soln. — pH 9.8. Dissolve 4 g H 3 B0 3 in
140 mL H 2 0. Add 5 mL NH 4 OH by pipet and then dropwise
to pH 9.8. Check daily.
(e) Ammonium thiocyanate. — \M. Dissolve 0.76 g NH 4 CNS
in 10 mL H 2 0.
(f) Zinc std so Ins. — (7) Stock soln. — 1000 ppm. Dissolve
1.000 g pure Zn metal in small amt HCI-HNO3 (1 + 1). Evap.
to small vol., add 3 mL HCI, and heat. Dil. to 1 L with H 2 0.
(2) Working soln. — 10 ppm. Dil. 10 mL stock soln to 1 L
with H 2 0.
(g) Sodium hydroxide soln. — 0.3N. Dissolve ca 1 .25 g NaOH
in 100 mLH 2 0.
B. Preparation of Resin Column
Wash 12 g new resin in 250 mL beaker with H 2 until
washings are neut. Introduce resin as slurry into 25 X 2.2 cm
chromatgc tube with fritted glass disk and stopcock at bottom.
Mark vol. levels on column at 10, 40, and 50 mL above packed
resin and on 250 mL separator at 90 mL. (Keep resin wet and
store under liq. when not in use.) Connect separator to top of
column thru stopper. Attach inverted U-shaped glass dispen-
sing tube to 250 mL vol. flask thru vented stopper or cork and
connect with Zn-free plastic tubing to stopcock of column with
stopcock grease. See Fig. 973.01.
Mount reservoir (aspirator bottle or carboy) contg > 1 L
0.005iV HCI high enough to effect back washing. Attach Zn-
free tubing and pinch clamp.
C. Flow Calibration
Use sweep sec hand of watch or stopwatch to establish flow
rates. Det. number drops/mL leaving dispensing tube. Re-
move separator and vol. flask; drain and then backwash resin
(see 973. 01E). Remove reservoir tubing, open stopcock, elute
40 drops from dispensing tube, and measure vol. Use this fac-
tor to convert 0.5 mL/min (required in Zn elution, 973. 01E)
to drops /sec.
D. Preparation of Sample
Remove separator and elution tubing from column. Activate
resin by draining column and adding 50 mL 0.5jV HCI. Drain
column to 40 mL mark.
Vented
Fig. 973.01 — Apparatus for elution of resin column
(a) Samples containing 0.14% or more zinc. — Dissolve 1 .000
g well-ground sample in 10 mL HCI and 5 mL HN0 3 in 250
mL beaker. Evap. to near dryness on hot plate. (Caution: Do
not bake.) Redissolve residue in ca 40 mL 0.5/V HCI, boiling
gently if necessary. Filter thru Whatman No. 41 paper into 100
mL vol. flask. Thoroly wash residue and dil. filtrate to vol.
with 0.5N HCI. Drain column to 10 mL mark. Tap column to
pack resin. Pipet aliquot contg 0.7-0.8 mg Zn onto column.
Elute sample soln at ca 5 sec/drop.
(b) Samples containing less than 0.14% zinc. — Weigh, to
nearest mg, sample contg 0.7-0.8 mg Zn into 250 mL beaker.
Digest and filter sample and prep, column as in (a). Tightly
attach open separator to column. Close stopcock. Transfer en-
tire sample soln to separator, rinsing with two 10 mL portions
0.5N HCI. Open stopcock. Elute sample soln at ca 5 sec /drop.
Remove empty separator, rinse twice with 20 mL 0.5yV HCI,
and add rinses to remaining soln in column.
E. Elution of Zinc
After sample soln passes thru resin, immediately rinse col-
umn with 0.5N HO at ca 1.5 sec. /drop until 1 mL eluate gives
clear, colorless soln with \M NH 4 SCN. If Fe +3 is present, soln
will turn brown. Drain resin and backwash with 0.005N HCI
from reservoir thru elution tubing, forcing out air bubbles from
tubing and column. Simultaneously, tap resin into suspension
as it is forced up. Close column stopcock when liq. reaches
50 mL mark on column. Attach dispensing tube to vol. flask.
Reopen stopcock, and raise flask until flow just stops. Con-
tinue ht adjustment until a drop remains in equilibrium at tip
of dispensing tube and neither rises nor falls. Secure flask.
Attach open separator with H 2 seal to column and close stop-
36
Fertilizers
AOAC Official Methods of Analysis (1990)
cock. Add 240 mL 0.0057V HCJ to separator and reopen stop-
cock. Open column stopcock until rate of ca 0.5 mL/min is
sustained 10 min. If rate decreases, increase rate slightly until
nearly const. Let elution continue overnight. Then, if >90 mL
remains in separator, readjust rate as above and continue elu-
tion to 90 mL mark. Finally, lower flask, fill to 250 mL mark
at convenient rate from dispensing tip, and mix. Detach hose
and separator from column, and drain all 3. Reactive resin,
and stopper column as in 973. 01D.
F. Determination
Pipet 20 mL eluate into 50 mL vol. flask contg small piece
litmus paper. Make alk. with 0.3JV NaOH, then just acidic
with 0.257V HC1. Pipet in 2 mL more acid, 5 mL buffer, and
3 mL zincon soln. DiL to vol. with H 2 0. Similarly prep. 0,
1, 2, and 3 ppm std solns, using 0, 5, 10, and 15 mL std Zn
working soln, resp. Using ppm std soln as blank, det. A at
620 nm 15-45 min after zincon addn. Plot std curve of ppm
against A .
% Zn = (C X F)/W
where C — ppm from std curve; W = g sample; and F =
0.0625 for samples contg <0. 14% Zn or 6.25/mL aliquot pi-
petted onto resin for samples contg >0.14% Zn.
Ref.: J AOAC 56, 846(1973).
CAS-7440-66-6 (zinc)
Table 983.05 Operating Parameters
936.01*
Acid-Forming
or Nonacid-Forming Quality of Fertilizers
Final Action
Surplus 1970
See 2.141-2.142, 11th ed.
983.05 Aluminum in Aluminum
Sulfate-type Soil Acidifiers
Atomic Absorption Spectrophotometric Method
First Action 1983
A. Apparatus and Reagents
(a) Atomic absorption spectrophotometer. — Perkin-Elmer
Model 303, or equiv. See 972.06A for typical operating pa-
rameters.
(b) Diluting soln.— Add 20 mL H 2 S0 4 and 2.5 g NaCl to
500 mL H 2 0. Dil. to J L with H 2 0.
(c) Aluminum std solns. — (I) Stock soln. — 1 mg Al/mL
(1000 ppm). Accurately weigh 1.000 g pure Al and dissolve
in ca 25 mL HC1. Evap. almost to dryness, add 500 mL FLO,
20 mL H 2 S0 4 , and 2.5 g NaCl, and dil. to 1 L with H 2 0. (2)
Working solns. — Dil. aliquots of stock soln with dilg soln,
(b), to make 4 std solns within range 50-150 ppm.
B. Preparation of Sample
Accurately weigh ca 1 g sample into 500 mL screw-cap er-
lenmeyer, add 250 mL H 2 0, and shake on wrist-action shaker
ca 15 min. Quant, transfer to 500 mL vol. flask and dil. to
vol. with H 2 0. Filter thru Whatman No. 2 paper. Dil. aliquot
as necessary (diln factor - F) with dilg soln, (b), for concn
range 50-150 ppm.
C. Determination
Set up app. as shown in Table 983.05, or use previously
established optimum settings for app. Zero app. while aspir-
ating dilg soln, (b). Det. A of std solns within 50-150 ppm
Wavelength, nm
Slit width, mm
309.3
1
Source, ma
30
N 2 0, aspirating
N 2 0, auxiliary and aspirating
C 2 H 2 fuel
Flame
Ht, burner to light path, in.
Sample uptake, mL/min
Optimum concn range, ^g/mL
4.5 (scale divisions)
5.5 (scale divisions)
6 (metal ball scale division)
reducing
4
50-150
range, alternating with sample soln readings. Flush burner with
dilg soln, (b), and check zero point between readings.
Det. Al content from std curve of A against \xg Al/mL.
% Al - (pig Al/mL) x F x (500/g sample) x 10" 4
Ref.: JAOAC 66, 946(1983).
CAS-7429-90-5 (aluminum)
PEAT
973.02 Sampling of Peat
Procedure 1973
(Moss, humus, and reed-sedge types)
Use slotted single or double tube or slotted tube and rod,
all with pointed ends and min. 1" diam. for loose materials.
Use cutting type core sampler, with plunger, for compressed
materials. Pennsylvania State Forage Sampler (NASCO, 901
Janesville Ave, Fort Atkinson, WI 53538) is satisfactory core
sampler.
Take representative sample from lot or shipment as follows:
(a) Packaged or baled peats. — Lay bag or bale horizontally
and remove core diagonally from end to end. From lots of 1-
10 bags, sample all bags; from lots of ^11, sample 10 bags.
Take 1 core from each bag sampled; except for lots of 1-4
bags, take diagonal cores from each bag and addnl cores to
total >5 cores.
(b) Bulk samples. — Draw >]0 cores from different re-
gions.
(c) Small containers (10 lb or less). — Take entire package.
Working rapidly to prevent moisture losses, reduce composite
sample to <500 g (by wt) or 2 L (by vol.) by mixing on clean
plastic or paper and quartering. Place sample in air-tight con-
tainer.
Sampling by random "grab" procedure is necessary if par-
ticle size range is to be detd or if representative sample cannot
be taken with core sampler as above.
Refs.: Book of ASTM Stds (1971) Pts 11, 22, and 30, ASTM
D2973-D2978, D2980, and D2944. JAOAC 56,
154(1973).
967.02 Preparation of Peat Sample
First Action 1967
Final Action 1978
Place representative field sample on square rubber sheet, pa-
per, or plastic. Reduce sample to amt required by quartering
and place in moisture-proof container. Work rapidly to prevent
moisture losses.
Ref.: JAOAC 50, 394(1967).
AOAC Official Methods of Analysis (1990)
Peat 37
967.03
Moisture in Peat
First Action 1967
Final Action 1978
A. Method J
Mix sample thoroly and place 10-12 g in ignited and weighed
(with fitted heavy-duty Al foil cover) Vycor or porcelain evapg
dish, ^75 mL capacity. Crush soft lumps with spoon or spat-
ula. Cover immediately with Al foil cover and weigh to nearest
mg. Dry, uncovered, 16 hr at 105°. Remove from oven, cover
tightly, cool, and weigh.
% Moisture (report to nearest 0.1%)
= (g as-received sample - g oven-dried sample)
X 100/g as-received sample
B. Method II
(Use when pH, N, fiber, etc., are to be detd.)
Mix thoroly and weigh 100-300 g representative sample,
967.02, and spread evenly on large flat pan. Crush soft lumps
with spoon or spatula and let come to moisture equilibrium
with room air >24 hr. Stir occasionally to maintain max. air
exposure of entire sample. When wt is const, calc. loss in wt
as % moisture removed by air drying. Grind representative
portion air-dried sample 1-2 min in high-speed blender; use
for moisture, ash, and N detns.
Mix air-dried, ground sample and weigh, to nearest mg,
equiv. of 10 g sample on as-received basis (g air-dried sample
equiv. to 10.0 g as-received sample = 10.0 — 1(10.0 X %
moisture removed)/ 100]). Place weighed sample in ignited and
weighed (with fitted heavy duty Al foil cover) Vycor or por-
celain evapg dish and proceed as in 967. 03 A.
% Moisture (report to nearest 0.1%)
- (10.0 - g oven-dried sample) X 10.0
Ref.: J AOAC 50, 394(1967).
973.03 Particle Size Range of Peat
Mechanical Analysis
First Action 1973
A. Apparatus
Mechanical sieve shaker. — With 8" diam., Nos. 8 and 20
sieves equipped with cover and bottom pan.
B. Preparation of Sample
Air-dry as in 967. 03B.
C. Determination
Mix thoroly and place 20.0 g air-dried sample on No. 8
sieve nested on No. 20 sieve. Secure sieves and shake at suit-
able speed 10 min. Remove and weigh foreign matter, such
as sticks, stones, and glass, from No. 8 fraction. Weigh frac-
tions of peat retained on Nos. 8 and 20 sieves and portion
collected in bottom pan. Convert fraction and sample wts to
as-received basis and calc. in terms of %. (If foreign matter
is absent, conversion to as-received basis is not necessary.)
% Foreign matter =
fraction removed from No. 8 sieve x 100
% Coarse fiber = fraction retained on No. 8 sieve x 100
% Medium fiber = fraction retained on No. 20 sieve x 100
% Fines = fraction collected in pan X 100
If mech. sieve shaker is not available, use hand sieving.
Conduct sieving by appropriate lateral and vertical motions ac-
companied by jarring action. Continue until no appreciable
change is noted in sieve fraction.
Refs.: Book of ASTM Stds(1971)Pts 11, 22, and 30, ASTM
D2973-D2978, D2980, and D2944. JAOAC 56,
154(1973).
973.04
pH of Peat
A. Apparatus and Reagents
(a) pH meter. — Battery-operated or on elec. line with volt-
age regulator.
(b) Carbon dioxide-free water. — See 964.24.
(c) Acid potassium phthalate buffer soln.— 0.05m. See
964.24(c).
(d) Phosphate buffer soln.— 0.025m. See 964.24(d).
(e) Calcium chloride solns (Method II only). — (7) Stock
soln. — \.0M. Dissolve 147 g CaCl 2 .2H 2 in H 2 in 1 L vol.
flask, cool, dil. to vol., and mix. Dil. 15 mL of this soln to
200 mL with H 2 in vol. flask and stdze by titrg 25 mL aliquot
dild soln. with std 0AN AgN0 3 , 941. 18C, using 1 mL 5%
K 2 CrC 4 as indicator. (2) Working soln.—OMM (pH 5.0-6.5).
Dil. 20 mL stock soln. to 2 L with H 2 0.
S. Determination
(a) Method I {in distilled water). — Weigh ca 3.0 g air-dried
peat or equiv. amt moist material into 100 mL beaker. Add
50 mL H 2 0. (Addnl H 2 may be needed for very fibrous ma-
terials such as sphagnum moss peat.) Let soak 30 min, with
occasional stirring. Read on pH meter.
(b) Method II (in 0.0IM calcium chloride soln). — Weigh
ca 3.0 g air-dried peat or equiv. amt moist material into 100
mL beaker. Add 50 mL 0.01M CaCl 2 . Let soak 30 min, with
occasional stirring. Read on pH meter. Report results as pH
in 0.0 1M CaCl 2 soln. (pH values in CaCl 2 soln are usually ca
0.5-0.8 units lower than those in H 2 0. Observed pH in CaCl 2
soln is virtually independent of initial amt salt present in soil,
whereas pH readings in H 2 can be modified by salts such as
fertilizer material.)
Refs.: Book of ASTM Stds(1971)Pts .11, 22, and 30, ASTM
D2973-D2978, D2980, and D2944. JAOAC 56,
154(1973).
967.04
Ash of Peat
First Action 1967
Final Action 1978
Place uncovered (retain cover for weighing) Vycor or por-
celain dish contg dried sample from moisture detn in furnace.
Gradually bring to 550° and hold until completely ashed. Cover
with retained Al foil cover, cool, and weigh.
% Ash (report to nearest 0. 1%) = g ash X 100/g as-received
sample taken for moisture detn. (If moisture Method 11 was
used, g as-received sample - 10.0.)
Ref.: JAOAC 50, 394(1967).
973.05 Sand in Peat
First Action 1973
A. Preparation of Sample
Air-dry as in 967.03B.
B. Determination
(Caution: See safety notes on chloroform.)
38
Fertilizers
AOAC Official Methods of Analysis (1990)
Place 25 g air-dried, ground sample into 125 mL tall-form
beaker, or equiv. Nearly fill beaker with CHCI3, stir briefly,
and let settle ca 1 min. With spoon, discard most floating org.
material, decant remaining org. material and CHC1 3 , taking
care not to disturb settled portion (sand), and air-dry to remove
residual CHC1 3 . (Stirring aids drying.)
When dry, weigh settled portion and calc. as % sand (in-
cludes other minerals present such as limestone, etc.).
% Sand = (g air-dried settled residue
x 100)/g air-dried sample
Refs.: Book of ASTM Stds (1971) Pts 11, 22, and 20, ASTM
D2973-D2978, D2980, and D2944. JAOAC 56,
154(1973).
967.05 Organic Matter in Peat
First Action 1967
Final Action 1978
% Org. matter = 100.0 - (% moisture + % ash)
Ref.: JAOAC 50, 394(1967).
973.06 Nitrogen (Total) in Peat
First Action 1973
Det. N as in 955.04, using well mixed, air-dried, ground
sample equiv. to 10.0 g sample on as-received basis.
Det. g air-dried sample equiv. to 10.0 g as -received sample
as in 967.03B
Refs.: Book of ASTM Stds (1971) Pts 1 1, 22, and 30, ASTM
D2973-2978, D2980, and D2944. JAOAC 56,
154(1973).
969.05 Water Capacity and Volumes
for Peat
First Action 1969
Final Action 1978
A. Apparatus
Dispensing apparatus. — 2 dispensing burets, 250 mL in 1
mL subdivisions, ±2 mL tolerance, pinchcock type; 1-hole
No. 6 rubber stopper; straight polyethylene drying tube with
serrated rubber tubing fittings, 15 cm long, 3 / 4 " od, 5 /s" id (No.
14782-2, Cenco, Inc., 2600 Kostner Ave, Chicago, IL 60623);
and stainless steel screen circle, ca 16 mesh and 28.7 mm diam.
Assemble dispensing app. as follows: Discard serrated rub-
ber tubing fittings from polyethylene drying tube and use tube
only. Center stainless steel screen on one end of tube and seal.
(Soldering iron is useful.) Adjust length of tube to match con-
venient graduation of buret; then scallop end without screen
to allow for H 2 drainage, and insert into dispensing buret
with screen side up.
B. Preparation of Sample
See 967.02.
C. Determination
Det. moisture content on sep. sample by 967. 03A or B.
Weigh buret fitted with plastic tube and screen. Working
rapidly to prevent moisture losses, mix sample thoroly, place
on top of No. 4 screen, and shake until sieving is complete.
Use only portion that has passed thru sieve for detn. Firmly
pack buret with 25 cm (10") of 4 mesh sample as follows:
Attach rubber stopper to delivery end of buret. Add ca 20 mL
portions, firmly tapping 3 times vertically from ht of 15 cm
(6") on rubber stopper, for final ht of 25 cm. (This will ensure
that ht of final wet vol. is 19-25 cm.) Remove stopper; weigh
buret to nearest g. Position buret to use sink as drain. Place
H 2 source (19 L (5 gal.) bottle) equipped with siphon device
above level of buret. Connect clamped rubber tubing of siphon
device to buret with glass tubing (ca 13 cm (5") long, con-
stricted at one end) inserted into one-hole rubber stopper fitting
tightly into top of buret. Attach rubber tubing with pinch clamp
to delivery end of buret. Open both clamps and pass H 2 thru
sample ^24 fir, maintaining water reservoir over sample at all
times. (Moss-type samples may float but gradually settle as
sample becomes wet.) After initial soaking, regulate H 2 flow
thru column by adjusting screw clamp at delivery end of buret.
(In-flow of H 2 should be ca equal to out- flow; flow of ca 1
drop/sec is suitable.) When sample is super satd, close both
clamps and let sample settle in H 2 ca 5 min. Top surface of
sample should be as level as possible.
Raise buret and replace rubber tubing on delivery end of
buret with 250 mL dispensing buret filled with H 2 O s using
rubber stopper for connection. Connect two burets tightly, with
no air leaks. Remove siphon device and open outlet clamps
of both burets to empty. (Suction created is equiv. to ca 38
cm (15") H 2 0. Check for air leaks to ensure that std suction
is exerted on sample. It is important to remove excess H 2 as
described.) Measure ht of wet peat. Ht should be 19-25 cm.
Record vol. in mL and weigh buret, plastic tube with screen,
and wet peat to nearest g.
Wet sample again as above >]. hr, drain by suction, record
vol., and weigh. Repeat until consistent results are obtained.
D. Calculations
(a) Saturated Volume Weights, g/mL
As-recd = g as-recd sample/mL wet vol.
Oven-dried = g dried sample/mL wet vol., where g dried
sample — g as-recd sample X [(100 — % moisture)/! 00]
Wet = g wet sample/mL wet vol.
(b) Water-Holding Capacity, %
(1) Weight basis:
As-recd
= Kg wet sample — g as-recd sample)
x 100]/g as-recd sample
Oven-dried
- [(g wet sample ~ g dried sample)
x 100] /g dried sample
(2) Volume basis:
Water vol.
= Kg wet sample — g dried sample)
x 100]/(mL wet vol. x 1.0)
(c) Dry Peat Volume, %
Dry peat vol. = (g dried sample
X 100)/(mL wet vol. X 1.5)
(d) Air Volume, %
Air vol. = 100 ~ (% water vol. + % dry peat vol.)
Refs.: JAOAC 51, 1296(1968); 52, 384(1969).
973.07
Volume, Peat
Alternative Method
First Action 1973
A. Principle
Method consists of dividing particles of peat from original
container by passing them thru 12.7 mm (0.5") sieve and al-
lowing them to fall into vol. -measuring container.
AOAC Official Methods of Analysis (1990)
Peat
39
S. Apparatus
(a) Sieve.— No. 7/ (12.7 mm).
(b) Measuring box. — Steel or wood, bound with metal hav-
ing one of the following sets of inner dimensions: (7) l /2 cu.
ft. - 12 X 12 x 12" with line scribed 6" from bottom; (2) 3 / 4
cu. ft. = 12 x 12 X 12" with line scribed 9" from bottom; (J)
1 cu. ft. - 12 x 12 X 12" box, 2 cu. ft. = 16 X 16 base x
13.5" ht, 5 cu. ft - 16 x 16 base x 33.75" ht.
C. Determination
(a) Loose peat. — Remove material from bag or container,
pass it thru l / 2 " sieve, and place directly into measuring box.
Pour contents from ca 60 cm (2') into measuring box. Det.
contents of bag or container only once. Fill corners of mea-
suring box by shaking with rotary motion, 1 rotation/sec for
5 sec, without lifting box from floor or surface. When filled,
level off by straightedge. Use ht of box to calc. vol. in cu. ft.
(b) Baled peat.- — Vol. baled material = ht X area of base.
Correct measurements for outside wrappers. Det. amt loose
peat in bale by passing thru ] / 2 ff sieve and measuring amt loose
peat, using 12 x 12 X 12" box as in (a). Report vol. of peat
in cu. ft.
Report total vol. of sieved peat from original container.
Refs.: Book of ASTM Stds(1971)Pts II, 22, and 30, ASTM
D2973-D2978, D2980, and D2944. JAOAC 56,
154(1973).
973.08 Volume Weight, Water-Holding
Capacity, and Air Capacity of Water-Saturated
Peat Materials
First Action 1973
A. Apparatus
(a) Hollow spray nozzle. — Monarch F-97-W, nozzle No.
4.6160 (Monarch Mfg. Works Inc., 2501 E Ontario St, Phil-
adelphia, PA 19134), or equiv.
(b) Pipe connection. — For installation of nozzle on H 2
faucet in sink.
(c) Containers. — Approx. 2 L (2 lb coffee cans are suita-
ble) fitted with plastic covers. Replace metal bottom of one
with No. 20 Cu screen (test container).
(d) Aluminum pie pans. — 20 cm (8") diam. Drill holes in
side walls of pan so that H 2 depth in pan remains ca 1.3 cm
(0.5").
B. Preparation of Sample
See 967.02.
C. Determination
Det. moisture content on sep. sample by 967. 03A or B.
Weigh test container fitted with plastic cover, screen, and
circle of filter paper (12.5 cm Whatman No. 4, or equiv.) which
is placed on screen. Thoroly mix equal wts of H 2 and peat
and place in container without pressure to ht of 10 cm (4");
record wt in g. (If peat is dried out, mix 1 part peat with 2
parts H 2 0. If wet, mix 2 parts peat with 1 part H 2 0.)
Place test container in Al pan filled with H 2 in sink >30
cm (12") directly under spray nozzle. Water ca 24 hr as mist
to prevent compression of peat. Place cover on container, seal
(tape is suitable) to prevent evapn, and let stand in Al pan,
maintaining 1.3 cm FLO head 2 days. Remove from pan and
drain 2 hr with container at 45° angle. Remove seal on cover,
and record wt and vol. Vol. can be detd by using container
identical to test container not fitted with screen, filling H 2 to
same ht as sample in test container, and transferring to grad-
uate with mL markings.
D. Calculations
(a) Saturated Volume Weights, g/mL
(1) As-recd = g as-recd sample/mL wet vol., where g as-
recd sample = g total sample/2; or g total sample/3 if 2 parts
H 2 used; or (g total sample X 2)/3 if 2 parts peat used.
(2) Oven-dried.— See 969. 05D
(3) Wet.— See 969.05D.
(b) Water -Holding Capacity, %.—See 969.051).
(c) Dry Peat Volume, %.—See 969.05D.
(d) Air Volume, %.—See 969. 05D
Refs.: Book of ASTM Stds(1971)Pts 11, 22, and 30, ASTM
D2973-D2978, D2980, and D2944. JAOAC 56,
154(1973).
973.09 Cation Exchange Capacity for Peat
Titrimetric Method
First Action 1973
Final Action 1978
AOAC-ASTM Method
A. Principle
Cation exchange capacity is measure of total amt exchange-
able cations that can be held by peat, expressed as mequiv./
100 g air-dried peat. Peat sample is shaken with 0.5/V HC1 to
remove bases and to sat. sorption complex with H + . Excess
acid is removed; absorbed H + is replaced with Ba H " 2 , titrd with
O.WNaOH, using phthln indicator, and calcd to mequiv./lOO
g air-dried peat.
B. Reagents
(a) Dilute hydrochloric acid. — 0.5/V. Dil. 42 mL HC1 to 1
L with H 2 0.
(b) Barium acetate soln. — 0.5/V. Dissolve 64 g Ba(OAc) 2
in H 2 and dil. to 1 L.
(c) Silver nitrate soln. — 1%. Dissolve 1 g AgN0 3 in 100
mL H 2 0.
(d) Sodium hydroxide std soln.— 0. IN. Prep, and stdze as
in 936.16
C. Preparation of Sample
See 967.02.
D. Determination
Thoroly mix air-dried ground peat sample and place 2.00 g
in 300 mL erlenmeyer. Add ca 100 mL 0.57V HC1; stopper
flask and shake vigorously periodically during 2 hr (or shake
mech. 30 min). Filter thru rapid paper (24 cm fluted, or equiv.)
in large powder funnel. Wash with 100 mL portions H 2 until
10 mL wash shows no ppt with ca 3 mL 1% AgN0 3 . Discard
filtrate. Immediately transfer moist peat to 300 mL erlen-
meyer, by puncturing apex of paper and forcing moist peat
thru funnel stem into erlenmeyer, using spray from wash bottle
contg ca 100 mL 0.5/V Ba(OAc) 2 . Stopper flask and shake vig-
orously periodically during 1 hr (or shake mech. 15 min). Fil-
ter, and wash with three 100 mL portions H 2 0. Discard peat,
and titr. washings with 0, IN NaOH, using 5 drops phthln, to
first pink.
Calc. mequiv./ 100 g air-dried peat
= (mL X normality NaOH x 100)/g sample.
Ref.: JAOAC 56, 154(1973).
3* Plants
Robert A- Isaac, Associate Chapter Editor
University of Georgia
922.01
Sampling of Plants
Final Action
When more than one plant is sampled, include enough plants
in sample to ensure that it adequately represents av. compo-
sition of entire lot of plants sampled. (This number depends
upon variability in composition of the plants.) Det. details of
sampling by purpose for which sample is taken.
Refs.: Botan. Gaz. 73, 44(1922). Proc. Am. Soc. Hort. Sci.
1927, p. 191. JAOAC 13, 224(1930); 16, 71(1933);
19, 70(1936).
922.02
Plants
Preparation of Sample
Final Action
(a) For mineral constituents . — Thoroly remove all foreign
matter from material, especially adhering soil or sand, but to
prevent leaching, avoid excessive washing. Air- or oven-dry
as rapidly as possible to prevent decomposition or wt loss by
respiration, grind, and store in tightly stoppered bottles. If re-
sults are to be expressed on fresh wt basis, record sample wts
before and after drying. When Cu, Mn, Zn, Fe, Al, etc. are
to be detd, avoid contaminating sample by dust during drying
and from grinding and sieving machinery.
(b) For carbohydrates . — Thoroly remove all foreign matter
and rapidly grind or chop material into fine pieces. Add weighed
sample to hot redistd alcohol to which enough pptd CaC0 3 has
been added to neutze acidity, using enough alcohol so that
final concn, allowing for H 2 content of sample, is ca 80%.
Heat nearly to. bp on steam or H 2 bath 30 min, stirring fre-
quently. (Samples may be stored until needed for analysis.)
Refs.: Botan. Gaz. 73, 44(1922). Proc. Am. Soc. Hort. Sci.
1927, p. 191. JAOAC 13, 224(1930); 16, 71(1933);
19, 70(1936).
930.04
Moisture in Plants
Final Action 1965
See 934.01, 920.36*, or 930.15.
930.05
Ash of Plants
Final Action 1965
See 900.02A, 900.02B, or 942.05.
920.08* Sand and Silica in Plants
Gravimetric Method
Final Action
Surplus 1989
See 3.005, 14th ed.
METALS
953.01 Metals in Plants
General Recommendations
for Emission Spectrographs Methods
Final Action 1988
(Applicable to aluminum, barium, boron, calcium, copper, iron,
magnesium, manganese, molybdenum, phosphorus, potas-
sium, sodium, strontium, and zinc)
(a) Instrumental technic. — If, because of equipment limi-
tations, described methods cannot be followed in detail, or if
detn of other elements is desired, following protocol is rec-
ommended: Det. experimentally, with available facilities, po-
tentials of various sample prepns and excitation conditions with
relation to element detectability and general concn require-
ments. Select analysis lines on basis of desirable intensity and
freedom from spectral interference by other elements, as detd
by prepg spectrum of each component element at av. concn at
which it occurs in samples to be analyzed. Line and phototube
characteristics are usually detd by instrument manufacturer.
(b) Precision. — Stdze all conditions of technic and det. re-
producibility of results by making ca 20 successive exposures
on sample of representative composition. For each element,
calc. std deviation of single exposure and divide by square root
of number of individual exposures that will be averaged in
practice to constitute 1 detn. From this est. of std deviation of
single detn, calc. coefficient of variation for each element.
Following upper limits for precision error of spectrographic
detns in analysis of plant material are satisfactory in relation
to other routine methods or to practical requirements: Coef-
ficients of variation (%) — Ca, Mg, Mn, and Mo, 3-7; B, Ba,
Cu, K, P, and Zn, 7-15; and Al, Fe, and Na, >15. Coeffi-
cients of variation vary from instrument to instrument for each
element; above values were obtained from std plant tissue by
11 different instruments.
(c) Accuracy. — Precise technic is essential but not only fac-
tor involved in accuracy. Reliability and appropriateness of stds
and judgment used in ref. method are of utmost importance.
Failure in any of these respects can result in serious calibration
error for otherwise satisfactory method.
Carefully prep, synthetic stds from highest grade H 2 0-free
analyzed chems, collectively blanked for minor and trace ele-
ments. Preferably confirm values assigned to natural stds by
results of >1 laboratory.
Matrix similarity between stds and samples, or closely con-
trolled correction system for matrix differences, is essential.
Check correction scales frequently against stds which closely
match particular types of plant materials being analyzed.
Precision error of technic applies to ref. exposures as well
as to samples. For this reason, base fiducial adjustments on as
many ref. exposures as may be feasibly included in each series
of samples.
Refs.: JAOAC 36, 41 1.(1953); 37, 721(1954); 58, 764(1975).
40
AOAC Official Methods of Analysis (1990)
980.03 Metals in Plants
Direct Reading Spectrographic Method
Final Action 1988
A. Apparatus
(a) Spark excitation source.
(b) Spectrograph. — 1.5 m grating spectrograph with spark
stand and disk attachment rotating at 30 rpm.
(c) Electrode sharpener.
(d) Disk electrode. — High purity graphite disk 0.492" diam.
and 0.200" thick.
(e) Upper (pin) electrode. — Point appropriate lengths of std
grade spectrographic C rods, 0.180" diam., in pencil sharpener
equipped with pin stop to produce Vig" diam. flat tip.
(f) Porcelain boat. — 60 mm long, 10 mm wideband 8 mm
high (Coors No. 2, or equiv.).
B. Reagents
(a) Buffer.— Dissolve 50 g Li 2 C0 3 in 200 mL HN0 3 , and
dil. to 1 L with H 2 0. (Caution: See safety notes on nitric acid.)
(b) Element stock std solns. — On basis of expected sample
concn range, prep, stock solns from individual pure nitrates,
chlorides, or carbonate salts, or metal of resp. elements, as
indicated in Table 980.03A.
(c) Mixed element std solns. — Prep. 5 std solns contg % or
ppm element indicated in Table 980. 03B as follows: Dissolve
Metals
41
50 g Li 2 C0 3 in 200 mL HN0 3 , pipet in indicated aliquots, and
dil. to 1 L with H 2 0. Prep, fresh every 6 months.
C. Preparation of Sample
Dry plant material 24 hr at 80° and grind in Wiley mill with
No. 20 stainless steel sieve. Store in air-tight containers or in
coin envelopes in dry atm.
Weigh 1.0 g prepd sample into 30 mL high form crucible
(porcelain is satisfactory). Ash >4 hr at 500° with crucible
resting on asbestos plate rather than on floor of furnace. Cool,
add 5.0 mL buffer soln, (a), stir, and let stand 30 min.
D. Determination
(a) Excitation. — Align and space electrodes 4 mm apart in
holders; position pin electrode over disk electrode. Set source
parameters to give uniform breakdown voltage at tandem air
gap with operating parameters at 4 breaks/cycle and 4 amp
(parameters may vary with source for best operating effi-
ciency).
Place aliquot of prepd soln in porcelain boat, set boat on
arc stand, and raise to immerse Vie" of disc in soln. Spark 10
sec to condition electrodes and photomultiplier tubes and then
spark addnl 30 sec for integration.
(b) Calibration. — Calibration technic varies with instru-
ment. Use mixed element std solns and known plant tissue stds
to calibrate spectrograph by same technic as for samples. Prep,
std curves to cover desired concn range, using ratio to internal
std and background correction for best results.
Table 980.03A Preparation of Stock Standard Solutions
Element Salt
Element,
9/L
Salt,
g/L Solvent
955.08*
Metals in Plants
K KCI
125
238.36 H 2
99.89 1 N HNO3
Direct Current Arc Excitation Method
Ca CaC0 3
40
Surplus 1970
Mg MgO
20
33.16 IA/HNO3
P H 3 P0 4
10
31 .64 H 2
See 44.003-44.006. 11th ed.
Na NaCl
10
25.42 H 2
Fe Fe metal
1
1.00 IA/HNO3
Mn MnO
1
1.29 1A/HN0 3
Al Al metal
1
1.00 1 N HN0 3
Zn Zn metal
1
1 .00 "\N HNO3
955.09*
Metals in Plants
Cu Cu metal
1
1 .00 1 N HNO3
Ba BaCI 2
1
1 .52 H 2
Alternating Current Spark Excitation Method
Sr SrC0 3
1
1.68 IA/HNO3
Surplus 1970
B H3BO3
1
5.72 H 2
Mo (NH 4 ) 6 Mo 7 24 .H 2
0.1
1.29 H 2
See 44.007-44.011, 11th ed.
Table 980.03B Preparation of Mixed Element Standard Solutions
Element, g/L
Standard Solution Number
Element
1
2
3 4
5
mL to give (%) a
K (CAS-7440-09-7)
125
2(0.5)
4(1.0)
8(2.0) 12(3.0)
20(5.0)
Ca (CAS-7440-70-2)
40
100(2.0)
50(1.0)
30(0.6) 10(0.2)
15(0.3)
Mg (CAS-7439-95-4)
20
100(1.0)
70(0.7)
50(0.5) 20(0.2)
10(0.1)
P (CAS-7723-14-0)
10
2(0.1)
4(0.2)
6(0.3) 10(0.5)
mL to give (ppm) a
14(0.7)
Na (CAS-7440-23-5)
10
1(50)
2(100)
10(500) 20(1000)
40(2000)
Fe (CAS-7439-89-6)
10
10(500}
4(200)
2(100) 1(50)
6(300)
Mn (CAS-7439-96-5)
10
0.4(20)
1(50)
2(100) 4(200)
10(500)
Ai (CAS-7429-90-5)
10
0.6(30)
1(50)
2(100) 4(200)
10(500)
Zn (CAS-7440-66-6)
1
2(10)
4(20)
6(30) 10(50)
20(100)
Cu (CAS-7440-50-8)
1
1(5)
2(10)
4(20) 10(50)
14(70)
Ba (CAS-7440-39-3)
1
20(100)
10(50)
4(20) 2(10)
1(5)
Sr (CAS-7440-24-6)
1
40(200)
20(100)
10(50) 6(30)
3(15)
B (CAS-7440-42-8)
1
1(5)
2(10)
4(20) 10(50)
14(70)
Mo (CAS-7439-98-7)
0.1
2(1)
4(2)
8(4) 12(6)
20(10)
a Concn based on 1 g sample taken up in 5 mL buffer.
42
Plants
AOAC Official Methods of Analysis (1990)
975.03 Metals in Plants
Atomic Absorption Spectrophotometry Method
First Action 1975
Final Action 1988
(Applicable to calcium, copper, iron, magnesium, manganese,
potassium, and zinc)
A. Apparatus and Reagents
Deionized H 2 may be used. See 965. 09 A and B, and fol-
lowing:
(a) Potassium stock soln. — 1000 |xg K/mL. Dissolve 1.9068
g dried (2 hr at 105°) KC1 in H 2 and dil. to 1 L. Use fol-
lowing parameters for Table 965.09: 7665 A, air-C 2 H 2 flame,
and 0.04-2 |mg/mL range.
(b) Calcium stock solas. — Prep. Ca stock soln and working
stds as in 965.09B.
(c) Cu, Fe, Mg, Mn, and Zn stock solns. — Prep, as in
965.09B(b), (c), (e), (f), and (g).
(d) Working std solns. — Dil aliquots of solns (c) with 10%
HC1 to make > 4 std solns of each element within range of
detn.
B. Preparation of Sample
(a) Dry ashing. — Accurately weigh 1 g sample, dried and
ground as in 922.02(a), into glazed, high-form porcelain cru-
cible. Ash 2 hr at 500°, and let cool. Wet ash with 10 drops
H 2 0, and carefully add 3-4 mL HN0 3 (1 + 1). Evap. excess
HN0 3 on hot plate set at 100-120°. Return crucible to furnace
and ash addnl 1 hr at 500°. Cool crucible, dissolve ash in 10
mL HC1 (1 + 1), and transfer quant, to 50 mL vol. flask.
(b) Wet ashing. — Accurately weigh 1 g sample, dried and
ground as in 922.02(a), into 150 mL Pyrex beaker. Add 10
mL HNO3 and let soak thoroly. Add 3 mL 60% HCIO4 and
heat on hot plate, slowly at first, until frothing ceases. (Cau-
tion: See safety notes on wet oxidation.) Heat until HN0 3 is
almost evapd. If charring occurs, cool, add 10 mL HN0 3 , and
continue heating. Heat to white fumes of HCIO4. Cool, add
10 mL HC1 (1 + 1), and transfer quant, to 50 mL vol. flask.
C. Determination
To soln in 50 mL vol. flask, add 10 mL 5% La soln, and
dil. to vol. Let silica settle, decant supernate, and proceed as
in 965.09D.
Make necessary dilns with 10% HC1 to obtain solns within
ranges of instrument.
D. Calcuiations
ppm Element = (|xg/mL) X F/g sample
% Element = ppm x 10~ 4
where F = (mL original diln x mL final diln)/mL aliquot if
original 50 mL is dild.
Ref.: JAOAC 58, 436(1975).
985.01 Metals and Other Elements in Plants
Inductively Coupled Plasma Spectroscopic Method
First Action 1985
Final Action 1988
(Applicable to B, Ca, Cu, K, Mg, Mn, P, and Zn)
A. Principie
Sample is dry-ashed, treated with HNO3, and dissolved in
HC1; elements are detd by ICP emission spectroscopy.
B. Reagents and Apparatus
(a) Stock solns. — 1000 |xg/mL. Weigh designated reagent
into sep. 1 L vol. flasks, dissolve in min. amt of dissolving
reagent,
and dil. to vol. with H 2 0.
Element
Reagent
g
Dissolving Reagent
B
H3BO3
5.7192
H 2
Ca
CaC0 3
2.4973
6N HCI
Cu
pure metal
1.0000
HNO3
K
KCI
1.9067
H 2
Mg
MgS0 4 .7H 2
10.1382
H 2
Mn
Mn0 2
1.5825
6N HCI
P
NH 4 H 2 P0 4
3.7138
H 2
Zn
pure metal
1.0000
6N HCI
(b) Std solns. — Pipet following vols of stock soln into 1 L
vol, flasks. Add 100 mL HCI and dil. to vol. with H,0.
Std Soln 1
Std Soln 2
Stock
Final
Stock
Final
Element
Soln, m
L Concn, M-g/ m L
Soln, mL Concn, ^g/mL
B
10
10
Ca
5
5
60
60
Cu
1
1
K
5
5
60
60
Mg
1
1
20
20
Mn
10
10
P
5
5
60
60
Zn
10
10
Make any needed subsequent dilns with 10% HCI (1 +9).
(c) ICP emission spectrometer. — Suggested operating pa-
rameters: forward power, 1.1 kilowatts; reflected power, <10
watts; aspiration rate, 0.85-3.5 mL/min; flush between sam-
ples, 15-45 s; integration time, 1-10 s.
Element
Wavelength, A
B (CAS-7440-42-8)
2496
Ca (CAS-7440-70-2)
3179
Cu (CAS-7440-50-8)
3247
K (CAS-7440-09-7)
7665
Mg (CAS-7439-95-4)
2795
Mn (CAS-7439-96-5)
2576
P (CAS-7723-14-0)
2149
Zn (CAS-7440-66-6)
2138
C. Dry Ashing
Accurately weigh 1 g sample, dried and ground as in
922.02(a), into glazed, high-form porcelain crucible. Ash 2 h
at 500°, and let cool. Wet ash with 10 drops of H 2 0, and care-
fully add 3-4 mL HN0 3 (1 + 1). Evap. excess HNO a on hot
plate set at 100-120°. Return crucible to furnace and ash addnl
1 h at 500°. Cool crucible, dissolve ash in 10 mL HCI (1 +
1), and transfer quant, to 50 mL vol. flask. Dil. to vol. with
H 2 0.
D. Determination
Elemental detn is accomplished by inductively coupled plasma
emission spectroscopy. Calibration of instrument is done thru
use of known calibration stds. After calibration is complete,
samples can be analyzed. Check calibration after every 10
samples. If instrument has drifted out of calibration (>3% of
original values), recalibrate.
Calc. concn for each element of each dild sample as jxg/
mL.
Ref.: JAOAC 68, 499(1985).
AOAC Official Methods of Analysis (1990)
Metals
43
INDIVIDUAL METALS
928.03 Aluminum and Iron in Plants
Titrimetric Method
Final Action
(Caution: See safety notes on sulfuric acid.)
Take aliquot of Soln /, 920.08, contg enough Fe and Al to
form ca 40 mg Fe- and A1P0 4 . Add few drops HN0 3 , Br-H 2 0,
or H 2 2 to oxidize Fe. If soln does not already contain excess
phosphate, add 0.5 g (NH 4 ) 2 HP0 4 , stir until dissolved, and dil.
to 50 mL with H 2 0. Add few drops thymol blue soln (0.1%:
dissolve 0.1 g thymol blue in H 2 0, add enough IN NaOH
to change color to blue, and dil. to 100 mL), and then add
NH 4 OH until soln just turns yellow. Add 0.5 mL HC1 and 25
mL 25% NH 4 OAc soln, and stir. Let stand at room temp, until
ppt settles (ca 1 hr). Filter, and wash 10 times with hot 5%
NH 4 NO^ soln. Ignite at 500-550° and weigh as FeP0 4 and
A1P0 4 .
Fuse ignited residue in Pt crucible with ca 4 g Na 2 C0 3 -K 2 C0 3
(1 + 1) mixt. When fusion is complete, let crucible cool, add
5 mL H 2 S0 4 , and heat until copious fumes of SO3 are evolved.
Cool, transfer to flask, add H 2 0, and digest until soln is clear.
Reduce Fe with Zn, cool, and titr. with 0.1 AT KMn0 4 . Correct
for blank and calc. as % Fe or % Fe 2 3 . Calc. to FeP0 4 and
subtract from total Fe- and A1P0 4 to obtain A1P0 4 . Correct
for blank and report as A1 2 3 .
Refs.: JAOAC 11, 203(1928); 16, 70(1933); 19, 70(1936).
CAS-7784-30-7 (aluminum phosphate)
CAS- 10045-86-0 (ferric phosphate)
910.01
Calcium in Plants
Titrimetric Macro Method
Final Action
Transfer aliquot of Soln I, 920.08, to 200 mL beaker, add
H 2 if necessary to vol. of 50 mL, heat to bp, and add 10 mL
satd (NH 4 ) 2 C 2 4 soln and drop Me red (dissolve 1 g Me red
in 200 mL alcohol). Almost neutze with NH 4 OH and boil until
ppt is coarsely granular. Cool, add NH 4 OH (1+4) until color
is faint pink (pH 5.0), and let stand 5=4 hr. Filter, and wash
with H 2 at room temp, until filtrate is oxalate-free. (Reserve
filtrate and washings for Mg detn, 920.09.)
Break point of filter with Pt wire, and wash ppt into beaker
in which Ca was pptd, using stream of hot H 2 0. Add ca 10
mL H 2 S0 4 (1 +4), heat to ca 90°, add ca 50 mL hot H 2 0, and
titr. with 0.05 N KMn0 4 . Finally add filter paper to soln and
complete titrn.
Refs.: J. Biol. Chem. 7, 83(1910). JAOAC 4, 392(1921); 16,
70(1933).
CAS-7440-70-2 (calcium)
921.01
Calcium in Plants
Titrimetric Micro Method
Final Action
Weigh 2 g sample into small crucible and ignite in furnace
at 500-550°. Dissolve ash in HCl (1+4) and transfer to 100
mL beaker. Add 5 mL HCl and evap. to dryness on steam
bath to dehydrate Si0 2 . Moisten residue with 5 mL HCl, add
ca 50 mL H 2 0, heat few min on steam bath, transfer to 100
mL vol. flask, cool quickly to room temp., dil. to vol., shake,
and filter, discarding first portion of filtrate.
Pipet 15 mL aliquot into conical-tip centrf. tube contg 2 mL
satd (NH 4 ) 2 C 2 4 soln and 2 drops Me red (dissolve 1 g Me
red in 200 mL alcohol). Add 2 mL HOAc (1+4), rotating tube
to mix contents thoroly. Add NH 4 OH (1+4), while intermit-
tently rotating tube, until soln is faintly alk.; then add few
drops of the HOAc until color is faint pink (pH 5.0). (It is
important at this point to rotate tube so that last bit of liq. in
conical tip has required color.) Let stand >4 hr; then centrf.
15 min. (Ppt should be in firm lump in tip of tube.) Remove
supernate, using suction device, Fig. 921.01, taking care not
to disturb ppt. Wash ppt by adding 2 mL NH 4 OH (1+49),
rotating tube to break up ppt. (It may be necessary to jar tube
sharply.) Centrf. 10 min, again remove supernate, and wash
with reagent as before. Repeat washing of ppt 3 times.
After removing last supernate, add 2 mL H 2 S0 4 (1+4) to
tube, break up ppt as before, heat on steam bath to 80-90°,
and titr. in tube with 0.02N KMn0 4 , rotating liq. during titrn
to attain proper end point. If tube cools to <60° during titrn,
as indicated by slow reduction of KMn0 4 , reheat in steam bath
few min and complete titrn. Perform blank on identical vol.
H 2 S0 4 in similar tube heated to same temp, to det. vol. KMn0 4
soln necessary to give end point color. Subtract this value from
buret reading. 1 mL 0.02N KMn0 4 - 0.000400 g Ca. Report
as % Ca.
Refs.: J. Biol. Chem. 47, 475(1921); 50, 527, 537(1922).
JAOAC 14, 216(1931); 16, 71(1933); 19, 71(1936).
CAS-7440-70-2 (calcium)
951.01
Cobalt in Plants
Nitrosocresol Method
Final Action 1965
(Caution: See safety notes on distillation, toxic solvents, car-
bon tetrachloride, and nitroaromatics.)
A. Reagents
(Make all distns in Pyrex stills with J joints. Store reagents in
g-s Pyrex bottles.)
(a) Redistilled water. — Distil twice, or pass thru column of
ion exchange resin (IR-IOOA, H-form, or equiv.) to remove
heavy metals.
FIG. 921.01 — Suction device used in micro method for deter-
mining calcium
44
Plants
AOAC Official Methods of Analysis (1990)
(b) Hydrofluoric acid. — 48%. Procurement in vinyl plastic
bottles is advantageous.
(c) Perchloric acid. — 60%. No further purification neces-
sary.
(d) Hydrochloric acid. — (1 + 1). Add equal vol. HC1 to distd
H 2 and distil.
(e) Ammonium hydroxide. — (1 + I). Distil coned NH 4 OH
into equal vol. redistd H 2 0.
(f) Ammonium hydroxide. — 0.02N . Add 7 mL of the NH4OH
(1 + 1) to 2.5 L redistd H 2 0.
(g) Carbon tetrachloride. — Distil over CaO, passing dis-
tillate thru dry, acid-washed filter paper. Used CC1 4 may be
recovered as in 941.03A(a).
(h) Dithizone. — Dissolve 0.5 g dithizone in 600-700 mL
CCI4 (tech. grade is satisfactory). Filter into 5 L separator contg
2.5-3.0 L 0.02 N NH 4 OH, shake well, and discard CC1 4 layer.
Shake with 50 mL portions redistd CC1 4 until CC1 4 phase as
it seps is pure green. Add 1 L redistd CC1 4 and acidify slightly
with the HC1 (1 + 1). Shake the dithizone into CC1 4 layer and
discard aq. layer. Store in cool, dark place, preferably in re-
frigerator.
(i) Ammonium citrate soln.— 40%. Dissolve 800 g citric acid
in 600 mL distd H 2 0, and, while stirring, slowly add 900 mL
NH 4 OH. Reaction is exothermic; take care to prevent spatter-
ing. Adjust pH to 8.5, if necessary. Dil. to 2 L and ext with
25 mL portions dithizone soln until aq. phase stays orange and
CC1 4 remains predominantly green. Then ext soln with CC1 4
until all orange is removed.
(j) Hydrochloric acid.— AN. Dil. 16.6 mL of the HC1
(1 + 1) to 1 L with redistd H 2 0.
(k) Hydrochloric acid,— 0.0} N. Dil. 100 mL of the 0.1 JV
HC1 to .1 L with redistd H 2 0.
(1) Sodium hydroxide soln. — IN. Dissolve 40 g NaOH in 1
L redistd H 2 0.
(m) Borate buffer.— pH 7.8. Dissolve 20 g H 3 B0 3 in 1 L
redistd H 2 0. Add 50 mL \N NaOH and adjust pH, if neces-
sary. Equal vols borate buffer and 0.01 N HC1 should give soln
ofpH7.9.
(n) Borate buffer.— pH 9.1. To 1 L borate buffer, pH 7.8,
add 120 mL IN NaOH and adjust pH, if necessary.
(o) Skelly solve B . — Essentially «-hexane. Purify by adding
20-30 g silica gel/L, let stand several days, and distil. Avail-
able from Getty Refining and Marketing Co., PO Box 1650,
Tulsa, OK 74102.
(p) Cupric acetate soln.-— Dissolve 10 g Cu(OAc) 2 .H 2 in
1 L redistd H 2 0.
(q) o-Nitrosocresol soln. — Dissolve 8.4 g anhyd. CuCl 2 and
8.4 g NH 2 OH.HCl in 900 mL H 2 0. Add 8 mL m-cresol (East-
man Kodak Co., practical grade) and stir vigorously while
slowly adding 24 mL 30% H 2 2 . Stir mech. 2 hr at room temp.
(Standing for longer periods results in excessive decomposi-
tion.) Add 25 mL HC1 and ext o-nitrosocresol with four 150
mL portions Skellysolve B, (o), in large separator. Then add
addnl 25 mL HC1 and again ext with four 150 mL portions
Skellysolve B. Wash combined Skellysolve B exts twice with
50-100 mL portions 0.17V HC1 and twice with 50-100 mL
portions redistd H 2 0. Shake <9-nitrosocresol soln with succes-
sive 50-100 mL portions 1% Cu(OAc) 2 soln until aq. phase
is no longer deep blood-red. When light purple is evident, extn
is complete. Discard Skellysolve B phase, acidify aq. soln of
Cu salt with 25 mL HC1, and ext reagent with two 500 mL
portions Skellysolve B; wash twice with 150-200 mL portions
0. 17V HC1 and several times with 150-200 mL portions redistd
H 2 0. Store c-nitrosocresol soln in refrigerator at ca 4°. Re-
agent is stable >6 months.
(r) Sodium o-nitrosocresol soln. — Ext 100 mL onitroso-
cresol by shaking with two 50 mL portions borate buffer, pH
9.1, in separator. (If this is carried out as 2 extns, resulting
reagent is more coned. It is important that total vol. o-nitro-
socresol soln equal total vol. buffer.)
(s) Cobalt std solns. — (J) Stock soln. — Heat CoS0 4 .7H 2
in oven at 250-300° to const wt (6-8 hr). Weigh exactly 0.263
g of the C0SO4 and dissolve in 50 mL redistd H 2 and 1 mL
H 2 S0 4 . Dil. to 1 L, (2) Working soln. — 0.5|xg/mL. Transfer
5 mL stock soln to 1 L vol. flask and dil. to vol. with redistd
H 2 0.
(t) Hydroxylamine acetate buffer. — pH 5. 1 ±0.1. Dissolve
10 g NH 2 OH.HCl and 9.5 g anhyd. NaOAc in 500 mL redistd
H 2 0.
B. Apparatus
(a) Platinum dishes. — Approx. 70 mL; for ashing.
(b) Automatic dispensing burets. — 100 mL; type that can
be fitted to ordinary 5 lb reagent bottle and filled by means of
aspirator bulb is most convenient.
(c) Wooden separator rack. — Twelve-unit 125 mL sepa-
rator size is convenient for dithizone extns. Rack is fitted across
top with removable bar padded with sponge rubber so all 12
separators can be shaken as unit.
(d) Racks,— Consisting of 5 x 5 x 65 cm (2 x 2 x 25")
wooden bars with holes drilled at close intervals to take 50 mL
centrf. tubes fitted with No. 13 ¥ glass stoppers. To make these
tubes, ream out necks of heavy- wall Pyrex centrf. tubes
(Rockefeller Institute type) with t C rod and grind to take ¥
stopper. Place tubes upright in one section, and place other
section (fitted with sponge rubber disks 13 mm thick in bottom
of holes) across their tops. Fasten 2 sections at ends with re-
movable rubber connectors made from ordinary tubing of con-
venient size, so that any number of tubes can be shaken as
unit. Use these tubes for reaction of Co with nitrosocresol,
extn of complex into Skellysolve B, and washing of Skelly-
solve B soln.
(e) Shaking machine. — Mech. shaker giving longitudinal
stroke of 5 cm at ca 180 strokes/min; use to make dithizone
extns and to ext Co complex, or shake by hand.
C. Cleaning of Glassware
Clean 120 mL Pyrex separators for dithizone extns by ini-
tially soaking 30 min in hot HN0 3 and rinsing several times
with H 2 0. As added precaution, shake with several portions
dithizone in CC1 4 . After use, clean by rinsing with H 2 0, drain,
and stopper to avoid contamination. It is not necessary to clean
every time with acid. Repeat HN0 3 cleaning if blanks are un-
usually high.
Clean 50 mL g-s Pyrex centrf. tubes by soaking 30 min in
HNO3 followed by several rinsings in H 2 0.
Completely submerge pipets in cylinder of chromic acid
cleaning soln overnight, rinse several times with H 2 0, and sus-
pend upright in rack to dry.
Wash all other glassware thoroly in detergent and rinse well
with tap H 2 followed by dip in chromic acid cleaning soln.
Rinse off cleaning soln with tap H 2 followed by several distd
H 2 rinses.
Clean Pt by scrubbing with sea sand followed by boiling in
HO (1+2) 30 min, and rinse several times with H 2 0.
D. Preparation of Sample
See 922.02(a). Oven-dry all plant material 48 hr and prep,
for ashing by either of following methods:
(a) Grind material in Wiley mill equipped with stainless steel
sieve, mix thoroly by rolling, and sample by quartering.
(b) Using stainless steel shears, cut material by hand fine
enough for convenient sampling.
E. Ashing of Samples
(Caution: See safety notes on distillation, hydrofluoric acid,
and perchloric acid.)
AOAC Official Methods of Analysis (1990)
Metals
45
Weigh 6 g dry plant tissue into clean Pt dish. Cover with
Pyrex watch glass and place in cool furnace; heat slowly to
500° and hold at this temp, overnight. Remove sample and
cool. Wet down ash carefully with fine stream redistd H 2 0.
From dispensing buret, slowly add 2-5 mL HC10 4 , dropwise
at first to prevent spattering. Add ca 5 mL HF, evap. on steam
bath, transfer to sand bath, and keep at medium heat until fum-
ing ceases.
Cover with Pyrex watch glass, return to partially cooled fur-
nace, heat gradually to 600°, and keep at this temp. 1 hr. Re-
move sample and cool. Add 5 mL HC1 (1 + 1) and ca 10 mL
redistd H 2 0. Replace cover glass and warm on steam bath to
dissolve. (Usually clear soln essentially free of insol. material
is obtained.) Transfer sample to 50 mL vol. flask, washing
dish several times with redistd H 2 0, dil. to vol., and mix tho-
roly. (Pt dishes can ordinarily be used several times between
sand and acid cleanings.)
F. Dithizone Extraction
(Caution: See safety notes on distillation, perchloric acid, and
carbon tetrachloride.)
Transfer suitable aliquot (2-3 g dry material) to 120 mL
separator (use petroleum jelly as stopcock lubricant). Add 5
mL NH 4 citrate soln and 1 drop phthln; adjust to pH 8.5 with
NH 4 OH (1 + 1). If ppt forms, add addnl NH 4 citrate. Add 10
mL dithizone in CC1 4 and shake 5 min. Drain CC1 4 phase into
100 mL beaker. Repeat as many times as necessary, using 5
mL dithizone soln and shaking 5 min each time. Extn is com-
plete when aq. phase remains orange and CC1 4 phase remains
predominantly green. Then add 1.0 mL CC1 4 , shake 5 min, and
combine with CC1 4 ext. Final 10 mL CC1 4 should be pure green.
If not, extn was incomplete and must be repeated.
Add 2 mL HC10 4 to combined CC1 4 exts, cover beaker with
Pyrex watch glass, and digest on hot plate until colorless. Re-
move cover glass and evap. slowly to dryness. (If sample is
heated any length of time at high temp, when dry, losses of
Co may occur. Heat only enough to evap. completely to dry-
ness. If free acid remains, it interferes with next step where
pH control is important.)
Add 5 mL 0.0 IN HC1 to residue. Heat slightly to assure
soln. If Cu is to be detd, transfer with redistd H 2 to 25 mL
vol. flask, and dil. to vol. Transfer 20 mL aliquot to 50 mL
g-s centrf . tube or 60 mL separator and reserve remainder for
Cu detn, 953. 03B. If Cu is not to be detd, transfer entire acid
soln with redistd H 2 to centrf. tube or separator.
G. Determination
Add 5 mL borate buffer, pH 7.8, and 2 mL freshly prepd
Na o-nitrosocresol soln to sample soln. Add exactly 5 mL
Skellysolve B and shake 10 min. Remove aq. phase by mod-
erate suction thru finely-drawn glass tube. To Skellysolve B
layer add 5 mL Cu(OAc) 2 soln and shake 1 min to remove
excess reagent. Again remove and discard aq. phase. Wash
Skellysolve B by shaking I min with 5 mL redistd H 2 0, re-
moving aq. layer as before; finally shake Skellysolve B 1 min
with 5 mL NH 2 OH -NaOAc buffer to reduce Fe. Transfer Skel-
lysolve B soln of the Co complex to 5 cm cell and read in
spectrophtr as close as possible to point of max. A, 360 nm.
H. Blanks and Standards
With each set of detns include ashing blank and Co stds of
0.0, 0.5, and 1.0 |xg. Beer's law holds for this range. A of
0.0 jULg point should be <0.05. If above, repurify o-nitroso-
cresol by transferring alternately to aq. phase as Cu salt and
to Skellysolve B phase as free compd after acidifying aq. phase.
It is also advisable to include std sample with each set of
samples to detect contamination or unusual losses of Co in
method. Com. buckwheat flour contg 0.05 ppm Co has proved
satisfactory for this purpose.
/. Calculations
Express results in terms of ppm Co, based upon dry wt of
sample.
ppm Co — (p,g Co/mL dithizone aliquot)
X (mL total soln/g dry sample)
Value for p,g Co is obtained from curve minus ashing blank.
Refs.: JAOAC 34, 710(1951); 36, 405(1953).
CAS-7440-48-4 (cobalt)
953.02
Cobalt in Plants
Nitroso-R-Salt Method
Final Action 1965
A. Reagents
Those listed in 951. 01 A, and following:
(a) N itr o so -R- salt soln. — 0.2%. Dissolve 2 g powd nitroso-
R-salt (Eastman Kodak Co., No. 1124) in redistd H 2 0,
951.01A(a), and dil. to 1 L.
(b) Dilute nitric acid. — (1 + 1). Dil. HNO3 with equal vol.
H 2 and redistil in Pyrex app. Store in Pyrex bottles.
(c) Bromine water. — Satd soln of Br in redistd H 2 0,
951.01A(a)
(d) Citric acid soln. — 0.2 N. Use special reagent grade Pb-
free citric acid.
B. Preparation and Ashing of Samples
Proceed as in nitrosocresol method, 951.010 and E, thru
"(Usually clear soln essentially free of insol. material is ob-
tained.)" except use 10 g instead of 6 g dry plant tissue.
C. Dithizone Extraction
Transfer entire soln to 120 mL separator, and proceed as in
951. 01F, thru "If free acid remains . . . pH control is impor-
tant.)" Dissolve in 1 mL citric acid soln, (d), transfer to 25
mL vol. flask, and dil. to vol. with redistd H 2 0, 951.01A(a).
D. Determination
Transfer suitable aliquot (ca 8 g dry material) of citric acid
soln, 953.02C, to 50 mL beaker. Evap. to 1-2 mL. Add 3
mL borate buffer, 951.01A(n), and adjust pH to 8.0-8.5 with
NaOH (check externally with phenol red). (Vol. <5 mL.) Add
I mL nitroso-R-salt soln slowly with mixing. Boil 1-2 min and
add 2 mL dil. HN0 3 . Boil 1-2 min, add 0.5-1.0 mL Br-H 2 0,
cover with watch glass, and let stand warm 5 min. Boil 2-3
min to remove excess Br (use effective fume removal device).
Cool, and dil. to 10 or 25 mL (depending on length of light
path in absorption cell). Transfer to cell and read at 500 nm
within 1 hr. Prep, stds contg 0.5, 1, 2, 3, and 4 |xg Co and
add 1 mL citric acid soln, 352.03A(d), to each. Proceed as
for unknowns, beginning "Evap. to 1-2 mL."
Ref.: JAOAC 36, 405(1953).
CAS-7440-48-4 (cobalt)
953.03
Copper in Plants
Colorimetric Method
Final Action 1965
A. Reagents
Those listed in 951.01 A, and following:
(a) Sodium diethyldithiocarbamate soln.-
prepd in redistd H 2 0, 951.01A(a).
-0.1%. Freshly
46
Plants
AOAC Official Methods of Analysis (1990)
(b) Copper std soln. — I |mg/mL. Dissolve 0.3929 g
CuS0 4 .5H 2 in redistd H 2 0, 951.01A(a), add 5 mL H 2 S0 4 ,
dil. to 1 L, and mix. Take 10 mL aliquot, add 5 mL H 2 S0 4 ,
dil to 1 L, and mix.
B. Determination
Transfer aliquot (0.5-1 g dry material) from soln obtained
from 951.01F or 953. 02C to 125 mL separator. Add 2 mL
NH 4 citrate soln, 1 drop phthln, 5 mL Na diethyldithiocar-
bamate soln, and NH 4 OH (1 + 1), 951.01A(e), until pink. Add
10 mL CC1 4 and shake 5 min. Drain CC1 4 , centrf. 5 min, trans-
fer to absorption cell, and read at max. A, ca 430 nm.
Prep, std curve with 0, 1, 5, 10, 15, and 20 |ULg Cu treated
as above.
Ref.: JAOAC 36, 405(1953).
CAS-7440-50-8 (copper)
937.03
Iron in Plants
Colorimetric Method
Final Action
120 g HO Ac to 1 L with H 2 0.
— 1%. Dissolve 1 g NH 4 citrate
A Reagents
(a) Acetic acid. — 2M. Oil. .
(b) Ammonium citrate soln.-
in H 2 and dil. to 100 mL.
(c) Bromophenol blue indicator. — 0.04%. Grind 0.1 g bro-
mophenol blue in mortar with 3 mL 0.05W NaOH, transfer to
vol. flask, and dil. to 250 mL with H 2 0.
(d) Buffer solns.-(l) pH 3.5.~~~Mix 6.4 mL 2M NaOAc
with 93.6 mL 2Af HO Ac and dil. to 1 L. (2) pH 4. 5. —Mix
43 mL 2M NaOAc with 57 mL 2M HOAc and dil. to 1 L.
(e) Hydroquinone soln. — Dissolve 1 g hydroquinone in 100
mL pH 4.5 buffer, (d)(2). Keep in refrigerator, and discard
when any color develops.
(f) o-Phenanthroline soln. — Dissolve 1 g o-phenanthro-
line.H 2 in H 2 0, warming if necessary, and dil. to 400 mL.
(g) Sodium acetate soln. — 2M. Dissolve 272 g NaOAc.3H 2
in H 2 and dil. to 1 L.
(h) Iron std soln. — 1 mg/mL. Dissolve 1 g electrolytic Fe
in 50 mL H 2 S0 4 (1+9), warming if necessary to hasten re-
action. Cool, and dil. to 1 L with H 2 0.
B. Preparation of Sample
(Caution: See safety notes on distillation, hydrofluoric acid,
and sulfuric acid.)
Use Soln I, 920.08, or if Soln I is not available, weigh sam-
ples of finely ground plant material (1-5 g, depending on Fe
content) into porcelain crucibles with smooth inner surfaces,
and ash overnight at 500-550° in furnace. Cool, add 5 mL
HC1 (1 + 1), and heat on steam bath 15 min to dissolve Fe and
to hydrolyze pyrophosphate. Filter into 100 mL vol. flask.
Transfer insoJ. residue to filter and wash 5 times with 3 mL
portions hot HO (1 + 100), then with hot H 2 until washings
are Cl-free. Ignite paper and any remaining C in Fe-free Pt
crucible. Cool, add 2 drops H 2 S0 4 and 1 mL HF, and carefully
evap. to S0 3 fumes. Cool, add few drops HC1 (1+1), and
warm. Filter and wash as before into same vol. flask, dil. to
vol., and mix.
C. Determination
Pipet identical aliquots of Soln 1, 920.08, or sample soln,
937. 03B, into 25 mL vol. flask and into test tube or small
erlenmeyer. Add 5 drops bromophenol blue indicator to ali-
quot in test tube, and titr. with 2M NaOAc soln until color
matches that of equal vol. of pH 3.5 buffer contg same amt
of indicator. Add 1 mL hydroquinone soln and 2 mL o-phen-
anthroline soln to aliquot in vol. flask, and adjust pH to 3.5
by adding same vol. NaOAc soln found necessary for aliquot
in test tube. If turbidity develops upon adjusting pH of aliquot
in test tube, add I mL NH 4 citrate soln to vol. flask before
adding the NaOAc soln. Dil. to vol., mix, and let stand .1 hr
for complete color development, and measure A at max., ca
510 nm.
Prep, curve relating A to mg Fe in 25 mL by treating series
of solns contg amts of Fe that cover usable range of instrument
exactly as described for unknowns, detg their respective read-
ings at max. A, ca 510 nm, and plotting these against corre-
sponding concns of Fe. H 2 may be used as ref., and blanks
detd to correct for amt Fe in reagents used, or blank soln itself
may be made basis of comparison.
Refs.: Ind. Eng. Chem. Anal. Ed. 9, 67(1937); 10, 13(1938).
JAOAC 25, 555(1942); 27, 526(1944).
CAS-7439-89-6 (iron)
936.02
Iron in Plants
Titrimetric Method
Final Action
Take appropriate aliquot of Soln I or of soln prepd as in
937.03B, and oxidize Fe by adding soln of KMn0 4 (1 + 1000)
dropwise until very faint permanganate color persists. Add 5
mL 10% NH 4 SCN and titr. with dil. TiCl 3 soln until red color
disappears. (To prep, appropriate TiCl 3 soln, boil 5-10 mL
20% TiCl 3 with 50 mL HC1 few min, cool, and dil. to 1 L.
Stdze against std Fe soln, keep in dark in well-filled container,
and restdze each time it is used, or every few hr when many
detns are being made. Discard when decomposition is indi-
cated by loss of color and increased titer against std.)
Refs.: JAOAC 19, 359(1936); 27, 526(1944).
CAS-7439-89-6 (iron)
920.09* Magnesium in Plants
Gravimetric Method
Final Action
Surplus 1989
See 3.039, 14th ed.
921.02
Manganese in Plants
Colorimetric Method
Final Action
To aliquot of Soln I, 920.08, contg 0.2-0.5 g ash, add 15
mL H 2 S0 4 and evap. to ca 30 mL. Add 5-10 mL HN0 3 and
continue evapn. (Do not evap. until dense fumes appear, be-
cause Fe 2 (S0 4 ) 3 then dissolves with difficulty. HN0 3 may be
present, but not HC1.) Add H 2 0, little at time, heat until Fe
salts dissolve, and dil. to ca 150 mL. Add 0.3 g KlG 4j or its
equiv. in HI0 4 , in small portions, boil few min or until color
of KM11O4 shows no further increase in intensity, and let cool.
Prep, std as follows: To vol. H 2 equal to sample add 15
mL H 2 S0 4 and enough pure Fe(N0 3 ) 3 , free from Mn, to equal
approx. amt of Fe in sample. Add measured vol. 0.1 N KMn0 4
until color is slightly darker than sample, then add 0.3 g K10 4 ,
and boil few min. When cool, transfer sample and std to 250
AOAC Official Methods of Analysis (1990)
Metals
47
mL vol. flasks and dil. to vol. with H 2 0. (If color is weak, it
may be necessary to dil. to <250 mL.) Measure A with pho-
tometer or spectrophtr set at max., ca 530 nm. Report as %
Mn.
Ref.: JAOAC 4, 393(1921).
CAS-7439-96-5 (manganese)
960.05 Molybdenum in Plants
Colorimetric Method
First Action 1960
Final Action 1965
A. Apparatus
Photoelectric colorimeter or spectrophotometer . — Capable
of isolating band at ca 465 nm. (Photometer equipped with
filter with max. T at 440-460 nm and J cm cells of 10 mL
capacity is suitable.)
0. Reagents
(a) Isoamyl alcohol. — Reagent grade 3-methyI-l-butanol,
bp 128-132°.
(b) Dilute hydrochloric acid. — (7) 20% soln. — Dil. coned
HCl to ca 20% HCl (1 + 1 .85). (2) 6N soln.—- Stdze to second
decimal place.
(c) Iron std soln. — 100 (xg/mL. Dissolve 0.7022 g
Fe(NH4) 2 (S0 4 > 2 .6H 2 in H 2 0, add 1 mL H 2 S0 4 , and dil. to
1 L.
(d ) Molybden am std solus . — (1 ) Stock soln . — 1 00 jxg /mL .
Dissolve 0.0920 g (NH 4 ) 6 Mo 7 24 .4H 2 in H 2 and dil. to 500
mL. (2) Working soln. — 5 fxg/mL. Dil. 25 mL stock soln to
500 mL.
(e) Potassium thiocyanate soln. — 20%. Dissolve 50 g KSCN
in H 2 and dil. to 250 mL.
(f) Sodium fluoride saturated soln. — Add 200 mL H 2 to
ca 10 g NaF. Stir until satd and filter.
(g) Stannous chloride solns. — (1) 20% soln. — Weigh 10 g
SnCl 2 .2H 2 into beaker, add 10 mL 20% HCl, (b)(7), and
heat until completely dissolved. Cool, add granule of metallic
Sn, dil. to 50 mL with H 2 0, and store in g-s bottle. (2) 0.8%
wash soln.— OH . 4 mL 20% soln to 100 mL with H 2 0.
C. Determination
{Caution: See safety notes on wet oxidation, nitric acid, and
perchloric acid.)
Weigh 1-5 g finely ground sample, contg <35 u-g Mo, into
200 mL tall-form Pyrex beaker. To 1, 2, or 5 g samples add
10, 15, or 35 mL HN0 3 , resp. Include 2 beakers for blanks.
Cover beaker with cover glass, and let stand ca 15 min; then
heat cautiously on steam bath or hot plate at ca 100°, avoiding
frothing over top. If froth approaches cover glass, remove beaker
from heat until frothing subsides; then continue heating. Di-
gest, usually ca 2 hr, until most of solids disappear.
Cool to room temp. If contents should go to dryness, add
few mL HN0 3 . Add 6 mL 70-72% HCIO4, cover beaker, place
on hot plate, and gradually raise temp, so that contents boil
vigorously but do not bump. Continue heating until digestion
is complete as indicated by liq. becoming colorless or pale
yellow. If necessary, make repeated addns of HN0 3 and HCIO4
and continue to digest until C is completely oxidized.
After digestion is complete, place cover glass slightly to one
side of top of beaker, or replace it with elevated watch glass,
and evap. just to dryness or until residue appears only slightly
moist. Remove beaker from hot plate, and cool. Wash down
sides of beaker and underside of cover glass with few mL H 2 0,
return to hot plate, and boil few min. Remove from hot plate,
cool, and again rinse sides of beaker and cover glass with small
amt H 2 0.
Add 2 drops Me orange and neutze with NH 4 OH. Add 67V
HCl, dropwise with stirring, until soln is just acid; then add
8.2 mL excess to give final concn of ca 3% HCl. Add 2 mL
satd NaF soln, and 1 mL Fe soln, if sample contains <100
jxg Fe.
Transfer soln to 125 mL separator and dil. to 50 mL with
H 2 0. Add 4 mL 20% KSCN soln, mix thoroly, and add 1.5
mL 20% SnCl 2 soln. Mix again, and from buret or pipet, add
exactly 15 mL isoamyl alcohol. Stopper separator and shake
vigorously 1 min, let phases sep., and drain and discard aq.
layer. Ext into alcohol without delay, since colored complex
is somewhat unstable in aq. soln.
Add 25 mL freshly prepd 0.8% SnCl 2 wash soln, and shake
gently 15 sec. Let phases sep., and drain and discard aq. layer.
Transfer isoamyl alcohol soln to centrf. tube, and centrf. 5 min
at ca 2000 rpm to remove H 2 droplets. If alcohol layer is not
clear, recentrf. Stopper tubes to prevent evapn, if A readings
cannot be made immediately.
Compare unknown solns with isoamyl alcohol at ca 465 nm
in spectrophtr, and make appropriate corrections in A readings
for Mo in blanks. Obtain Mo concn from calibration curve
relating A readings to conens of series of solns of known Mo
content.
Prep, calibration curve for instrument used, as follows: Place
aliquots of working std soln contg 0, 5, 10, 15, 20, 25, 30,
and 35 |xg Mo, resp., into 200 mL tall-form beakers and carry
them thru entire detn, beginning with digestion with HNO3 and
HCIO4. Plot A against corresponding Mo conens.
Refs.: JAOAC 36, 412(1956); 41, 309(1958); 43, 510(1960).
CAS-7439-98-7 (molybdenum)
925.01* Potassium and Sodium in Plants
Gravimetric Method
Final Action
Surplus 1974
■See 3.015, 11th ed.
956.01 Potassium and/or Sodium in Plants
Flame Photometric Method
Final Action 1965
A. Reagents
(a) Potassium stock soln. — 1000 ppm K. Dissolve L907 g
dry KCl in H 2 and dil. to 1 L.
(b) Sodium stock soln. — 1000 ppm Na. Dissolve 2.542 g
dry NaCl in H 2 and dil. to 1 L.
(c) Lithium stock soln. — 1000 ppm Li. Dissolve 6.108 g
LiCl in H 2 and dil. to 1 L. (Needed only if internal std method
of evaluation is to be used.)
(d) Ammonium oxalate stock soln. — 0.24/V. Dissolve 17.0
g (NH 4 ) 2 C 2 4 .H 2 in H 2 and dil. to 1 L.
(e) Extracting solns . — (1 ) For potassium . — For internal std
method, dil. required vol. LiCl stock soln to 1 L; otherwise
use H 2 0. (2) For sodium. — To 250 mL NH 4 oxalate stock soln
add required vol. LiCl stock soln (if internal std method is
used) and dil. to 1 L. If internal std requirements are same for
both Na and K detns, this reagent may be used as common
extg soln.
48
Plants
AOAC Official Methods of Analysis (1990)
B. Preparation of Standard Solutions
Dil. appropriate aliquots of stock solns to prep, series of stds
contg K and/or Na in stepped amts (including 0) to cover in-
strument range, and Li and NH 4 oxalate (if required) in same
concns as in corresponding extg solhs. (If common extg soln
is used, 1 set of stds contg both K and Na suffices.)
C. Sample Extraction
Transfer weighed portion of finely ground and well mixed
sample to erlenmeyer of at least twice capacity of vol. of extg
soln to be used. Add measured vol. extg soln, stopper flask,
and shake vigorously at frequent intervals during 2=15 min.
Filter thru dry, fast paper. If paper clogs, pour contents onto
addnl fresh paper and combine filtrates. Use filtrate for detn.
Note: Do not make exts more coned than required for in-
strument because there is tendency toward incomplete extn as
ratio of sample wt to vol. extg soln increases. Prep. sep. exts
for K and Na when their concns in sample differ greatly. For
K, use wt sample <0. 1 g/50 mL extg soln; for low Na concns
use >1.0 g/50 mL extg soJn; and for higher concns, prep.
weaker exts by reducing ratio of sample to extg soln rather
than by dilg stronger exts.
D. Determination
(Caution: See safety notes on flame photometer.)
Rinse all glassware used in Na detn with dil. HN0 3 , fol-
lowed by several portions H 2 0. Protect solns from air-borne
Na contamination.
Operate instrument according to manufacturer's instruc-
tions. Permit instrument to reach operating equilibrium before
use. Aspirate portions of std solns toward end of warm-up pe-
riod until reproducible readings for series are obtained.
Run stds, covering concn range of samples involved, at fre-
quent intervals within series of sample soln detns. Repeat this
operation with both std and sample solns enough times to re-
sult in reliable av. reading for each soln. Plot curves from
readings of stds, and calc. % K and/or Na in samples.
Ref.: J AOAC 39, 419(1956).
CAS-7440-09-7 (potassium)
CAS-7440-23-5 (sodium)
935.03* Potassium in Plants
Platinic Chloride Method
Final Action
Surplus 1974
See 3.020, ilth ed.
936.03*
Potassium in Plants
Perchloric Acid Method
Surplus 1974
See 3,021, 11th ed.
935.04* Potassium in Plants
Rapid Method for Potassium Only
Surplus 1974
See 3.022, llth ed.
929.03* Sodium in Plants
Uranyl Acetate Method
Final Action
Surplus 1989
See 3.052-3.053, 14th ed.
941.03
A. Reagents
Zinc in Plants
Mixed Color Method
Final Action 1965
(Redistil all H 2 from Pyrex. Treat all glassware with HN0 3
(1 + 1) or fresh chromic acid cleaning soln. Rinse repeatedly
with ordinary distd H 2 and finally with Zn-free H 2 0.)
(a) Carbon tetrachloride. — Use ACS grade without puri-
fication. If tech. grade is used, dry with anhyd. CaC1 2 and
redistil in presence of small amt CaO. (Used CC1 4 may be
reclaimed by distn in presence of NaOH (1 + 100) contg small
amts of Na 2 S 2 3 , followed by drying with anhyd. CaCI 2 and
fractional distn in presence of small amts of CaO.) (Caution:
See safety notes on distillation and carbon tetrachloride.)
(b) Zinc std solns. — (I) Stock soln. — 1 mg/mL. Place 0.25
g pure Zn in 250 mL vol. flask. Add ca 50 mL H 2 and 1
mL H 2 S0 4 ; heat on steam bath until all Zn dissolves. Dil. to
vol. and store in Pyrex vessel. (2) Working soln. — 10 p,g/mL.
Dil. 10 mL stock soln to 1 L. Store in Pyrex vessel.
(c) Ammonium hydroxide soln. — IN. With all-Pyrex app.
distil NH 4 OH into H 2 0, stopping distn when half has distd.
Dil. distillate to proper concn. Store in g-s Pyrex vessel.
(d) Hydrochloric acid. — IN. Displace HC1 gas from HC1
in glass flask by slowly adding equal vol. H 2 S0 4 from drop-
ping funnel that extends below surface of the HC1. Conduct
displaced HC1 gas thru delivery tube to surface of H 2 in re-
ceiving flask (no heat is necessary). Dil. to proper concn. Use
of 150 mL each of HC1 and H 2 S0 4 will yield 1 L purified HC1
soln of concn >IN.
(e) Diphenylthiocarbazone (dithizone) soln. — Dissolve 0.20
g dithizone in 500 mL CC1 4 , and filter to remove insol. matter.
Place soln in g-s bottle or large separator, add 2 L 0.027V NH 4 OH
(40 mL IN NH 4 OH dild to 2 L), and shake to ext dithizone
into aq. phase. Sep. phases, discard CC1 4 , and ext ammoniacal
soln of dithizone with 100 mL portions CC1 4 until CC1 4 ext is
pure green. Discard CC1 4 after each extn. Add 500 mL CC1 4
and 45 mL IN HC1, and shake to ext dithizone into CC1 4 . Sep.
phases and discard aq. phase. Dil. CC1 4 soln of dithizone to
2 L with CC1 4 . Store in brown bottle in dark, cool place.
(f) Ammonium citrate soln. — 0.5M. Dissolve 226 g
(NH 4 ) 2 HQH 5 7 in 2 L H 2 0. Add NH 4 OH (80-85 mL) to pH
of 8.5-8.7. Add excess dithizone soln (aq. phase is orange-
yellow after phases have been shaken and sepd), and ext with
100 mL portions CC1 4 until ext is full green. Add more dithi-
zone if necessary. Sep. aq. phase from CC1 4 and store in Pyrex
vessel.
(g) Carbamate soln. — Dissolve 0.25 g Na diethyldithio-
carbamate in H 2 and dil to 100 mL with H 2 0. Store in re-
frigerator in Pyrex bottle. Prep, fresh after 2 weeks.
(h) Dilute hydrochloric acid.— 0.02N '.Dil. lOOmLlJVHCl
to5L.
B. Preparation of Solutions
To reduce measuring out reagents and minimize errors due
to variations in composition, prep. 3 solns in appropriate amts
from reagents and store in Pyrex vessels, taking care to avoid
AOAC Official Methods of Analysis (1990)
Metals
49
loss of NH 3 from Solns I and 2. Discard solns after 6-8 weeks
because Zn increases slowly with storage. Det. std curve for
each new set of reagents. Following amts of Solns 1 and 2 and
2 L dithizone soln are enough for 100 detns:
(1) Soln jf.— Dil.. 1 L 0.5M NH 4 citrate and 140 rnL \N
NH 4 OH to 4 L.
(2) Soln 2.— Dil. 1 L 0.5 M NH 4 citrate and 300 mL IN
NH 4 OH to 4.5 L. Just before using, add 1 vol. carbamate soln
to 9 vols NH3-NH4 citrate soln to obtain vol. of Soln 2 im-
mediately required.
Note: If Zn-free reagents have been prepd, they can be used
to test chemicals for Zn. Certain lots of NH 4 OH and HC1 are
sufficiently free of Zn to be used without purification.
C. Ashing
Ash 5 g finely ground, air-dried plant material in Pt dish in
furnace at 500-550°. Include blank detn. Moisten ash with
little H 2 0; then add 10 mL IN HCI (more if necessary) and
heat on steam bath until all substances sol. in HCI are dis-
solved. Add 5-10 mL hot H 2 0. Filter off insol. matter on 7
cm paper (Whatman No. 42, or equiv., previously washed with
two 5 mL portions hot IN HCI, then washed with hot H 2
until HCl-free), and collect filtrate in 100 mL vol. flask. Wash
filter with hot H 2 until washings are not acid to Me red. Add
1 drop Me red (dissolve 1 g Me red in 200 mL alcohol), to
filtrate in 100 mL flask; neutze with \N NH 4 OH and add 4
mL 17V HCI. Cool, and dil. to vol. with H 2 0.
D. First Extraction
(Sepn of dithizone complex-forming metals from ash soln)
Pipet aliquot of ash soln contg ^30 jxg Zn into 125 mL
Squibb separator. Add 1 mL 0.2 N HCI for each 5 mL ash
soln <10 mL taken, or 1 mL 0.27V NH 4 OH for each 5 mL
>10 mL taken. (10 mL aliquot is usually satisfactory in anal-
ysis of plant materials.) Add 40 mL Soln 1 and 10 mL dithi-
zone reagent. Shake vigorously 30 sec to ext from aq. phase
the Zn and other dithizone complex-forming metals that may
be present; then let layers sep. At this point excess dithizone
(indicated by orange or yellow-orange aq. phase) must be pres-
ent. If excess dithizone is not present, add more reagent until,
after shaking, excess is indicated. Shake down the drop of CC1 4
ext from surface, and drain CC1 4 ext into second separator as
completely as possible without letting any aq. layer enter stop-
cock bore. Rinse down CC1 4 ext from surface of aq. layer with
1-2 mL clear CCl 4 ; then drain this CC1 4 into second separator
without letting aq. phase enter stopcock bore. Repeat rinsing
process as often as necessary to flush ext completely into sec-
ond separator. Add 5 mL clear CCl 4 to first separator, shake
30 sec, and let layers sep. (CC1 4 layer at this point will appear
clear green if metals that form dithizone complexes have been
completely extd from aq. phase by previous extn.) Drain CC1 4
layer into second separator and flush ext down from surface
and out of separator as directed previously. If last ext does not
possess distinct clear color, repeat extn with 5 mL clear CC1 4
and flushing-out process until complete extn of dithizone com-
plex-forming metals is assured; then discard aq. phase.
E. Second Extraction
(Sepn of Cu by extn of Zn into 0.02 W HCI)
Pipet 50 mL 0.02N HCI into separator contg CC1 4 soln of
metal dithizonates. Shake vigorously 1.5 min, and let layers
sep. Shake down drop from surface of aq. phase, and as com-
pletely as possible drain CC1 4 phase contg all Cu as dithizon-
ate, without letting any aq. phase, which contains all the Zn,
enter stopcock bore. Rinse down CC1 4 ext from surface of aq.
phase, and rinse out stopcock bore with 1-2 mL portions clear
CCI4 (same as in first extn) until all traces of green dithizone
have been washed out of separator. Shake down drop of CC1 4
from surface of aq. phase, and drain CC1 4 as completely as
possible without letting any aq. phase enter stopcock bore. Re-
move stopper from separator and lay it across neck until small
amt of CCI4 on surface of aq. phase evaps.
F. Final Extraction
(Extn of Zn in presence of carbamate reagent)
Pipet 50 mL Soln 2 and 10 mL dithizone soln into 50 mL
0.027V HCI soln contg the Zn. Shake 1 min and let phases sep.
Flush out stopcock and stem of separator with ca 1 mL CC1 4
ext; then collect remainder in test tube. Pipet 5 mL ext into
25 mL vol. flask, dil. to vol. with clear CC1 4 , and measure A
with spectrophtr set at absorption max., ca 525 nm. (Caution:
Protect final ext from sunlight as much as possible and read
within 2 hr.)
Det. Zn present in aliquot from curve relating A and concn,
correct for Zn in blank, and calc. % Zn in sample.
G. Standard Curve
Place 0, 5, 10, 15, 20, 25, 30, and 35 mL Zn working std
soln in 100 mL vol. flasks. To each flask add 1 drop Me red
and neutze with 1 N NH 4 OH; then add 4 mL 1 N HCI and dil.
to vol. Proceed exactly as for ash solns, beginning with first
extn, and using 10 mL aliquots of each of the Zn solns (0, 5,
10, 15, 20, 25, 30, and 35 fxg Zn, resp.). Construct std curve
by plotting |xg Zn against A.
Refs.: Ind. Eng. Chem. Anal. Ed. 13, 145(1941). JAOAC
24, 520(1941).
CAS-7440-66-6 (zinc)
953.04
Zinc in Plants
Single Color Method
Final Action 1965
A. Reagents
See 941. 03A and B plus following:
vol. dithizone soln,
(a) Dilute dithizone soln. — Dil.
941.03A(e), with 4 vols CC1 4 .
(b) Carbamate soln. — Dissolve 1.25 g Na diethyldithio-
carbamate in H 2 and dil. to 1 L. Store in refrigerator and
prep, fresh after long periods of storage.
(c) Dilute ammonium hydroxide. — Dil. 20 mL IN NH 4 OH,
941.03A(c), to 2 L.
B. Ashing
Weigh 2 g sample finely ground plant material into well-
glazed porcelain, Vycor, or Pt crucible, include crucible for
blank detn, and heat in furnace at 500-550° until ashing is
complete. Cool, moisten ash with little H 2 0, add 10 mL \N
HCI (more if necessary to ensure excess of acid), and heat on
steam bath until all sol. material dissolves. Add few mL hot
H 2 and filter thru quant, paper into 200 mL vol. flask. Wash
paper with hot H 2 until washings are not acid to Me red.
Add 2 drops Me red soln to filtrate, neutze with \N NH 4 OH,
add exactly 3.2 mL IN HCI, dil. to vol. with H 2 0, and mix.
C. Formation of Zinc Dithizonate
(Removal of interferences and sepn of excess dithizone)
Pipet aliquot of ash soln contg ^15 jxg Zn into 125 mL
amber glass separator. (25 mL aliquot is usually satisfactory.)
If necessary to use different vol., add 0.4 mL 0.2N HCI for
50
Plants
AOAC Official Methods of Analysis (1990)
each 5 mL less, or 0.4 mL 0.2 TV NH 4 OH for each 5 mL more,
than 25 mL taken. If <25 mL of the soln is taken, add H 2
to 25 rnL.
Add 10 mL dithizone reagent, 941.03A(e), to aliquot in sep-
arator and shake vigorously 1 min. Let layers sep. and discard
CC1 4 layer. Add 2 mL CC1 4 to aq. so]n, let layers sep., and
discard CC1 4 . Repeat this rinsing once. Then add 5 mL CC1 4 ,
shake vigorously 15 sec, let layers sep., and discard CC1 4 .
Rinse once more with 2 mL CC1 4 as above. Discard CC1 4 layer
and let CC1 4 remaining on surface of soln in funnel evap. be-
fore proceeding.
Add 40 mL NH 4 citrate Soln 1 , 941.03B(1), 5 mL carbamate
soln, 954.04A(b), and 25 mL dil. dithizone reagent,
954.04A(a). Accurately add carbamate and dithizone reagents
from pipet or buret. Shake vigorously 1 min. Let layers sep.
and draw off aq. layer thru fine tip glass tube connected to
aspirator with rubber tubing. To remove excess dithizone from
CC1 4 layer, add 50 mL 0.017V NH 4 OH and shake vigorously
30 sec.
D. Determination
Dry funnel stem with pipestem cleaner and flush out with
ca 2 mL of the Zn dithizonate soln. Collect adequate portion
of remaining soln in 25 mL erlenmeyer, or other suitable con-
tainer, and stopper tightly. (Amber glass containers are con-
venient, but colorless glassware will suffice if solns are kept
in dark until A readings are made.)
Measure A of each soln against CC1 4 with spectrophtr set at
absorption max., ca 535 nm. Correct for Zn in blank detns.
Calc. amt Zn present in soln from curve relating concn and A.
E. Standard Curve
Into 200 mL vol. flasks place 0, 2, 4, 6, 8, 10, 12, and 14
mL, resp., Zn working std soln. To each flask add 2 drops
Me red soln, neutze with 17V NH 4 OH, add 3.2 mL 17V HC1,
and dil. to vol. with H 2 0. Pipet 25 mL aliquots of each of
these solns, contg 0, 2.5, 5, 7.5, 10, 12.5, 15, and 17.5 jxg
Zn, resp., into amber glass separators, and proceed as for ash
solns, 954. 04C, beginning with second par. Det. A of each
soln and plot values against corresponding amts Zn.
Ref.: JAOAC 36, 397(1953).
CAS-7440-66-6 (zinc)
NONMETALS
930.06
Arsenic in Plants
Titrimetric Method
Final Action
Prep, soln as in 963. 21 C. Proceed as in 963. 2 ID, or take
aliquot and det. as in 925.02, beginning ". . . add 3 mL H 2 S0 4
CAS-7440-38-2 (arsenic)
958.03 Boron in Plants
Quinalizarin Method
First Action 1958
Final Action 1965
A. Reagents
(a) Dilute sulfuric acid. — 0.367V. Dil. 10 mL H 2 S0 4 to
1 L.
(b) Calcium hydroxide saturated soln. — Filter before use.
(c) Quinalizarin soln.- — Dissolve 45 mg quinalizarin in 1 L
95-96% H 2 S0 4 .
(d) Boron std soln. — 0.5 mg B/mL. Dissolve 2.860 g H 3 B0 3
and dil. to 1 L with H 2 0. Prep, working stds by further diln
with H 2 0.
B. Determination
Place 1.00-2.00 g dry, ground plant material in Pt or Si0 2
dish. Add 5 mL satd Ca(OH) 2 soln and dry at 105°. Carefully
volatilize over burner, ash in furnace 1 hr at 600°, and cool.
Add exactly 10 or 15 mL 0.36 A' H 2 S0 4 , break up ash with
glass rod, stir gently, and filter. Transfer 2 mL filtrate to col-
orimeter tube, add an exact amt (e.g., 15 mL) quinalizarin
reagent, stopper, and mix by swirling gently. Let stand at room
temp. 24 hr (or until both unknowns and stds have cooled to
same temp.). Shake tube again immediately before reading in
photoelec. colorimeter (620 nm filter).
Adjust colorimeter to 100% T with blank soln prepd as above
but using 2 mL H 2 in place of sample soln. Prep, std curve
with series of stds contg 0.5-10 fxg B/mL.
Ref.: JAOAC 41, 304(1958).
CAS-7440-42-8 (boron)
928.04*
Chloride in Plants
Gravimetric Method
Final Action
Surplus 1989
See 3.069-3.070, 14th ed.
915.01
Chloride in Plants
Volumetric Method I
Final Action
(Since precision of this titrn is considered to be ±0.2 mg CI,
accuracy of 1.0% requires samples contg >20 mg.)
A. Reagents
(a) Silver nitrate std soln. — 1 mL = 0.00355 g CI. Prep,
soln slightly stronger than 0.1 TV, stdze as in 941. 18E, and ad-
just to exactly 0. IN.
(b) Ammonium or potassium thiocyanate std soln. — 0.17V.
Prep, soln slightly stronger than 0.17V, stdze as in 941.18D(b),
and adjust to exactly 0.1 TV.
(c) Ferric indicator. — Satd soln of FeNH4(S0 4 )2.12H 2 0.
(d) Nitric acid. — Free from lower oxides of N by dilg HNO3
with ca V4 vol. H 2 0, and boiling until perfectly colorless.
B. Determination
To prepd soln, 928. 04A, add known vol. std AgN0 3 soln
in slight excess. Stir well, filter, and wash AgCl ppt thoroly.
To combined filtrate and washings add 5 mL ferric indicator
and few mL HNO3, and titr. excess Ag with thiocyanate std
soln to permanent light brown. From mL AgN0 3 used, calc.
%C1.
Refs.: Sutton, "Systematic Handbook of Volumetric Analy-
sis," 11th ed., 1924, p. 146. J. Am. Chem. Soc. 37,
1128(1915).
CAS-7782-50-5 (chlorine)
AOAC Official Methods of Analysis (1 990)
NONMETALS
51
935.05
Chloride in Plants
Volumetric Method II
Final Action
A. Reagents
(a) Potassium iodide std soln. — I mL - 1 mg CI. Weigh
4.6824 g pure (ACS) KI, dried to const wt at 105-150°, dis-
solve in H 2 0, and dil. to 1 L.
(b) Silver nitrate stock soln. — Approx. 0.37V. 1 mL = ca
10 mg CI. Dissolve 48 g AgNO, in H.O, filter, and dil. to
1 L.
(c) Silver nitrate std soln. — Dil. 100 mL reagent (b) to ca
900 mL and adjust by stdzg against reagent (a) so that 1 mL
= 1 mg CI.
(d) Chloride-free starch indicator. — For each 100 mL final
soln take 2.5 g sol. starch and make to paste with cold H 2 0.
Stir out lumps, add 25-50 mL more cold H 2 0, and stir or
shake 5 min. Centrf. , decant, and discard liq. Repeat extn 3
times and finally transfer residue to flask contg proper amt of
boiling H 2 0. Stir again, heat to bp, cover with small beaker,
and cool under tap, shaking occasionally.
(e) Dilute sulfuric acid. — Add 35 mL H 2 S0 4 to each 1 L
H 2 0, boil 5-10 min, and cool to room temp.
(f) Iodine indicator. — To ca 20 g I in 500 mL g-s bottle
add 400 mL dil. H 2 S0 4 , (e), and shake 10 min. Decant and
discard first soln, since it may contain iodides. Repeat process
and store soln in small g-s bottles.
(g) Potassium permanganate soln. — Dissolve 60 g KMn0 4
in 400 mL warm H 2 (ca 50°) and dil. to 1 L.
(h) Potassium sulfate-copper sulfate mixture. — Thoroly mix
16 parts K 2 S0 4 and 1 part CuS0 4 .5H 2 0.
(i) Wash soln.— Mix 980 mL H 2 and 20 mL HN0 3 .
B. Determination
(Caution: See safety notes on wet oxidation, nitric acid, and
permanganates.)
Weigh sample contg 10-40 mg CI into beaker. (If >4 g is
taken, use proportionately more HN0 3 and KMn0 4 soln.) Add
10 mL 0.3iV AgN0 3 and stir until sample is thoroly soaked,
adding little H 2 or warming if necessary. Add 25 mL HN0 3 ,
stir, add 5 mL KMn0 4 soln, and stir until frothing stops. Place
mixt. in H 2 bath or on hot plate and keep just below bp. Stir,
and wash down sides of beaker at intervals with min. FLO.
After 20 min, or when reaction stops, add addnl KMn0 4 soln,
little at time, until color begins to fade slowly. Dil. to ca 125
mL with boiling H 2 and heat 10 min longer. (Beaker may
stand in bath or on hot plate until ready to filter.)
Filter while hot thru Whatman No. 5, or equiv. paper, with
suction as follows: Place disk of 30-mesh stainless steel wire
gauze or No. 40 filter cloth in bottom of 3" (7.6 cm) Hirsch
funnel. Fold 9 cm paper over bottom of No. 11 rubber stopper,
shaping it to funnel by making 9-10 folds up side of stopper.
Place paper in funnel and apply strong suction. Wet paper and
keep wet while fitting into funnel so as to avoid double thick-
nesses of paper. Wash paper thoroly, first with H 2 and then
with wash soln. Discard washings and rinse out flask. Decant
thru filter and transfer ppt and sample residue to filter. If fil-
trate is not turbid, or if it is only slightly opalescent, wash ppt
thoroly, applying wash soln very gently, but keeping strong
suction on filter. If combined filtrate and washings are clear,
test for Ag. If turbid, reheat and pass thru filter, repeating until
clear, and finally wash as above. If filtrate does not give def-
inite test for Ag, repeat detn on smaller sample.
Place paper and contents in Kjeldahl flask and add such amts
of K 2 S0 4 -CuS0 4 mixt. and H 2 S0 4 as would be appropriate for
protein detn on same kind and amt of sample, and digest sim-
ilarly. (For 2 g grass, 8 g sulfate mixt. and 20 mL acid are
enough.) When digest is cool, add 175 mL H 2 0, boil 5-10
min, and cool to room temp. Titr. the Ag 2 S0 4 in Kjeldahl flask
with KI std soln, using 5 mL starch indicator and 30 mL I
indicator. (Add latter just before titrn.) Rinse neck of flask
after each addn of KI when near end point and titr. until soln
stays blue after shaking. If <30 mg CI is present, add starch
and I so Ins at beginning. If larger but unknown amt is present,
add 2 mL starch and 10 mL I indicator at beginning and titr.
until end point approaches. Shake vigorously to coagulate ppt,
add rest of starch and I solns, and proceed to end point. If
known large amt is present, titr. to within 2 mL of end point,
shake as above, add indicator reagents, and continue titrn. If
end point is overrun, add 5 mL std AgN0 3 soln and titr. again.
Blank detns are not necessary after testing reagents. If blank
made by using pure sugar as sample is >0.05 mg, examine
filter paper, distd H 2 0, and various reagents for CI.
Refs.: JAOAC 18, 379(1935); 19, 72(1936).
CAS-7782-50-5 (chlorine)
975.04 Fluoride in Plants
Potentiometric Method
First Action 1975
Final Action 1988
(Rinse all plastic and glass containers with HCl (1+3) and H 2
before use. Perform analyses in laboratory free from F; prep.
samples in another laboratory.)
A. Princ'tpie
F is extd from dry, pulverized foliage with HN0 3 followed
by aq. KOH. Slurry is adjusted to pH 5.5, and complexing
agent and background F are added. Potential is measured with
ion selective electrode and compared against calibration curve.
Method is applicable to 10-2000 |ULg F/g dry wt leaf tissue
not exposed to unusual amts of Al or other F-binding agents;
it is not applicable to insol. inorg. F or F in org. combinations.
Between-laboratory precision of individual analyses is ±20%
at 30 ppm F; ±10%, ^100 ppm F. Accuracy is 90-100%.
B. Apparatus
Electrometer. — Range ±200 mv with readability of 0. 1 mv
(Model 701 or 701 A (replacement model SA720) digital pH/
mv meter, Orion Research Inc., or equiv.) or expanded scale
pH meter with mv mode of operation, with F ion selective
electrode (No. 94-09 single electrode, Orion Research Inc.,
or equiv.) and reference electrode (No. 90-01 single junction,
Orion Research Inc., or equiv.). Check system at intervals to
assure adherence to following performance criteria: Using
technic of 975.04D, system should reach equilibrium (A E <0.2
mv/min) within 5 min with each F working std soln, checked
in following sequence: 0.1, 0.2, 0.5, 2.0, and 10.0 ppm F.
Replicate std solns should differ by ^1 mv. Calibration curve
should be linear between 0.2 and 10.0 ppm and slope should
be 57 ±2 mv per 10-fold change in F concn. If any parameter
is not obtained, check electrodes, reagents, and electrometer.
Maintain temp, control of ±1°.
C. Reagents
(Store all solns in tightly closed, plastic bottles.)
(a) Nitric acid.—U) ION.— Add 63 mL HN0 3 to H 2 0, cool,
and dil. to 100 mL. (2) 0.2N.— Dil. 5.0 mL 107V to 250 mL.
(3) 0.05N.—DW. 5.0 mL 10/V to 1 L.
52
Plants
AOAC Official Methods of Analysis (1990)
(b) Potassium nitrate so In, — 0.4M. Dissolve 4.0 g KN0 3
in H 2 anddiJ. to 100 mL.
(c) Sodium citrate soln. — 0.8M. Dissolve 58.8 g Na ci-
trate. 2H 2 in 200 mL H 2 0, adjust to pH 5.5 by drop wise addn
of \0N HN0 3 , using pH meter, and dil to 250 mL with H 2 0.
(d) Sodium citrate with fluoride soln. — 0AM citrate with 1
ppm F. Dil. 125 mL 0.8M Na citrate soln and 25.0 mL 10
ppm F std soln to 250 mL with H 2 0.
(e) Fluoride std solns. — (/) Stock soln. — 100 ppm F. Dry
ca 1 g NaF 2 hr at 110°. Accurately weigh 0.221 g NaF, dis-
solve in H 2 0, and dil. to 1 L. (2) Intermediate soln, — 10 ppm
F. Dil. 10.0 mL stock soln to 100 mL with H 2 0. (3) Working
soln. — Prep, as in Table 975.04 in 100 mL vol, flasks. Prep.
0.2 and 0.1 ppm solns fresh as needed.
D. Preparation of Calibration Curve
Place 25.0 mL 0.1 ppm F working std soln into plastic con-
tainer contg stirring bar. Insert electrodes ca 12 mm into soln
and stir mag. Record mv readings at 1 min intervals until change
is <0.2 mv/min. Remove electrodes, blot lightly with ab-
sorbent paper, and repeat reading with 0.2, 0.5, 2.0, and 10.0
ppm std solns. Place electrodes in 0.2 ppm std soln until sam-
ples are analyzed. (10 ppm std soln may be omitted if samples
are known to contain <400 ppm F.)
Plot potential (mv) on vertical arithmetic axis and F concn
((jig/mL; ppm) on horizontal (logarithmic) axis of 2-cycle
semilog graph paper.
E. Preparation of Sample
Dry foliage 48 hr at 80°. Grind to pass No. 40 sieve and
store in clean, dry, tightly closed plastic bottle. Rotate bottle
to mix sample thoroly before removing aliquots.
F. Determination
Accurately weigh ca 0.25 g powd sample, and place in 75-
100 mL wide-mouth plastic container. Add 20 mL 0.057V HNO3
and place on rotating shaker or stir mag. 20 min. Add 20 mL
AN KOH (5.6 g/L) and agitate addnl 20 min. Add 5.0 mL
Na citrate soln contg 1 ppm F, adjusted to pH 5.5, and 5.0
mL 0.2jV HNO3. (Samples may be stored covered <4 hr at
this point.) Det. mv readings as in 975. 04D and prep, cali-
bration curve before and after each series of samples.
If sample series contains mixt of high and low samples,
make preliminary estn of F content after 2 min. Then det. F
concn in samples contg <40 ppm first and in higher ones last.
ppm F (p,g/g) = (C - 0.10) x 50/w
where C = ppm F from curve; 0.10 = ppm background F in
final soln; 50 = mL final soln; and w = g sample.
Ref.: JAOAC58, 1129(1975).
CAS-7782-41-4 (fluorine)
984.02 Fluoride in Plants
Willard-Winter Distillation Method
Final Action
See 944.08.
978.03 Fluoride in Plants
Semiautomated Method
First Action 1978
ASTM-lntersociety Committee-AOAC Method
A. Principle
Dried and ground plant material is ashed, fused with alkali,
and dild to vol. In case of leaf samples, F on external surfaces
Table 975.04 Preparation of Working Standard Solutions
mL soln to be dild to 100 mL
Concn,
0.4 M
0.8 M Na 100 ppm 10 ppm
ppm
KNO3
citrate F soln F soln
10
10.0
5.0 10.0 0.0
2
10.0
5.0 2.0 0.0
0.5
10.0
5.0 0.0 5.0
0.2
10.0
5.0 0.0 2.0
0.1
10.0
10.0 mL Na citrate soln contg 1 ppm F
may be washed off sep. Digest and H 2 S0 4 are pumped into
microdistn app. maintained at 170°. Stream of air carries acidi-
fied sample to fractionation column where F and H 2 are distd
into condenser, and condensate passes into small collector.
Distillate is mixed continuously with alizarin F blue-lanthanum
reagent, colored stream passes thru tubular rlowcell of color-
imeter, and A is measured at 624 nm.
Interfering metal cations and inorg. phosphate are not distd,
and org. substances are destroyed by ashing. Interference from
remaining volatile inorg. anions is reduced with high concn
acetate buffer with some reduction in sensitivity. Very large
arnts solid matter, particularly silicates, retard distn. There-
fore, smallest sample consistent with obtaining suitable amt F
should be used. Conditions must be carefully controlled, since
accurate results depend upon obtaining same degree of effi-
ciency of distn from samples as from std F solns used for cal-
ibration.
Acid concn during distn is maintained at const value by us-
ing specific amts CaO and NaOH for ashing and fusion and
HCIO4 for transfer of fused samples. Any marked change in
vac. (>0.2" Hg or 5 torr) over short time indicates either leak
or block in system. Distil at same vac. each day and maintain
proper ratio between air flow on line drawing liq. and solid
wastes from distn coil and on line drawing HF and H 2 vapor
from distn unit (Fig. 978. 03 A). Adjust flowmeters to keep this
ratio const and to maintain higher vac. on HF line, C i9 so that
min. is diverted to waste line.
Method can detect 0.1 |xg F/mL. Normal range is 0.1-4.0
p,g F/mL. Dil. higher concns with NaOH-HC10 4 soln, (k). If
digested samples routinely exceed 4.0 jmg/mL, modify anal.
portion of pump manifold to reduce sensitivity, or use smaller
sample aliquot (preferred). Most accurate results are obtained
in middle or upper part of calibration curve. For example, to
decrease sensitivity, pump sample thru 0.081" tube (2.5 mL/
min) and dil. with H 2 pumped thru 0.065" tube (1.6 mL/
min) before sample enters distn app. Total vol. sample and
diluent should approx. original vol. used (4.1 mL/min).
If air-borne contaminants are present in laboratory, attach
small drying bulb contg CaC0 3 granules to air inlet tube of
microdistn unit. Teflon distn coil of microdistn unit must be
cleaned periodically to avoid accumulation of solids which re-
duce sensitivity.
Coefficient of variation of 20-100 ppm F is generally <10%.
Samples with large amt of Si (orchard grass) or Al may present
special difficulties. There should be no significant deviation
from linearity with different amts sample and with different
amts added F.
B. Apparatus
(Cat. Nos refer to current Technicon equipment, except where
indicated. Corresponding equipment under previous Cat. Nos
is satisfactory.)
(a) Automatic analyzer. — (Fig. 978. 03B) Auto Analyzer,
Technicon Instruments Corp., or equiv. (7) Sampler. — Sam-
pler IV with rotary stirrer and 8.5 mL plastic sample cups.
Use 10 or 20 /hr cam with 1:3 sample-to-wash ratio (No. 171-
AOAC Official Methods of Analysis (1990)
NONMETALS 53
FIG. 978.03A— Schematic drawing of air flow system used in semiautomated analysis for fluoride
AO 15-07). (2) Colorimeter. — With 15 mm tubular flowcell and
624 nm interference filter (199-A001-05). (3) Recorder.— Ra-
tio type with 2-100 mv full scale range (01 1-A1 15-01). (4)
Multichannel proportioning pump and manifold cartridge. —
With assorted pump tubes, nipple connectors, and glass con-
nectors (pump III 113-A014-08; cartridge 116-8340-01).
(b) Pulse suppressors. — For sample and color reagent
streams. Coil 10' length of 0.035" id Teflon std tubing around
2.5" diam. tube. Force outlet end into short length of 0.045"
id Tygon tubing which is then sleeved with piece of 0.081"
Tygon tubing. Slip sleeved end over "h" fitting which joins
sample and reagent streams. (Pulse suppressor included with
manifold cartridge.)
(c) Voltage stabilizer.— 161- AQQ7-Q1 (also part of 199-A001-
05).
(d) Rotary vacuum and pressure pump. — With continuous
oiler.
(e) Micro distillation apparatus. — (Fig. 978. 03C) Major
components are (Cat. Nos. are those of Lurex Scientific, ex-
cept as noted); (Y) Bottom only of jacketed 1 L resin reaction
flask with conical flange (JR-5130), modified by evacuating
space between inner and outer walls and sealing off port (a);
(2) resin reaction flask top with conical flange (JR-7935) mod-
ified to have one ¥ 29/42 center joint and four ~§ 24/40 side
joints; (3) resin reaction flask clamp (JR-92 10-0000); (4) vari-
able high-speed stirrer (S-6362) (d); (5) stainless steel, heavy
Tube Size
(incnesl sampler 11
(7) O 090 Wcter ( 2 90 ml/mi
®o
056 Water (I 20 ml/mm)
-0=
© 0090Somple (2 90 ml/mm) \
-Q>
0035 Air (0 42 ml/mm)
Microdistiilotion
device
©
-®-
© 0.081 At" 1 1250-
Gioss Bead Mixer
■— ®-
"Qj) 008i Impinqer (250 ml/mm}
-^
PULSE
suppressors Waste -*-
@ 0073(S) A-C
_^\ Reagent ( I 69 ml/mm)
— ®-
0.045 (S) (0 70 ml /mm)
Time Delay Coil
Bubble
trap
Vacuum
pump
£
PROPORTIONING
PUMP
^y
□
COLORIMETER RECORDER
'5 mm Tubular f c
624 m „ flUeti
FIG. 978.03B — Flow diagram for semiautomated analysis for fluoride
54
Plants
AOAC Official Methods of Analysis (1990)
FIG. 978.03C— Schematic drawing of microdistillation appara-
tus
duty stirrer stuffing box with ¥ 29/42 and shredded Teflon
packing (JS-1 160-0102 and JS-3050-0000); (6) 10 mm diam.
stainless steel stirrer rod with propeller to fit stuffing box; (7)
therm ometer-thermoregulator, range 0-200° (c); (8) electronic
relay control box; (9) low drift immersion heater, 750 watts
{b); (10) 30' length coil of flexible Teflon TFE tubing, Vs" id,
3 /j 6 " od, 0.030" wall, on rigid support of such diam. that com-
pleted coil will fit into resin reaction flask (avoid kinking of
tubing) (<?); (77) 2 flowmeters with ranges 0. 15-1 .00 and 0.6-
5.0 L/min, both with needle valve controls (Dwyer lnstru-
Points pushed
into glass
This shoulder should be
rounded, not abrupt
6 mm od
FIG. 978.03D— Microdistillation column
ments, Inc., PO Box 373, Michigan City, IN 46360); (72) vac.
gage with range 0-10" Hg or 0-254 torr (mm Hg); (73) frac-
tionation column of borosilicate glass (g; see also Fig. 978. 03D;
(14) distillate collector; (75) H 2 0-jacketed condenser (h); (16)
Dow-Corning 200 fluid (100 centistokes at 25°) (/); and (17)
condenser (j).
(f) Crucibles.— Inconel, Ni, or Pt, 40-50 mL.
(g) Airflow system. — (Fig. 978. 03 A) Draw air thru air inlet
tube, (a), before Teflon microdistn coil, (b). Air sweeps thru
(b) to fractionation column, and is diverted into 2 channels.
In channel c f , air passes thru H 2 0-jacketed condenser, (d),
sample trap, (<?), to waste bottle, (/). Air then passes thru Vs"
id glass tube directed against surface of H 2 S0 4 in waste bottle,
(g). Partially dehydrated air passes thru gas drying tower, (h),
contg 450 g indicating silica gel. Emerging air passes thru T -
tube, (/), connected to vac. gage, (j) (0-10" Hg or 0-254
torr), thru flowmeter, (k) (0-5 L/min), thru T-tube, (/), and
then to vac. pump, (m).
In channel c 2 , air passes thru H 2 0-jacketed waste trap, (ri),
to waste bottle, (o). Air leaving waste bottle flows thru drying
bulb, (/?), filled with indicating silica gel, and the dry air then
passes thru flowmeter, (q) (0-1 L/min). Air stream then con-
nects thru T-tube, (/), with air from first channel.
C. Reagents
(Caution: See safety notes on perchloric acid and sulfuric
acid.)
(Deionized H 2 may be used. CaO for ashing and NaOH for
fusion must be low in F.)
(a) Sulfuric acid.— (1 + 1). Mix 500 mL H 2 S0 4 with 500
mL H 2 and cool before use.
(b) Acetate buffer.— 2 A4M (pH 4.0). Dissolve 60 g
Na0Ac.3H 2 in 500 mL H 2 0, add 100 mL HOAc, and dil.
to 1 L with H 2 0. Stable at 25°.
(c) Alizarin fluorine blue color reagent stock soln. — 0.0 1M.
Suspend 0,9634 g reagent (alizarin complexone, alizarin com-
plexan; 3-amino-ethylalizarin-Af,/V-diacetic acid; Burdick &
Jackson Laboratories, Inc.) in ca 100 mL H 2 in 250 mL vol.
flask. Add 2 mL NH 4 OH and shake until completely dis-
solved. Add 2 mL HOAc and dil. to vol. with H 2 0. Stable
indefinitely at 4°.
(d) Lanthanum nitrate stock soln. — 0.02A7. Dissolve 8.6608
g La(N0 3 ) 3 .6H 2 in H 2 and dil. to 1 L in vol. flask.
(e) Wetting soln. — 30% soln (w/v) polyoxyethylene lauryl
ether in H 2 (Brij-35, Technicon No. T21-0110). Soln is sta-
ble at 25°.
(f ) Working reagent. — Mix, in order listed, 300 mL acetate
buffer, 244 mL H 2 0, 300 mL acetone, 100 mL /m-butanol,
36 mL alizarin fluorine blue stock soln, 20 mL La (N0 3 ) 3 stock
soln, and 2 mL wetting soln. Unused reagent is stable 7 days
at 4°. Before using reagent, place under vac. 10 min to remove
air bubbles from soln.
(g) Fluoride std solns. — (7) Stock soln. — 100 jjig F/mL.
Dissolve 0.2207 g NaF in H 2 and dil. to 1 L. (2) Working
solns. — Prep. 7 solns contg 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, and
4.0 |xg F/mL. Before dilg to vol., add 6 g NaOH and 20 mL
70% HC10 4 for each 100 mL final working soln so that stds
have same composition as sample solns. Dil. with H 2 only
for analysis of H 2 samples or air samples absorbed in H 2 0.
Store working solns at 4° in polyethylene bottles; stable in
presence of NaOH.
(h) EDTA solns. — 1%. Dissolve I g Na 4 EDTA in 99 mL
H 2 0. Prep. 0.05% and 0.01% solns by mixing 5 mL 1% soln
with 95 mL H 2 and 1 mL 1% soln with 99 mL H 2 0, resp.
(i) Phenolphthalein soln. — Dissolve 1 g phthln in 50 mL
absolute alcohol or isopropanol and add 50 mL H 2 0.
AOAC Official Methods of Analysis (1990)
NONMETALS
55
(j) Detergent. — Alconox (Alconox, Inc., 215 Park Ave S,
New York, NY 10003); available from laboratory supply firms.
(k) Sodium hydroxide-perchloric acid soln. — Dissolve 6 g
NaOH in H 2 0, add 40 mL 70% HC10 4 (1 + 1), and dil. to 100
mL with H 2 0. Use to dil. samples when F in unknown sample
exceeds std curve.
D. Preparation of Sample
(a) Leaves. — If it is necessary to remove surface F, wash
sample with aq. soln contg 0.05% detergent and 0.05%
Na4EDTA in polyethylene container 30 sec with gentle agi-
tation. Remove, drain 3-4 sec, and rinse 10 sec in each of 3
beakers of H 2 0. Discard solns after use.
(b) Fresh plant tissues. — Dry 24-48 hr in 80° forced-draft
oven, and grind as in (c).
(c) Dry plant tissues. — Grind in semimicro Wiley mill to
pass No. 40 sieve, and store in plastic container.
E. Ashing and Fusion
Accurately weigh 0.1-2.0 g well mixed dried plant tissue
into crucible. Add 100±10 mg low-F CaO, enough H 2 to
make loose slurry, and 4 drops phthln soln. Mix thoroly with
polyethylene policeman. Final mixt. should be purple and re-
main purple during evapn to dryness.
Place crucible on cold hot plate and under IR lamp. Evap.
under lamp to dryness, turn on hot plate, and char 1 hr. Trans-
fer crucible to furnace at 600° and ash 2 hr. (Caution: To avoid
flaming, place crucibles at front of furnace with door open ca
5 min to further char samples; then reposition in furnace.)
Remove crucibles, add 3.0 ±0.1 g NaOH pellets, and re-
place in furnace with door closed to melt NaOH. (Caution:
Avoid creeping of molten NaOH.) Remove crucibles individ-
ually and swirl to suspend particulate matter until melt is par-
tially solidified. Let cool until addn of small amt H 2 does
not cause spattering. Wash down inner walls with 10-15 mL
H 2 0. Suspend melt with polyethylene policeman and transfer
with H 2 to plastic tube graduated at 50 mL. Rinse crucible
with 20~.0 mL 70% HC10 4 (1 + 1), add rinse to tube, and dil.
to 50 mL with H 2 0. Solns can be stored at this point if tightly
capped.
Analyze blank contg all reagents with each set of ca 10
samples.
Clean crucibles as soon as possible after each use. Boil In-
conel crucibles I hr in 10% NaOH soln. Rinse with hot tap
H?0, detergent, and then distd H 2 0. Immerse crucibles which
held samples contg >100 (xg F in AN HC1 45 min before boil-
ing in NaOH soln. Perform blank analyses on these crucibles
before addnl use to check for contamination. Scrub Ni and Pt
crucibles with detergent and hot H 2 and rinse thoroly with
H 2 0. Briefly rinse crucibles which held samples contg >100
fjLg F in 4 7V HC1 before rinsing with H 2 0.
F. Analytical System
Place F std solns, ashed and fused samples, or impinged air
samples in 8.5 mL plastic cups in sample module. Actuate
sampler and pump from cup at net rate of 2.48 mL/min with
air segmentation of 0.42 mL/min after sampler crook, and pump
into microdistn device thru sample inlet (7, Fig. 978.03C), us-
ing 0.051" id Teflon tubing. Pump H 2 S0 4 at 2.5 mL/min thru
acid inlet (m, Fig. 978. 03C). Cool and discard acid and solids.
Pump distillate from sample trap at 2.0 mL/min thru 0.051"
Teflon tubing, add color reagent at 1.69 mL/min, and mix in
4" length of Vs" id glass tubing packed with pieces of 20 mesh
broken Pyrex glass. Pass colored stream thru time delay coil
of 15' of 0.035" Teflon spaghetti tubing, thru debubbler fitting
where small portion of stream and bubbles are removed to waste
bottle at rate of 0.70 mL/min, and thru 15 mm tubular flowcell
of colorimeter. A is measured at 624 nm and plotted on re-
corder. Lag time from sampling to appearance of peak is ca
5 min. Time between samples is 6 min with sampling rate of
10/hr and 3 min at 20/hr.
G. Start-Up
Turn on H 2 to condenser and cooling jacket. Turn on col-
orimeter. Engage manifold on proportioning pump and start
pump. Turn on stirring motor of microdistn unit, vac. pump
adjusted for full vac, and heater of microdistn unit. Connect
lines to H 2 S0 4 , color reagent, and H 2 bottles. Sampling tube
of sampler unit should be in H 2 reservoir. Equilibrate app.
until silicone oil in microdistn unit reaches 1.70 ±2°. Check that
all connections are secure. Adjust distn flowmeter (k, Fig.
978.03C) to 2.5-3 L/min; adjust waste flowmeter (q) to 0.3
L/min. Distillate should now fill sample trap. Readjust flow-
meter (k) to give reading on vac. gage of 5-6" Hg (127-150
torr). (Satisfactory setting for app. must be detd by trial and
error. Once detd, use each day.) No air bubbles should be in
anal, system beyond point where color reagent and distillate
streams are joined. Turn on recorder, adjust baseline to desired
level, and run several min to assure that all components are
operating properly. Baseline should be reasonably smooth and
straight.
Transfer F std solns to 8.5 mL plastic cups and place in
sampler. Sep. last std soln from sample solns with cup of H 2 0.
Program sampler for 10 samples/hr (90 sec sampling period,
270 sec washout period) or 20 samples/hr (45 sec sampling
period, 135 sec washout period).
Prep, std curve, 978. 03 J, before and after each day's set of
samples. Net A of 0.7-0.9 should be obtained with std soln
contg 4 (xg F/mL. A of each std soln should be reproducible
within 10% from day to day and std curve should be linear
from 0.2 to >3.2 u.g/mL.
H. Shut-Down
Turn off chart recorder. Disconnect H 2 S0 4 line and place in
H 2 0. Disconnect color reagent line and place in 0.01% EDTA
soln ca 1 min; then transfer line to H 2 and let H 2 pass thru
system ca 5 min. Clean Teflon distn coil as in 978.031(a).
Turn off heater and stirrer of microdistn unit. Turn off vac.
pump. Release pump tube manifold. Turn off H 2 to con-
denser and cooling trap.
/. Maintenance
(a) Cleaning of Teflon distillation coil. — (After use with
samples contg particulate matter.) Briefly insert Tygon tube
connected to air inlet line of microdistn unit into 0.01% EDTA
soln. After all deposited material has been removed, wash with
3—4 five mL portions distd H 2 0.
(b) Pump tubes. — Replace after 200 working hr or earlier
if hard and inflexible or flattened. Always leave in relaxed po-
sition when not in use. Remove dirt and grease from pump
plates and rollers after each day of use.
(c) Indicating silica gel. — Regenerate when ca 2 /s has lost
normal blue color.
(d) Cleaning tubing. — Clean tubing contg reagent after each
daily run with 0.01% EDTA soln followed by distd H 2 0.
(e) Monthly checks. — Oil proportioning pumps monthly.
Check gain on recorder monthly and adjust.
J. Calibration and Standards
Before and after each day's set of samples, prep, std curve
by transferring aliquots of each working std soln to 8.5 mL
sample cups and proceed with analysis. Draw straight line con-
necting baseline before and after analysis. Record A of each
peak and subtracts of baseline at peak. Plot netv4 against jxg
F/mL.
56
Plants
AOAC Official Methods of Analysis (1990)
K. Calculations
ppm F in sample = (F x V X D)/W
where F = (xg F/mL sample from std curve; V = mL sample,
usually 50; D = diln factor used only when F of sample ex-
ceeds std curve = mL final vol. to which original aliquot was
dild with NaOH-H00 4 soln, (k)/mL original aliquot taken;
and W = g sample taken for analysis.
L. Check Procedure
(a) Contamination. — Perform reagent and equipment blank
with crucibles and reagents but without sample to detect con-
tamination from previous samples, contaminated furnace, and
reagents. Blank values >5 jxg F are evidence of contamina-
tion. Perform 2 blank detns with each set of 20-40 samples.
Usual blanks are 1-3 jxg.
(b) Recoveries .- — Occasionally add known amts F std soln
from microburet to aliquots of low F tissue. Recovery of added
F should be 100± 10%. Low values indicate loss of F, possibly
during pretreatment; high values indicate contamination.
(c) Linearity. — Occasionally analyze different amts (0.1-
2.0 g) plant sample contg 50-65 ppm F. Linear relationship
should exist between F found and amt tissue taken. Nonline-
arity may indicate that some component of tissue is retarding
distn or interfering with color development.
(d) Calibration curves. — Prep, at least twice daily.
M. Trouble Shooting
(a) Irregular baseline. — May result from: (1) excessive pulse
pressures — check for faulty pump tubes, absence of surge sup-
pressors, or improperly made or placed suppressors; (2) air
bubbles in flowed . I — check for absence of debubbler bypass,
blockage in reagent pump tube, or periodic emptying of sam-
ple trap (latter results if air flow to distn trap becomes too
great); (3) excessive H 2 S0 4 carry-over — check for too high
temp, in oil bath, improper H 2 S0 4 concn, or too high vac. on
system; (4) air flow imbalances — check flowmeter settings,
trapped air in tubing, or leak or block in system; (5) high F
content in samples (baseline may not return to normal between
samples) — dil. or check sampling speed and sample-to-wash
ratio.
(b) Irregular peaks. — Asymmetrical or double peaks or peaks
with shoulders may result from: (1) baseline irregularities; (2)
interfering substances from sample or impure reagents; (3) in-
adequate buffer concn; or (4) excessive amts solids in distn
coil. Presence or accumulation of solids may be due to insuf-
ficient flow of H 2 S0 4 , too large sample, excess CaO or NaOH
in sample, inadequate suspension of particles in samples, or
lack of proper air segmentation in sample tubing.
(c) Poor reproducibility. — Check for: improper sample
pickup; faulty pump tubes; inadequate washing of distn coil
between samples; large deviations in acid concn, temp., or air
flow in distn coil; or changes in vac. on waste system.
Refs.: JAOAC 55, 991(1972); 61, 150(1978).
CAS-7782-41 -4 (fluorine)
966.01 Phosphorus in Plants
Gravimetric Quinolinium Molybdophosphate Method
Final Action 1974
A. Preparation of Solution
Accurately weigh ca 2 g plant sample in porcelain dish, and
add 7.5 mL Mg(N0 3 ) 2 soln (dissolve 950 g P-free
Mg(N0 3 ) 2 .6H 2 in H 2 and dil. to 1 L). Dry in oven 2 hr at
110-115° (or until dry). Ignite carefully over Fisher burner,
or equiv., until bubbling and smoking cease. Complete ashing
in furnace 4 hr at 550-600°. Dissolve ash in few mL HO
(2+1) and evap. to dryness on steam bath. Take up residue in
10-15 mL HC1 (1 +9) and filter thru coarse paper into 200 mL
vol. flask. Wash paper thoroly with FLO and let filtrate cool
to room temp. Dil. to vol. with H 2 0.
B. Determination
Pipet 40 mL aliquot into 300 or 500 mL erlenmeyer and
proceed as in 962. 02C.
Ref.: JAOAC 49, 284(1966).
933.01 • Phosphorus in Plants
Macro Method
Final Action
Surplus 1974
See 3.065, 13th ed.
931.01
Phosphorus in Plants
Micro Method
Final Action
A. Reagents
(a) Phosphorus std soln. — 0.025 mg P/mL. Dissolve 0.4394
g pure dry KH 2 P0 4 in H 2 and dil. to 1 L. Dil. 50 mL of this
soln to 200 mL.
(b) Ammonium molybdate soln. — Dissolve 25 g NH 4 mo-
lybdate in 300 mL H 2 0. Dil. 75 mL H 2 S0 4 to 200 mL and
add to NH 4 molybdate soln.
(c) Hydroquinone soln. — Dissolve 0.5 g hydroquinone in
100 mL H 2 0, and add 1 drop H 2 S0 4 to retard oxidn.
(d) Sodium sulfite soln. — Dissolve 200 g Na 2 S0 3 in H 2 0,
dil. to 1 L, and filter. Either keep this soln well stoppered or
prep, fresh each time.
B. Preparation of Solution
To 1 or 2 g sample in small porcelain crucible add 1 mL
Mg(N0 3 ) 2 soln (dissolve 950 g P-free Mg(N0 3 ) 2 .6H 2 in H 2
and dil. to 1 L), and place on steam bath. After few min,
cautiously add few drops HC1, taking care that gas evolution
does not push portions of sample over edge of crucible. Make
2 or 3 further addns of few drops HC1 while sample is on bath
so that as it approaches dryness it tends to char. If contents
become too viscous for further drying on bath, complete drying
on hot plate. Cover crucible, transfer to cold furnace, and ig-
nite 6 hr at 500°, or until even gray ash is obtained. (If nec-
essary, cool crucible, dissolve ash in little H 2 or ale. -glyc-
erol, evap. to dryness, and return uncovered to furnace 4-5
hr longer.) Cool, take up with HC1 (1+4), and transfer to 100
mL beaker. Add 5 mL HC1 and evap. to dryness on steam
bath to dehydrate Si0 2 . Moisten residue with 2 mL HC1, add
ca 50 mL H 2 0, and heat few min on bath. Transfer to 100 mL
vol. flask, cool immediately, dil. to vol., mix, and filter, dis-
carding first portion of filtrate.
C. Determination
To 5 mL aliquot filtrate in 10 mL vol. flask add 1 mL NH 4
molybdate soln, rotate flask to mix, and let stand few sec. Add
1 mL hydroquinone soln, again rotate flask, and add 1 mL
Na 2 S0 3 soln. (Last 3 addns may be made with Mohr pipet.)
Dil. to vol. with H 2 0, stopper flask with thumb or forefinger,
and shake to mix thoroly. Let stand 30 min, and measure A
with spectrophtr set at 650 nm. Report as % P.
Refs.: JAOAC 14, 216(1931). J. Biol. Chem. 59, 255(1924).
CAS-7723-14-0 (phosphorus)
AOAC Official Methods of Analysis (1990)
NONMETALS
57
936.04*
Selenium in Plants
Gravimetric Method
Final Action
Surplus 1974
(Applicable to materials contg >2 ppm Se)
See 3.073, 11th ed.
969.06 Selenium in Plants
Fluorometric Method
First Action 1969
Final Action 1974
{Caution: See safety notes on photofluoro meter, wet oxida-
tion, nitric acid, and perchloric acid.)
A. Apparatus
(a) Micro-Kjeldahl flasks. — 30 mL Pyrex, ca 170 mm total
length with ¥ 12/18 outer joint at mouth.
(b) Air condensers . — 1 x 1 40 mm Pyrex tubes with IT 2 / 1 8
inner joint.
(c) Micro-Kjeldahl digestion unit with glass fume duct. —
Fit rack to hold flasks and attached air condensers in nearly
upright position during early stages of digestion. Use in fume
hood.
(d) Fluorometer. — Capable of illuminating sample at 369
nm and measuring fluoresced light at 525 nm. Spectrofluo-
rometer set to above wavelengths is also satisfactory.
B. Reagents
(Use deionized H 2 distd in glass for prepg solns and dilns.)
(a) Nitric acid. — Redistd in glass.
(b) Hydroxylamine-ethylenediaminetetraacetic acid soln.-—
Add ca 20 mL H 2 to 1.9 g EDTA (acid form). Slowly add
ca 5N NH 4 OH with stirring until EDTA just dissolves. Some
excess NH 4 OH is not harmful. Dissolve 6 g NH 2 OH.HCl in
100 mL H 2 0. Combine solns and diJ. to 250 mL with H 2 0.
(c) Cresol red indicator. — Dissolve 0.1 g cresol red in 10
mL H 2 and 1 drop 50% NaOH soln. Dil. to 50 mL with H 2 0.
(d) Selenium std soln. —0.3 fig Se/mL. Add 10 mL HNO3
to 30.0 mg Se (purity >99%) and warm to dissolve. Dil. to
100 mL with H 2 0, mix well, and transfer exactly 1 mL to
micro-Kjeldahl flask. Add 2 mL 70% HCIO4 and 1 glass bead.
Boil gently to HCIO4 fumes and cool. Add 1 mL H 2 and 1
mL HC1 (1+4); heat 30 min in boiling H 2 bath. Transfer to
1 L vol. flask and dil. to vol. with ca \N HC1. Store in all-
glass container. Soln is stable several months at room temp.
(e) Decalin. — Eastman Kodak No. 1905 decahydronaph-
thalene, or equiv.
(f) 2 ,3-Diaminonaphthalene (DAN) soln. — Prep, soln in
semidarkened room or in room with only yellow light at time
of detn. Protect from light and prep, fresh for each set of detns.
Add 50 mL ca 0.1 N HC1 to 0.05 g DAN (No. 13,653-0, Aid-
rich Chemical Co.). Place in 50° H 2 bath in dark 15 min.
Cool to approx. room temp, and ext twice with 10 mL decalin,
shaking vigorously each time and discarding decalin. Filter thru
paper satd with H 2 0. For >8 detns, prep, larger amt.
C. Preparation of Samples
Grind air-dried samples to pass No. 18 or finer sieve. Cut
fresh or wet samples finely with scissors or knife, or grind in
food chopper to assure representative sample.
Some plants (e.g.. Astragalus hisulcatus, A. racemosus,
Stanley a bipinnata, and Oonopsis condensatd) contain Se in
volatile form that is lost during drying. Analyze these plants
without drying. With usual agricultural crops, this is not a
problem if drying is performed at 60-70°.
D. Preparation of Fluorometric Blanks and Standard
(a) Blank. — Place 1 mL H 2 in micro-Kjeldahl flask. (For
samples contg <0.1 ppm, carry 10 mL HN0 3 as blank thru
entire detn.)
(b) Std. — Place 1.0 mL Se std soln in micro-Kjeldahl flask.
Add 2 mL 70% HC10 4 to each flask and continue as in detn,
beginning "Mix contents of flasks ..."
E. Determination for Low Level Samples
For samples containing <4 ppm selenium. — Weigh <l g
sample (air-dried wt basis) contg <0.4 |mg Se into micro-Kjel-
dahl flask. Add 1 glass bead, previously cleaned with HNO3.
Add 10 mL HN0 3 and let stand at room temp. >4 hr. (Use 5
mL HNO3 for samples <0.5 g.) Affix air condenser and place
flask in nearly upright position on micro-Kjeldahl digestion
unit. Heat ca 15 min with low flame and then increase heat
until HNO3 condenses in lower part of condenser. Heat 10 min
longer, turn off burner, and let cool 5 min. Wash down sides
of flask with 2 mL 70% HCIO4 thru air condenser. Swirl flask
and continue refluxing 15 min. Remove condenser and con-
tinue heating, drawing off fumes in fume duct, until HCIO4
fumes appear and then 15 min longer. Cool, add 1 mL H 2 0,
and again heat to HCIO4 fumes and 1-2 min longer. Cool,
and add 1 mL H 2 0.
Mix contents of flasks and add 1 mL HC1 (1+4) to each.
Place in boiling H 2 bath 30 min. Cool to ca room temp.
To each flask add 5 mL NH 2 OH-EDTA soln and 2 drops
cresol red indicator. Neutze to yellow with ca 5 N NH 4 0H and
add HC1 (1+4) to orange-pink. From this point, perform all
operations in semidarkened room or room with yellow light
only. Prep. DAN soln, add 5 mL to each flask, and dil. to
neck with ca 0.1 TV HC1. Mix and place in 50° H 2 bath in
dark 25 min.
Remove flasks from H 2 bath and cool to ca room temp,
in pan of H 2 0. Pour solns into 125 mL separators with Teflon
stopcocks and contg 10.0 mL decalin. Shake vigorously >30
sec, let stand ca 1 min, and drain and discard lower layer.
Wash decalin twice by shaking vigorously >15 sec with 25
mL ca 0.1 TV HC1. (VirTis, Rt 208, Gardiner, NY 12525, Ex-
tractomatic shaker with 100 mL separators may be substituted.
When used, shake ext 5 min and wash I min periods.) Trans-
fer decalin layer to 12 mL centrf. tubes and centrf. 2 min at
moderate speed. Pour decalin soln into fluorometer tubes, zero
fluorometer against decalin, and read all tubes at 525 nm within
5 min. Correct std and unknown readings for blank.
ppm Se — 0.3 x sample reading/std reading x g sample
F. Determination for Higher Level Samples
For samples containing >4ppm selenium . — Proceed as above ,
thru second par. Dil. digest to adequate vol. and take aliquot
contg ca 0.3 jxg Se for detn. Alternatively, digest sample in
10 vols HNO3 2 hr on steam bath. Dil. to definite vol., and
carry appropriate aliquot thru detn. Latter method is especially
applicable when proper sampling requires large sample. Do
not dil. decalin soln contg piazselenol, as this introduces er-
rors.
Ref.: JAOAC 52, 627(1969).
CAS-7782-49-2 (selenium)
58
Plants
AOAC Official Methods of Analysis (1990)
920.10
Sulfur in Plants
Sodium Peroxide Method
Final Action
(Caution: See safety notes on sodium peroxide.)
A. Preparation of Solution
Place 1.5-2.5 g sample in ca 100 mL Ni crucible and add
5 g anhyd. Na 2 C0 3 . Mix thoroly, using Ni or Ptrod, and mois-
ten with ca 2 mL H 2 0. Add Na 2 2 , ca 0.5 g at time, mixing
thoroly after each addn, and continue until mixt. becomes nearly
dry and quite granular (ca 5 g Na 2 2 ). Place crucible over S-
free flame or elec. hot plate and heat carefully, stirring oc-
casionally, until contents are fused. (If material ignites, detn
is worthless.)
After fusion, remove crucible, let cool somewhat, and cover
hardened mass with more Na 2 2 to depth of ca 5 mm. Heat
gradually and finally with full flame until fusion again takes
place, rotating crucible occasionally to bring any particles ad-
hering to sides into contact with oxidizing material. Continue
heating 10 min after fusion is complete. Cool somewhat, place
warm crucible and contents in 600 mL beaker, and carefully
add ca 100 mL H 2 0. After initial violent action ceases, wash
material out of crucible, make slightly acid with HO (adding
small portions at time), transfer to 500 mL vol. flask, cool,
dil. to vol., and filter.
B. Determination
Dil. aliquot of prepd soln to ca 200 mL with H 2 and add
HC1 until ca 0.5 mL free acid is present. Heat to bp and add
10 mL 10% BaCl 2 soln dropwise with constant stirring. Con-
tinue boiling ca 5 min, and let stand >5 hr in warm place.
Decant thru ashless paper or ignited and weighed gooch. Add
15-20 mL boiling H 2 to ppt, transfer to filter, and wash with
boiling H 2 until filtrate is Cl-free. Dry ppt and filter, ignite,
and weigh as BaS0 4 . Wt ppt x 0.1374 - S.
Refs.: USDA Bur. Chem. Bull. 105, p. 151; 116, p. 92; 137,
p. 30.
CAS-7704-34-9 (sulfur)
923.01 Sulfur in Plants
Magnesium Nitrate Method
Final Action
A. Preparation of Solution
Weigh 1 g sample into large porcelain crucible. Add 7.5
mL Mg(N0 3 ) 2 soln (dissolve 950 g P-free Mg(N0 3 ) 2 .6H 2 in
H 2 and dil. to 1 L), so that all material comes in contact with
soln. (It is important that enough Mg(N0 3 ) 2 soln be added to
ensure complete oxidn and fixation of the S present. For larger
samples and for samples with high S content, proportionally
larger vol. of this soln must be used.) Heat on elec. hot plate
(180°) until no further action occurs. Transfer crucible while
hot to furnace (<500°) and let it remain until sample is thoroly
oxidized. (No black particles should remain. If necessary, break
up sample and return to furnace.) Remove crucible and let cool.
Add H 2 0; then HO in excess. Bring soln to boil, filter, and
wash thoroly. If preferred, transfer soln to 250 mL vol. flask
before filtering and dil. to vol. with H 2 0.
B. Determination
Dil. entire filtered soln, 923.01A, to 200 mL, or take 100
mL aliquot of the measured vol., dil. to 200 mL, and proceed
as in 920.10B.
Ref.: JAOAC 6, 415(1923).
OTHER CONSTITUENTS
931.02 Sugars in Plants
Preparation of Sample
Final Action
A. Preparation of Solution
(a) General method. — Prep, fresh sample as in 922.02(b).
Pour ale. soln thru filter paper or extn thimble, catching filtrate
in vol. flask. Transfer insol. material to beaker, cover with
80% alcohol, warm on steam bath 1 hr, let cool, and again
pour ale. soln thru same filter. If second filtrate is highly col-
ored, repeat extn. Transfer residue to filter, let drain, and dry.
Grind residue so that all particles will pass thru 1 mm sieve,
transfer to extn thimble, and ext 12 hr in Soxhlet app. with
80% alcohol. Dry residue and save for starch detn. Combine
ale. filtrates and dil. to vol. at definite temp, with 80% al-
cohol.
For dried materials, grind samples finely, and mix well.
Weigh sample into beaker, and continue as above, beginning
"... cover with 80% alcohol, ..."
(b) Applicable when starch is not to be determined, — Prep.
fresh sample as in 922.02(b), but boil on steam bath 1 hr.
Decant soln into vol. flask, comminute solids in high-speed
blender with 80% alcohol. Boil blended material on steam bath
0.5 hr, cool, transfer to vol. flask, dil. to mark with 80% al-
cohol at room temp., filter, and take aliquot for analysis.
Grind dry material to pass No. 20 sieve or finer, transfer
weighed sample to vol. II ask, and add 80% alcohol and enough
CaC0 3 to neutze any acidity. Boil 1 hr on steam bath, cool,
adjust vol. at room temp, with 80% alcohol, filter, and take
aliquot for analysis.
B. Clarification with Lead (1)
Place aliquot ale. ext in beaker on steam bath and evap. off
alcohol. Avoid evapn to dryness by adding H 2 if necessary.
When odor of alcohol disappears, add ca 100 mL H 2 and
heat to 80° to soften gummy ppts and break up insol. masses.
Cool to room temp, and proceed as in (a) or (b):
(a) Transfer soln to vol. flask, rinse beaker thoroly with H 2 0,
and add rinsings to flask. Add enough said neut. Pb{OAc) 2
soln to produce flocculent ppt, shake thoroly, and let stand 15
min. Test supernate with few drops of the Pb(OAc) 2 soln. If
more ppt forms, shake and Jet stand again; if no further ppt
forms, dil. to vol. with H 2 0, mix thoroly, and filter thru dry
paper. Add enough solid Na oxalate to filtrate to ppt all the
Pb, and re filter thru dry paper. Test filtrate for presence of Pb
with little solid Na oxalate.
(b) Add twice min. amt of satd neut. Pb(OAc) 2 soln re-
quired to cause complete pptn, as found by testing portion of
supernate with few drops dil. Na oxalate soln. Let mixt. stand
only few min; then filter into beaker contg estd excess of Na
oxalate crystals. Let Pb ppt drain on filter and wash with cold
H 2 until filtrate no longer gives ppt in oxalate soln. Assure
excess of oxalate by testing with 1 drop Pb(OAc) 2 . Filter and
wash pptd Pb oxalate, catching filtrate and washings in vol.
flask. Dil. to vol. with H 2 and mix.
C. Clarification with ton-Exchange Resins (2)
Place aliquot ale. ext, 931. 02A, in beaker and heat on steam
bath to evap. alcohol. Avoid evapn to dryness by adding H 2 0.
When odor of alcohol disappears, add ca 15—25 mL H 2 and
heat to 80° to soften gummy ppts and break up insol. masses.
Cool to room temp. Prep, thin mat of Ceiite on filter paper in
buchner or on fritted glass filter and wash until H 2 comes
thru clear. Filter sample thru Ceiite mat, wash mat with H 2 0,
dil. filtrate and washings to appropriate vol. in vol. flask, and
mix well.
AOAC Official Methods of Analysis (1990)
Other Constituents
59
Place 50.0 mL aliquot in 250 mL erlenmeyer; add 2 g Am-
berlite I R- 120(H) analytical grade cation (replaced by REXYN
101(H) resin, Fisher Scientific Co.) and 3 g Duolite A-4(OH)
anion ion exchange resins. Let stand 2 hr with occasional
swirling. Take 5 mL aliquot de ionized soln and det. reducing
sugars as glucose as in 959. 11B.
Refs.: (1) JAOAC 14, 73, 225(1931); 15, 71(1932).
(2) JAOAC 36, 402(1953).
933.02
See 959.11B.
Glucose in Plants
Micro Method
Final Action 1965
960.06 Fructose in Plants
Somogyi Micro or Munson-Walker Method
First Action 1960
Final Action 1961
A. Reagents
(a) Glucose oxidase preparation. — Add slowly, stirring
constantly, 100 mL H 2 to 5 g glucose oxidase prepn ("DeeO
L-750" code 4633000, Miles Laboratories, Inc., 1127 Myrtle
St, PO Box 70, Elkhart, IN 46514). Stir ca 1 min and centrf.
or filter to obtain clear soln. Add ca 1 mL CHC1 3 and refrig-
erate. Soln is stable >:! month.
(b) Mcllvaine' $ citrate-phosphate buffer. — Dissolve 214.902
g Na 2 HP0 4 .12H 2 and 42.020 g citric acid in H 2 and dil. to
1 L.
B. Determination
To suitable aliquot add V-i its vol. of buffer to give pH ca
5.8. Add 30% as much glucose oxidase prepn as estd glucose
content (for 500 mg glucose add 150 mg glucose oxidase, i.e.,
3 mL soln), and few drops 30% H2O2 (omit if Somogyi method
is to be used in detn). Let stand overnight at room temp.
Det. fructose by Somogyi micro method, 959.1 IB, or by
Munson-Walker method, 906.03, using Table 960.06. Check
equivs in range of interest, using pure fructose as std, and cor-
rect as necessary.
Refs.: JAOAC 41, 307, 681(1958); 42, 650(1959); 43,
512(1960); 44, 267(1961).
921.03* Sugars (Reducing) in Plants
Quisumbing-Thomas Method
Final Action
Surplus 1970
See 31.048-31.049, 11th ed.
930.07
Sucrose in Plants
Inversion Methods
Final Action
A. Hydrochloric Acid inversion
Using aliquot of cleared soln, 931.02B, proceed as in 925.05.
B. invertase inversion
(a) For plants giving hydrolysis end point within 2 hours. —
Pipet aliquot of cleared soln, 931. 02B, into 400 mL Pyrex
beaker and make slightly acid to Me red with HO Ac. Add 3
drops 1 % soln of Wallerstein invertase scales. Let mixt. stand
at room temp. 2 hr. Add reagents as in 923. 09B, and det.
reducing power. Calc. results as invert sugar. Deduct reducing
power of original soln, also expressed as invert sugar, and
multiply difference by 0.95.
(b) For plants giving slower hydrolysis end point. — Place
aliquot of soln, 931.02B, in small vol. flask. Make slightly
acid to Me red with HO Ac. Add 3 drops \% soln of Wall-
erstein invertase scales and few drops toluene. Stopper flask
and let stand overnight or longer at room temp. Dil. to vol.
with H 2 and use aliquot for reducing power as above.
CAS-57-50-1 (sucrose)
930.09
See 920.39B.
930.10
See 962.09
Ether Extract of Plants
Gravimetric Method
Final Action
Fiber (Crude) in Plants
Digestion Method
Final Action
906.01 Sugars (Reducing) in Plants
Munson-Walker General Method
Final Action
See 906.03.
Table 960.06 Abbreviated Munson and Walker Table for Cal-
culating Fructose
(From Official and Tentative Methods of Analysis, AOAC, 5th Ed., 1940)
Cuprous Oxide,
Fructose,
Cuprous Oxide,
Fructose,
mg
mg
mg
mg
10
4.5
300
148.6
50
23.5
350
174.9
100
47.7
400
201.8
150
72.2
450
229.2
200
97.2
490
253.9
250
122.7
—
~
978.04 Nitrogen (Total) (Crude Protein)
in Plants
Kjeldahl Methods
First Action 1976
Final Action 1978
A. Kjeldahl Method for Nitrate-free Samples
See 955.04.
B. Kjeldahl Method for Nitrate-Containing Samples
See 968.01.
977.02 Nitrogen (Total) (Crude Protein)
in Plants
First Action 1977
A. Automated Method
See 976.05.
60
Plants
AOAC Official Methods of Analysis (1990)
B. Semiautomated Method
See 976.06.
948.02
Starch in Plants
Titrimetric Method
Final Action 1965
A. Reagents
(a) Iodine-potassium iodide soln. — Grind 7.5 g 1 and 7.5 g
KI with 150 mL H 2 0, dil. to 250 mL, and filter.
(b) Alcoholic sodium chloride soln.— Mix 350 mL alcohol,
80 mL H 2 0, and 50 mL 20% NaCl soln, and dil. to 500 mL
with H 2 0.
(c) Alcoholic sodium hydroxide soln. — 0.257V. Mix 350 mL
alcohol, 100 mL H 2 0, and 25 mL 5/V NaOH, and dil. to 500
mL with H 2 0.
(d) Dilute hydrochloric acid. — 0.77V. Dil. 60 mL HC1 to 1
L with H 2 0.
(e) Somogyi phosphate sugar reagent. — Dissolve 56 g an-
hyd. Na 2 HP0 4 and 80 g Rochelle salt in ca 1 L H 2 0, and add
200 mL 1.00/V NaOH. Then slowly add, with stirring, 160
mL 10% CuS0 4 .5H 2 soln. Dissolve 360 g anhyd. Na 2 S0 4 in
this soln, transfer to 2 L vol. flask, and add exactly 200 mL
QAN KI0 3 soln (3.5667 g/L). Dil. to vol., mix well, let stand
several days, and filter thru dry paper into dry flask, discarding
first 50 mL filtrate. Store reagent at 20-25°. It is 0.01N with
respect to KI0 3 ; 5.00 mL is equiv. to 10 mL 0.005W Na 2 S 2 3 .
Det. glucose factor of reagent as follows: Accurately weigh
150 mg NBS glucose SRM into 1 L vol. flask, dissolve in
H 2 0, dil. to vol., and mix well. Transfer 5 mL aliquot to 25
x 200 mm Pyrex test tube, add exactly 5 mL Somogyi re-
agent, stopper with size 00 crucible, and heat (together with
several blanks contg 5 mL H 2 and 5 mL reagent) exactly 15
min in boiling H 2 bath. Titr. as in detn. From difference
between blank and std titrns, calc. mg glucose equiv. to 1 mL
exactly 0.005/V Na 2 S 2 3 . Effective range for detn is 0.05-1.0
mg glucose in 5 mL aliquot.
(f) Sodium thiosulfate std soln. — 0.005 jV. Dissolve 2.73 g
Na 2 S 2 3 .5H 2 in H 2 and dil. to 2 L. Stdze daily as follows:
Add 1 mL KI soln, (g), and 3 mL 1.5 N H 2 S0 4 to 5 mL Som-
ogyi sugar reagent. Let stand 5 min, and titr. with Na 2 S 2 3
soln, adding starch indicator, (h), just before end point.
(g) Potassium iodide soln. — 2.5%. Stabilize with little
Na 2 C0 3 .
(h) Starch indicator. — Make 1.5 g sol. starch into paste with
few mL H 2 0, and add slowly, with stirring, to 300 mL boiling
H 2 0.
B. Determination
Select sample as in 922.01, remove all foreign matter, dry,
and grind to pass No. 80 sieve. Accurately weigh 0.1-1.0 g
powd sample contg ca 20 mg starch into 25 x 1 50 mm Pyrex]
test tube. Add ca 200 mg fine sand and 5 mL H 2 0, and mix
well with stirring rod to wet sample. Heat tube in boiling H 2
bath 15 min to gelatinize starch. Cool to room temp., and place
in 22-25° bath. Add 5 mL 60% HC10 4 rapidly with const ag-
itation. Grind tissue against lower wall of tube with stirring
rod for approx. min at time. Repeat grinding frequently during
30 min; then without delay, transfer quant, to 100 mL vol.
flask with H 2 0. Add 3 mL 5% uranyl acetate soln to ppt pro-
tein, dil. to vol. with H 2 0, mix well, and centrf. portion of
mixt. Pipet 10 mL clear supernate into 25 X 150 mm test
tube. Add ca 100 mg Celite, 5 mL 20% NaCl soln, and 2 mL
I-KI reagent, and mix well. Let stand overnight, centrf., and
decant.
Wash starch-I ppt by suspending it in 5 mL ale. NaCl soln,
centrf., and decant. Add 2 mL ale. NaOH soln to packed ppt.
Gently shake and tap tube until ppt is no longer blue. (Do not
use stirring rod; allow ample time for complex to decompose.)
Wash walls of tube with 5 mL ale. NaCl soln, centrf. liberated
starch, and wash with 5 mL ale. NaCl soln as before. Add 2
mL 0.7 N HC1 to ppt. Stopper tube loosely with size 00 cru-
cible, and heat 2.5 hr in boiling H 2 bath. (Bath should have
cover with holes to accommodate tubes; holes not occupied by
tubes must be covered.) Cool, and transfer quant, to 25 mL
vol. flask. Add drop phenol red, 941. 17A, and neutze with
1 N NaOH. Discharge color with 0.1 N oxalic acid, dil. to vol.,
and mix well. Transfer 5 mL aliquot to 25 X 200 mL Pyrex
test tube, add exactly 5 mL Somogyi reagent, and stopper tube
with size 00 crucible. Heat together with several blanks contg
5 mL H 2 and 5 mL Somogyi reagent in vigorously boiling
H 2 bath exactly 15 min. Remove tube from bath and cool to
25-30°. Add 1 mL 2.5% KI soln down wall of tube without
agitation and then add 3 mL 1.5 7V H 2 S0 4 rapidly with agita-
tion. After all Cu 2 dissolves, titr. soln with 0.005 N Na 2 S 2 3 ,
adding starch indicator, (h), just before end point is reached.
Treat blank solns similarly.
% Starch = |'50(mL blank - mL sample) x 0.90/mg sample]
x (jV/0.005) X G x 100
where 50 = diln factor, 0.90 = factor glucose to starch, N —
actual normality Na 2 S 2 3 soln, and G = mg glucose equiv. to
1 mL 0.005 Af Na 2 S 2 6 3 .
Refs.: Anal. Chem. 20, 850(1948). JAOAC 39, 423(1956).
932.01
Lignin in Plants
Direct Method
Final Action 1965
A. Preparation of Sample
Grind sample in mill to pass No. 80 sieve and dry at 105°.
Ext weighed sample (5-10 g) 30 hr in Soxhlet app. with al-
cohol-benzene soln (32 parts alcohol and 68 parts benzene by
wt). Dry material in oven to free it from solvs and place in
flask of suitable size. Add 150 mL H 2 0/g sample, and reflux
3 hr. Filter mixt, while still hot, preferably thru weighed fritted
glass crucible, and transfer extd material to flask. Add 1% HO
(111 g coned HC1 + 3890 mL H 2 0) in proportion of 150 mL
acid soln/g plant material, and reflux 3 hr. Filter mixt. while
still hot thru fritted glass crucible previously used, wash with
H 2 until acid-free, dry at 105°, and weigh. Calc. % total loss
due to successive extn with alcohol-benzene soln, hot H 2 0,
and 1% HC1. (With samples not especially rich in carbohy-
drates and proteins, extn with hot H 2 may be omitted.)
B. Apparatus
App., Fig. 932.01, consists of: (1) 1 .5 L bottle, A, to which
is attached by 2-hole rubber stopper 250 mL dropping funnel,
C. having lower end of stem bent as illustrated and placed
close to bottom of A; (2) Drechsel gas-washing bottle, D; (3)
3 Pyrex test tubes, 38 X 300 mm diam., G, G f , G", connected
in parallel by device, O, and immersed in wooden box, L,
filled with crushed ice, //; and (4) bottle contg H 2 for ab-
sorption of excess HC1, K. G, G\ and G" are provided with
2-hole rubber stoppers; glass tube with right angle bend ex-
tends thru 1 hole nearly to bottom of test tube, and similar
tube extending ca 10 mm into test tube passes thru other hole.
Rubber connections and stopcocks for regulating flow of gas
are provided as indicated in diagram. A is filled with ca 500
mL H 2 S0 4 and C with HO; HO flowing thru stopcock B into
A generates HO gas, which is dried by H 2 S0 4 in D, and flows
into G, G' , and G" contg samples and fuming HO reagent.
AOAC Official Methods of Analysis (1990)
Other Constituents
61
FIG. 932.01 — Apparatus for determining Hgnin
C. Reagent
Fuming hydrochloric acid. — (Caution: See safety notes on
fuming acids.) Density 1 .212-1.223 at 15°. To 500 g NaCl in
1 L g-s Pyrex distg flask, add cold soln of 250 mL H 2 in
450 mL H 2 S0 4 . Connect side tube of distg flask to glass tube
passing thru H 2 S0 4 wash bottle, and connect outlet tube of
H 2 S0 4 wash bottle to another glass tube, immersed in flask
contg 3 L HO. Surround flask contg HC1 with crushed ice.
Heat distg flask with small flame and pass HC1 gas into acid
soln until it attains sp gr of 1.212-1.223 at 15°. Keep reagent
refrigerated at <0°. If only few detns are to be made, prep.
correspondingly smaller amt.
D. Determination
Weigh three 1 g samples of extd and dried sample in weigh-
ing bottle and place in 3 large test tubes, G, G\ and G". Add
20 mL of the reagent to each tube, using this acid to wash
down any particles clinging to sides. When all material is wet
with reagent, add addnl 30 mL reagent. Add ca 3 drops capryl
alcohol to minimize foaming. Place test tubes, G, G', and G",
in wooden box, L, and surround with crushed ice. Lubricate
tubes F, F\ and F" with drop of glycerol so that they move
easily thru holes in rubber stoppers. Lead dry HC1 gas from
generator into reaction mixts thru tubes F, F' , and F" (F' and
F" are shown in top view), which reach nearly to bottom of
tubes G, G\ and G" . Regulate flow of gas thru reaction mixts
in G, G\ and G" by stopcocks shown in top view, continuing
passage of gas 2 hr. (At first rather slow stream of gas passes
in, but during last 15 min, flow is fairly rapid.)
After reaction period, discontinue flow of gas, and discon-
nect long tubes F, F\ and F" and outlet tubes of test tubes G,
G\ and G" from O and P. Pull tubes F, F r , and F" just above
surface of reaction mixt., and close with short pieces of rubber
tubing having one end plugged with short piece of glass rod.
Similarly close off outlet tubes, N, N f , and N fr . Place tubes
contg reaction mixt. in cold room or refrigerator (8-10°) 24
hr.
Transfer contents of G ( G\ and G" to 1 L erlenmeyers, tak-
ing care to remove any material adhering either to inside or
outside of tubes F, F' , and F". Dil. reaction mixts to 500 mL
with H 2 0. Connect flasks to reflux condensers and boil 1 hr.
Prep. 3 gooches in usual manner, dry at 105°, and weigh. Ig-
nite one of weighed crucibles, X, over Bunsen burner, cool in
desiccator, and reweigh. Let contents of flasks cool to room
temp, and filter thru weighed gooches. Wash ppts collected in
gooches with hot H 2 0, dry at 105°, and weigh in weighing
bottles. Ignite crude lignin in crucible X over Bunsen flame
and det. wt ash. Place one of other 2 gooches in wide-neck
Kjeldahl flask and det. % N in crude lignin as in 955.04. If
methoxyl in lignin is to be detd, collect ppt from one of flasks
in dried (105°) fritted glass crucible and proceed as in 956.07C.
Wt lignin
= wt crude lignin - wt ash — wt crude protein (N x 6.25)
Calc. % lignin in original dry unextd material.
Refs.: JAOAC 15, 124(1932); 18, 386(1935); 19, 107(1936).
CAS-9005-53-2 (lignin)
949.04
Lignin in Plants
Indirect Method
Final Action 1965
(Caution: See safety notes on asbestos.)
Ext 1 g sample with alcohol-benzene (1+2) 4 hr in Soxhlet
or comparable app. (extn vessel may be either coarse porosity
Alundum or paper thimble, closed at top with filter paper or
plug of cotton). Wash sample in thimble with suction, using
2 small portions alcohol followed by 2 small portions ether.
Heat at 45° in nonsparking oven to drive off ether, and transfer
sample to 250 mL wide-mouth erlenmeyer. Add 40 mL 1%
soln of pepsin in 0.1 N HCl, wetting sample well by adding
small portion reagent, stirring or shaking thoroly, and finally
washing down sides of flask with remaining soln. Incubate at
40° overnight.
Add 20-30 mL hot H 2 and filter, using filter stick. (Filter
62
Plants
AOAC Official Methods of Analysis (1990)
sticks are made with Pyrex fritted glass disk, 30 mm diaim,
medium porosity. Thin layer of pre-ashed diat. earth (Hyflo
Super-CeJ, or similar filter-aid) is sucked onto disk from H 2
suspension. This is usually enough for easy filtration; if not,
add extra Super-CeJ to material being filtered. Some sticks fil-
ter slowly with some samples. It is advisable to obtain more
than needed and discard slow-filtering ones. It is convenient
to arrange filter sticks in set of 12 attached to vac, manifold
by rubber tubing.)
Repeat washing twice and then wash residue into flask by
forcing 7-8 mL 5% (w/w) H 2 S0 4 downward thru filter stick,
using air pressure. Wash stick further with the H 2 S0 4 , finally
adding enough to bring total vol. to ca 150 mL. Reflux vig-
orously on hot plate 1 hr, adding H 2 occasionally to maintain
original vol. Filter off acid. Wash residue with three 20-30
mL portions hot H 2 0, two 15—20 mL portions alcohol, and
two 1.5 mL portions ether. Leave vac. on tew min to dry res-
idue, and transfer from stick to flask by tapping and brushing.
Heat to drive off any residual ether. If disk formed upon drying
is difficult to break up into finely divided state (sometimes in
case of immature plant samples), disperse residue in ether in
flask and then boil off ether on steam bath. Add 20 mL 72%
(w/w) H 2 S0 4 at 20° to residue and hold 2 hr at 20°, stirring
occasionally. Add 125 mL H 2 0, filter, wash once with 20 mL
hot H 2 0, and filter again. Wash residue from filter stick and
reflux as before 2 hr, using 150 mL 3% (w/w) H 2 S0 4 . Filter
residue onto gooch with asbestos pad and wash with hot H 2
until acid-free. Dry at 105-110° and det. lignin by loss in wt
on ignition at 600°.
Ref.: JAOAC 32, 288(1949).
CAS-9005-53-2 (lignin)
Weigh 1-5 g into mortar and add ca 0. 1 g CaC0 3 or Na 2 C0 3 .
Macerate tissue with pestle, add quartz sand, and grind short
time; then add 85% acetone, little at time, and continue grind-
ing until tissue is finely ground. Transfer mixt. to funnel, filter
with suction, and wash residue with 85% acetone. Return res-
idue to mortar with more 85% acetone and grind again. Filter
and wash as before. Repeat procedure until tissue is devoid of
any green, and washings are colorless. (It is advisable to grind
residue at least once with undild acetone and then to add enough
H 2 at end to bring acetone concn to 85%. High-speed blender
may be used instead of mortar to macerate and ext tissue (see
942. 04C), but each investigator should satisfy himself that de-
vice used exts tissue completely.) When extn is complete,
transfer filtered ext to vol. flask of appropriate size and dil.
to vol.
Measure T of soln with photoelec. colorimeter, and read amt
of chlorophyll present from curve relating T and concn. Ex-
press chlorophyll values as mg/g tissue, or in other convenient
manner.
Calibrate photoelec. colorimeter as follows: Ext sample of
fresh, green leaf material with 85% acetone, filter, wash res-
idue, and dil. ext to vol. as above. Make series of dilns of ext
and measure T of original and of each of dild solns with in-
strument in same manner as when chlorophyll prepn is being
used as calibration std. Transfer aliquot of original ext to ether
and evaluate total chlorophyll spectrophtric as in 942.04C(b)
and (c). From value thus obtained, calc. chlorophyll content
of original ext and that of each of dild solns, and construct
curve relating concn of chlorophyll with T ot A.
Refs.: Ind. Eng. Chem. Anal. Ed. 12, 148(1940); 15, 524
(1943).
CAS- 1406-65-1 (chlorophyll)
PIGMENTS
940.03 Chlorophyll in Plants
Photoelectric Colorimetric Method for
Total Chlorophyll Only
Final Action
A. Apparatus
(a) Mortar and pestle. — Deep glass mortar ca 10 cm id with
well-defined lip.
(b) Photoelectric colorimeter. — Calibrate for chlorophyll,
using plant ext as in 940. 03C and light filters with max. T
near 660 nm. (Combination of Kopp Glass Works filters K2408
and K3965 is suitable.)
(c) Wash bottles. — Type fitted with rubber bulb, permitting
operation with one hand.
(d) High-speed blender. — Waring Blendor, or equiv.
B. Reagents
(a) Acetone . — (1) Undild acetone and (2) 85% aq. soln by
vol. Com. acetone, tech. grade, is satisfactory.
(b) Quartz sand. — Acid- washed and dried.
C. Determination
(Caution: See safety notes on blenders, toxic solvents, and
acetone.)
Select field material carefully to ensure representative sam-
ple. Remove representative portion from field sample, and if
fresh, cut finely with hand shears and mix as thoroly as pos-
sible. Grind dried material in mill and mix thoroly.
942.04 Chlorophyll in Plants
Spectrophotometry Method for Total Chlorophyll
and the a and b Components
Final Action
A. Apparatus
Use app. in 940. 03 A (except photoelec. colorimeter), plus
following:
(a) Scrubbing tubes for washing ether solns. — Open tubes
ca 20 mm diam. to one end of each of which is sealed tube
of smaller diam. drawn to fine jet at lower end.
(b) Spectrophotometer . — Capable of isolating spectral re-
gion of ca 3 nm near 660 nm with negligible stray radiation.
Tubulated cells with tightly fitting glass stoppers are recom-
mended for work with ether.
B. Reagents
Those listed in 940. 03B and following:
Ether. — Com. grade is satisfactory without further purifi-
cation.
C. Determination
(Wash glassware with coned Na 3 P0 4 soln to remove traces of
acid that may decompose chlorophyll.)
(a) Extraction of chlorophyll from tissue. — Select and prep,
sample as in 940. 03C. Disintegrate weighed portion (2-10 g,
depending on chlorophyll content) of fresh plant tissue in blender
cup that contains ca 0.1 g CaC0 3 , or by use of mortar as in
940. 03C. After tissue is thoroly disintegrated, filter ext thru
buchner fitted with quant, paper. Wash residue with 85% ace-
AOAC Official Methods of Analysis (1990)
Pigments
63
tone, 940.03B(a), and if necessary, use little ether to remove
last traces of pigment. If extn is incomplete, return residue and
paper to blender container with more 85% acetone and repeat
extn. Filter and wash, as before, into flask contg first filtrate.
Transfer filtrate to vol. flask of appropriate size and dil. to
vol. with 85% acetone.
Pipet aliquot of 25-50 mL into separator contg ca 50 mL
ether. Add H 2 carefully until it is apparent that all fat-sol.
pigments have entered ether layer. Drain and discard H 2 layer.
Place separator contg ether soln in upper rack of support. Add
ca 100 mL H 2 to second separator placed in rack below first.
Set scrubbing tube in place, and let ether soln run thru it to
bottom of lower separator and rise in small droplets thru the
H 2 0. When all soln has left upper separator, rinse it and scrub-
bing tube with little ether added from medicine dropper. Place
scrubbing tube in upper separator and exchange its place in
support with separator now contg ether soln. Drain and discard
H 2 in upper separator, add similar portion of fresh H 2 to
lower separator, and repeat washing process. Continue wash-
ing ether soln until all acetone is removed (5-10 washings).
Then transfer ether soln to 100 mL vol. flask, dil. to vol., and
mix.
(b) Spectrophotometric measurements . — Add ca teaspoon-
ful (ca 5 mL) anhyd. Na 2 S0 4 to 60 mL reagent bottle, and fill
it with ether soln of pigment. When this soln is optically clear,
pipet aliquot into another dry bottle and dil. with enough dry
ether to give A value of 0.2-0.8 at wavelength to be used.
(Most favorable value is near 0.6 at 660 nm, since such soln
yields satisfactory value at 642.5 nm.)
Fill 2 clean g-s absorption cells with dry ether from pipet
and polish outside surfaces of each, first with cotton wet with
alcohol and then with dry cotton. Place cells in instrument,
and det. whether each gives same galvanometer deflection. If
not, clean again or select cells that do, and do this daily. Empty
one cell, fill it with the dried ether soln, and place in instru-
ment. Adjust entrance and exit slits until spectral region iso-
lated is 3-4 nm at 660.0 nm.
Det. whether instrument is in proper adjustment for wave-
length by taking A readings thru soln against solv. at 1 nm
intervals from 658 to 665 nm. Highest value should be at 660.0
nm; if not, adjust instrument until it is, or make 660.0 nm
readings at wavelength setting that gave highest A. With grat-
ing instrument, apply same correction at 642.5 nm; however,
with prism instrument, correction at 642.5 nm must be ob-
tained from wavelength calibration curve for particular instru-
ment in use. Calibrate instrument for wavelength in this way
often enough to ensure that it remains in proper adjustment.
Det. A at 660.0 and 642.5 nm (or corrected settings) for each
unknown soln.
(c) Calculation of chlorophyll concentration. — Calc. total
chlorophyll and each of a and b components (mg/L) as fol-
lows:
(1) Total chlorophyll - 7.12A 660 . + 16.8A 6425
(2) Chlorophyll a - 9.93 A 660 . - 0.777 A M2 _ 5
(3) Chlorophyll b = 17.6 A 642 . 5 - 2.81 A 660 .
D. Supplementary information
Factors involved in spectrophtric analysis of chlorophyll
system have been discussed in detail (Plant Physiol. 17, 198
(1942)). These authors used Beer's law in form:
c = Qogiolo/0/a X t [== A/a X t\
where / is intensity of light transmitted by solv. -filled cell; /
is intensity of light transmitted by sol n-fi lied cell; c is concn
of chlorophyll (g/L); a is absorptivity; / is thickness of soln
layer in cm; and A is absorbance.
Table 942.04 Absorption Constants Used in Analysis
(after Cornar and Zscheile)
Wavelength,
Absorptivities (for Ether Solns)
nm
Chlorophyll a
Chlorophyll b
660.0
102
4.50
642.5
16.3
57.5
600.0
9.95
9.95
581.0
8.05
8.05
568.0
7.11
7.11
613.0
15.6
8.05
589.0
5.90
10.3
Since, at given wavelength, observed A value of soln having
2 components represents sum of A values of each of compo-
nents, following equation holds in case of chlorophylls a and
b at given wavelength:
(4) ^observed ~ A a + A b
If 1 cm cell is used, this equation may be expressed as:
(5)
= a a c a + a b c b
Concns of chlorophylls a and b in given ether soln can now
be calcd by equation (5) as follows:
(a) Det. A for soln at 2 different wavelengths (660.0 and
642.5 nm have been found advantageous for this purpose).
(b) From Table 942.04, select proper absorptivities corre-
sponding to wavelengths used.
(c) Substitute observed A value and absorptivities in equa-
tion (5) for each of the 2 wavelengths used as illustrated for
660.0 and 642.5 nm in equations (6) and (7). Solve these 2
equations simultaneously for 2 unknowns, the concns of chlo-
rophylls a and b.
(6)
(7)
= 102 c a + 4.50 c h
16.3 c a + 57.5 c h
Equations (1), (2), and (3) were derived this way.
Criterion for accuracy of chlorophyll values detd by spec-
trophtric method is agreement between analytical results detd
from measurements at different wavelengths. Measurements at
660.0 and 642.5 nm are convenient for routine analysis (Plant
Physiol. 17, 198 (1942)); however, readings may be made at
other wavelengths to check these values. Absorptivities for
chlorophylls a and b in ether soln that may be used for this
purpose are presented in Table 942.04.
These values may be used for calcns as follows:
(a) Values for total chlorophyll and % composition may be
calcd from A at 660.0 and 642.5 nm as described.
(b) Check values for total chlorophyll may be calcd from
A at intersection points 600.0, 581.0, and 568.0 nm.
(c) Check values for % composition may be calcd from A
for each of points 613.0 and 589.0 nm in combination with
value of total concn obtained from (a) or (b).
Refs.: Ind. Eng. Chem. Anal. Ed. 14, 877(1942). Plant Phys-
iol. 17, 198(1942).
CAS- 1406-65-1 (chlorophyll)
955.10
See 941.15.
Carotenes in Plants
Final Action
64
Plants
AOAC Official Methods of Analysis (1990)
TOBACCO
966.02 Moisture in Tobacco
Gravimetric Method
First Action 1966
Final Action 1968
A. Apparatus
(a) Drying oven. — Forced-draft, regulated to 99.5±0.5°.
Suggested dimensions: 19 x 19 x 19" (48 cm). Approx. oven
settings: fresh air intake vent Vs open; air control damper 1 / 4
open; air exhaust vent } / 3 open.
(b) Moisture dish. — Al, diam. 45-65 mm, depth 20-45 mm,
with tight-fitting cover.
B. Determination
Accurately weigh ca 5 g sample (ground to pass <1 mm
screen) into weighed moisture dish and place uncovered dish
in oven.
Do not exceed 1 sample/ 10 sq in. (650 sq cm) shelf space,
and use only 1 shelf. Dry 3 hr at 99. 5 ±0.5°. Remove from
oven, cover, and cool in desiccator to room temp, (ca 30 min).
Reweigh to nearest 1 mg and calc. % moisture.
Ref.: JAOAC 49, 525(1966).
963.05 Chiorides in Tobacco
Potentiometric Method
First Action 1963
Final Action 1964
A. Reagents
Silver nitrate std soln. — 0.1 N. Stdze against KC1 as in detn.
B. Apparatus
(a) pH meter. — Leeds and Northrup, Sumneytown Pike, N
Wales, PA 19454, Beckman Instruments, or equiv., equipped
with Ag and glass electrodes.
(b) Buret. — 10 mL, graduated in 0.05 or 0.02 mL, pref-
erably reservoir-type.
C. Determination
Accurately weigh ca 2 g sample, ground to pass No. 40
sieve, into 250 mL electrolytic beaker. Add 100 mL H 2 0, small
amt at first to thoroly wet sample; then remainder. Let stand
>5 min at room temp. , stirring intermittently. Pipet 5 mL HN0 3
(1+9) into mixt. and insert clean electrodes. Start mag. stirrer
and continue stirring thruout titrn at rate that produces vigor-
ous agitation without spattering. Titr. with std 0.1 N AgN0 3
soln to potential previously established as equivalence point.
Det. equivalence point potential graphically by making several
titrns on one or more tobacco samples. Recheck occasionally,
and redet. when either electrode is replaced. Record vol. of
titrant and calc:
% CI = mL AgN0 3 x normality x 3.5453/g sample
Ref.: JAOAC 46, 415(1963).
959.04 Nitrogen in Tobacco
Kjeldahl Method for Samples
Containing Nitrates
Final Action 1964
(For nitrate-free samples, omit salicylic acid and thiosulfate
treatment.)
A. Reagents
See 920.02A and the following:
(a) Sodium hydroxide -thiosulfate soln. — Dissolve 500 g
NaOH pellets and 40 g Na 2 S 2 3 .5H 2 in H 2 and dil. to 1 L.
(b) Indicators. — (7) Dissolve 1 g Me red in 200 mL al-
cohol; or (2) prep, mixed indicator by dissolving 0.8 g Me red
and 0.2 g methylene blue in 500 mL alcohol.
B. Apparatus
See 920.02B.
C. Determination
Place weighed sample (1-2 g) in digestion flask. Add vol.
H 2 S0 4 (contg 2 g salicylic acid/40 mL) corresponding to wt
sample (35 mL for 1 g, 40 mL for 2 g for N0 3 -contg samples;
20 and 25 mL, resp., for N0 3 -free samples). Shake until tho-
roly mixed; let stand >30 min with occasional shaking; then
add 5 g Na 2 S 2 3 .5H 2 0. Shake, let stand 5 min, and heat care-
fully until frothing ceases. Turn off heat, add 0.7 g HgO (or
metallic Hg) and 15 g K 2 S0 4 , and boil briskly 1-1.5 hr after
soln clears.
Cool, add ca 200 mL H 2 0, cool to ca room temp. , and add
few Zn granules. Tilt flask and carefully add 50 mL NaOH-
thiosulfate soln without agitation. Immediately connect flask
to distn bulb on condenser whose tip is immersed in 50 mL
std 0.1 N acid in receiving flask. Then rotate digestion flask
carefully to mix contents. Heat until >150 mL distillate col-
lects, and titr. excess acid with std base, using Me red or mixed
indicator. Correct for blank detn on reagents.
Ref.: JAOAC 42, 302(1959).
CAS-7727-37-9 (nitrogen)
966.03 Potassium in Tobacco
Flame Photometric Method
First Action 1966
Final Action 1968
A. Reagents
(a) Potassium std so Ins. — (I) Stock soln. — 1000 ppm K.
See 956.01A(a). (2) Working solns.— Place 0, 5, 10, 15, 20,
25, and 30 mL stock soln in seven 1 L vol. flasks, add 40 mL
3 N HC1 to each, and dil. to vol. with H 2 0.
(b) Diatomaceous earth. — Celite 545, acid-washed.
B. Apparatus
(a) Flame photometer. —Natural gas-air fuel, or equiv., ad-
equate for K analysis.
(b) Chromatographic tube. — 20 x 150 mm with coarse
fritted disk.
C. Preparation of Sample Solution
Accurately weigh ca 0.5 g tobacco dust into ca 40 mL
weighing dish. Add ca 1 g Celite and mix intimately with spat-
ula. Transfer quant, thru powder funnel into chromatgc tube.
Add addnl Celite thru funnel into tube until 2.5 cm layer ac-
cumulates on top of sample-Celite mixt. Compact sample and
Celite by tapping tip of tube on table top, and insert tip of tube
into neck of 1 L vol. flask. Add 40 mL 3N HCl into tube by
pipet or dispenser, washing down sides, and let elute into vol.
flask. When liq. level reaches top of Celite, add 25 mL H 2
and let elute. Add second 25 mL portion of H 2 0, let elute by
gravity, or force thru rapidly with compressed air. Rinse tip
of tube into vol, flask, dil. to vol. with H 2 0, and mix well.
AOAC Official Methods of Analysis (1990)
Tobacco
65
D. Determination
Det. % T for sample eluate and K stds as specified in in-
struction manual of instrument. See also 956. 01D.
Prep, calibration curve and det. ppm K of sample from curve.
% K = ppm KxO.l/g sample
% K 2 - ppm K x 0.1205/g sample
Ref.: JAOAC 49, 521(1966).
CAS-7440-09-7 (potassium)
971.02 Glycerol, Propylene Glycol,
and Triethylene Glycol in Cased Cigarette Cut Filler
and Ground Tobacco
Gas Chromatographic Method
First Action 1971
Final Action 1985
(Caution: See safety notes on pipets and methanol.)
A. Apparatus
(a) Gas chromato graph. — With programmed temp, oven
and W hot wire detector; F&M Model 720 (current models
5700 series; Hewlett-Packard Co.), or equiv. Conditions: De-
tector bridge 140 ma; temps (°): injection 265, detector 280,
column 90-240 at 15°/min; He 60 mL/min adjusted, if nec-
essary, to facilitate sepns; attenuation 4, adjusted according to
sensitivity to yield peaks of sufficient size for accurate mea-
surement (use same attenuation for all stds and samples); chart
speed, 12"/hr.
(b) Column. — 42 (105 cm) x 3 / 16 " Cu tubing packed with
5% Carbowax 20M-terephthalic acid (TPA) on 60-80 mesh
Chromosorb G AW-DMCS (Hewlett-Packard Co., No. 8501-
6223 or Applied Science No. 04388). Prep, packing by plac-
ing 30.0 g Chromosorb in 500 mL 1" r-b flask. Add soln of
1.50 g Carbowax 20M-TPA in 150 mL CHC1 3 , and slurry.
Remove CHC1 3 under vac. in rotary evaporator and air dry
overnight at room temp. Condition new column 2 hr at 240°;
then inject three 30 jjlL samples tobacco ext before analyzing
samples. Recondition columns removed from app. before use.
B. Reagents
(a) Extracting soln. — Dil. 20.0 mL 1 ,3-butylene glycol stock
std soln, (b), to 2 L with anhyd. MeOH.
(b) 1,3-Butylene glycol stock std soln. — Accurately weigh
20.00 g USP 1,3-butanediol into 100 mL vol. flask and dil.
to vol. with anhyd. MeOH.
(c) Glycerol stock std soln. — Accurately weigh 10.00 g USP
glycerol into 100 mL vol. flask and dil. to vol. with extg soln.
(d) Propylene glycol stock std soln. — Accurately weigh 5.00
g USP propylene glycol into 100 mL vol. flask and dil. to vol.
with extg soln.
(e) Triethylene glycol stock std soln. — Accurately weigh 5.00
g triethylene glycol into 100 mL vol. flask and dil. to vol.
with extg soln.
(f) Humectant std solns. — Into each of four 100 mL vol.
flasks, pipet 1.0, 2.0, 3.0, and 4.0 mL, resp., glycerol, pro-
pylene glycol, and triethylene glycol stock std solns. Dil. to
vol. with extg soln. Each soln contains (in mg/100 mL):
Propylene
Triethylene
Soln
Glycol
Glycerol
Glycol
1
50
100
50
2
100
200
100
3
150
300
150
4
200
400
200
C. Extraction
Place 10.00 g sample in 250 mL ¥ erlenmeyer. Pipet 100
mL extg soln into flask and stopper. Shake mech. 1 hr and let
settle few min until supernate is clear. Alternatively, shake
mech. 30 min and let stand overnight.
D. Determination
Prime column by injecting two 30 jjlL aliquots supernate ext.
Then alternately inject 30 (jlL supernate exts and a humectant
std soln until all samples and stds have been run, repeating
ext injections, if necessary. (Sequence is exti, ext,, std, , ext],
std 2 , ext 2 , std 3 , ext 3 , std 4 , ext 4 , std 1? ext 5 , std 2 , etc. if <4 exts
are available, distribute ext injections among those available
so that sequence thru std 4 is run.) Det. peak hts and calc. ratios
of propylene glycol, glycerol, and triethylene glycol to butyl-
ene glycol for each std and sample soln. Plot peak ht ratios
against polyol concn for std solns and construct std curve for
each humectant. Det. concn in mg/100 mL for propylene gly-
col, glycerol, and triethylene glycol in sample soln from resp.
std curves.
% Humectant - (mg/100 mL) X 0.01
Ref.: JAOAC 54, 560(1971).
CAS-56-81-5 (glycerol)
CAS-57-55-6 (propylene glycol)
CAS-1 12-27-6 (triethylene glycol)
960.07 Alkaloids (Total
As Nicotine) in Tobacco
Distillation Method
First Action 1960
Final Action 1964
A. Apparatus
(a) Distillation apparatus. — 500 mL Kjeldahl flask fitted
with inlet tube for steam, trap bulb, and condenser; Griffith
still (Tobacco Sci. 1, 130(1957), available from Lab Glass,
Inc., PO Box 610, Vineland, NJ 08360); or other suitable steam
distn app.
(b) Spectrophotometer. — Beckman Instruments Model DU,
24, or 25 (replacement model DU-64), or other instrument ca-
pable of accurately measuring A in 200-300 nm range, equipped
with 1 cm quartz cells.
B. Reagents
(a) Alkali-salt soln.— Dissolve 300 g NaOH in 700 mL H 2
and sat. with NaCl.
(b) Silicotungstic acid soln (for gravimetric determina-
tion).— Dissolve 120 g Si0 2 .12W0 3 .26H 2 in H 2 and dil.
to 1 L. (Soln should be clear and free from green color.)
C. Standardization
(Caution: Nicotine is very toxic. Avoid contact with skin and
eyes. See safety notes on distillation and vacuum.)
Purify best grade of nicotine com. available by successive
vac. distns until center cuts from 2 successive distns have same
a at 259 nm (ca 34.3). Accurately weigh ca 0.2 g purified
nicotine; dissolve in and dil. to 1 L with ca 0.05 N HC1. Dil.
10 mL aliquot of this soln to 100 mL with ca 0.05 W HC1.
Det. A at 259 nm and calc. a = A/(c x b), where c is concn
of nicotine in g/L and b is cell length in cm.
D. Distillation
Accurately weigh 2-5 g tobacco sample and transfer to distn
flask or app. (If final detn of nicotine is gravimetric, use sam-
66
Plants
AOAC Official Methods of Analysis (1990)
pie contg ^0.1 g alkaloids; if spectrophtric, use >2 g sample.)
(If Griffith still is used, use 0.05-0.2 g sample.) Place 25 mL
HC1 (1+4) in receiver (1 L vol. flask is desirable) and place
receiver so that condenser tube dips into acid. (With Griffith
still, use 10 mL HC1 (1+4) in 250 mL vol. flask.) Add 50 mL
alkali-salt soln to distn flask so that sample is rinsed into bot-
tom of flask. (With Griffith still, use 5 mL alkali-salt soln.)
If large vol. of liq. is required for proper function of still, add
more alkali-salt soln; do not dil. Connect flask to app. im-
mediately and steam distil with as rapid current of steam as
can be condensed efficiently. Effluent condensate should not
be above room temp. Apply heat to distn flask from burner,
mantle, or other heat source to keep vol. in flask approx. const.
Collect ca 900 mL condensate (or distil addnl 100 mL after
condensate shows no nicotine by silicotungstic acid test). (With
Griffith still, collect 225 mL.) Dil. distillate to vol.
E. Determination
(a) Spectrophotometries — Dil. aliquots of distillate (if nec-
essary) with 0.05 7V HC1 so that A at 259 nm is 0.5-0.8 and
read A at 236, 259, and 282 nm. Calc. corrected A' 259 = 1.059
x [observed A 259 - Vs (^ 236 + ^282)] a f ter correcting all ob-
served A values to original distillate vol. basis. Concn, c, of
alkaloids as nicotine in g/L is given by c ~ A' 259 /(a X /?),
where a is absorptivity at 259 nm, and b is ceil length in cm.
% alkaloid (as nicotine) = c
X vol. distillate (L) X 100/g sample
(b) Gravimetric. — Det. alkaloids in distillate as in 920.35B,
but double amt of silicotungstic acid specified, i.e., 2 mL/
each 10 mg alkaloids expected.
Ref.: JAOAC 43, 524(1960).
CAS-55-11-5 (nicotine)
960.08 Alkaloids (Total As Nicotine)
in Tobacco
Cundiff-Markunas Method
First Action 1960
Final Action 1964
(Total alkaloids (as nicotine), tertiary alkaloids (as nicotine),
and secondary alkaloids (as nornicotine))
A. Reagents
(a) Benzene-chloroform soln.— Mix equal parts by vol. of
benzene and CHCl 3 and sat. with H 2 0.
(b) Sodium hydroxide soln. — 36%. Dissolve 500 g NaOH
in H 2 and dil. to 1 L.
(c) Dilute acetic acid. — 5%. Dil. 50 mL HOAc to 1 L with
H 2 0.
(d) Crystal violet indicator. — Dissolve 0.5 g crystal violet
in 100 mL HOAc.
(e) Perchloric acid std soln. — 0.025 7V. Add 4.7 mL 72%
HCIO4 to freshly opened 5 lb bottle HOAc and mix. {Caution:
See safety notes on acetic acid, acetic anhydride, and perchlo-
ric acid.) Stdze as follows: Accurately weigh 0.1 g KH phthal-
ate (NIST) into 125 mL erlenmeyer, add 50 mL HOAc, and
heat to dissolve. Cool, add 2 drops indicator, and titr. to blue-
green end point. Perform blank titrn on 50 mL HOAc and 2
drops indicator soln, and correct vol. of titrant.
N = wt KH phthalate x 4.896/mL HC10 4 soln
B. Determination
Accurately weigh 2.5 g finely ground tobacco into 250 mL
erlenmeyer. Add 15 mL 5% HOAc and swirl until tobacco is
thoroly wetted. Pipet 100 mL benzene-CHCl 3 soln into flask,
and then 10 mL 36% NaOH soln. Stopper flask tightly and
shake 20 min, using wrist-action shaker. Add 4.5-5 g (2 tea-
spoonfuls) Filter-Cel, mix, and filter most of benzene layer
thru Whatman No. 2 paper into second flask. If filtrate has
any turbidity, add 2-2.5 g (1 teaspoonful) addnl Filter-Cel and
ref i Iter thru Whatman No. 2 paper. Filtrate must be clear.
Pipet 25 mL aliquots of filtrate into each of two 125 mL
erlenmeyers. Pass stream of air over surface of soln in first
flask 5 min, add 2 drops indicator, and titr. to green end point
with 0.025 TV HCIO4. Add 1.0 mL Ac 2 to second flask and
let stand >15 min. Add 25 mL HOAc and 2 drops indicator,
and titr. to blue-green end point with 0.025 N HCIO4. Take
first appearance of blue-green thruout soln as end point. For
each series of analyses perform blank titrn s and correct re-
spective vols of titrant.
Calc. % alkaloids as follows: % total alkaloids (as nicotine)
= Vj X TV X 32.45/wt sample; % tertiary alkaloids (as nico-
tine) = (2V 2 ~ Vi) X N X 32.45/wt sample; % secondary
alkaloids (as nornicotine) - 2{V X - V 2 ) X 29.64/wt sample;
where V x = vol. titrant for nonacetylated aliquot; V 2 — vol.
titrant for acetylated aliquot; and N - normality HCIO4.
Ref.: JAOAC 43, 524(1960).
CAS-55-11-5 (nicotine)
CAS-494-97-3 (nornicotine)
979.01 Nicotine on Cambridge Filter Pads
Gas Chromatographic Method
First Action 1979
Final Action 1984
A. Apparatus and Reagents
(a) Gas chromato graph. — With flame ionization detector,
heated injection port, and thermostated column oven. Follow-
ing conditions have been found satisfactory: Column, 1.8 m
(6') x Vs" stainless steel; packing, 2% KOH and 10% Car-
bowax 20M (based on final packing wt) on 45-60 mesh cal-
cined diat. earth (such as Chromosorb W, or equiv.), resieved
before use to mesh range to remove fines and lumps; temps
(°): column 165, detector and injection port 200-250; carrier
gas flow, ca 40 mL/min. Adjust H and air Hows for max.
sensitivity and stability. Under these conditions, column should
have ht equiv. to theoretical plate (HETP) <1 mm and reso-
lution of >2, calcd with nicotine and anethole.
(b) Measuring system. — Measure peak areas with elec-
tronic integrator or other system with resolution of 2: 1 count/
mv-sec.
(c) Mechanical shaker. — Capable of extg ^99% nicotine.
Burreil Wrist- Action shaker has been found satisfactory.
(d) Extracting soln. — 2-Propanol contg 1 mg anethole/mL
as internal std for nicotine. If H 2 is also to be detd, add 20
mg EtOH/mL 2-propanol as addnl internal std.
(e) Nicotine std solns. — (/) Stock soln. — Weigh 2.500 g
nicotine, 960.07C, or equiv. amt of nicotine salt. Transfer quant.
into 100 mL vol. flask, and dil. to vol. with extg soln. (2)
Working std solns. — Pipet 1, 2, 3, 4, and 5 mL stock soln
into five 100 mL vol. flasks, and dil. to vol. with extg soln
(0.25, 0.50, 0.75, 1.00, and 1.25 mg nicotine/mL).(CawfK>/i;
See precaution in 960. 07C.)
B. Extraction
Place Cambridge filter material in flask or serum bottle ac-
commodated by shaker used, add 10.00 mL extg soln, stopper,
and shake until >99% of nicotine is extd (usually ca 15 min).
AOAC Official Methods of Analysis (1990)
Tobacco
67
C. Standardization
Prime column with aliquots of 1.25 mg/mL std soln. Let
baseline stabilize, inject 1 |iL each std soln in succession, and
repeat sequence 3 times. Det. area ratio (nicotine:anethole) for
each injection, and calc. slope and intercept of response curve,
preferably by method of least squares (See Definition of Terms
and Explanatory Notes). Correlation coefficient should be >0.99
and intercept <0.05 mg/mL.
D. Determination
Prime column with aliquots of ext, 979.01B. Let baseline
stabilize, and inject 1 jjlL of each sample soln. Calc. nicotine
concn in soln (C, mg/mL) = mx + b, where m = slope of
stdzn curve, b = intercept, and x — area ratio of nicotine to
anethole.
Nicotine yield /cigarette = (C X 10.00)/(No. cigarettes /pad)
Ref.: J AOAC 62, 229(1979).
CAS-55-11-5 (nicotine)
968.02 Menthol in Cigarette Filler
Colorimetric Method
First Action 1968
Final Action 1970
A. Apparatus and Reagents
(a) Distillation apparatus .—See Fig. 968.02.
(b) Spectrophotometer. — With matched cells; capable of
measuring A at 550 nm.
(c) Menthol std. soln. — 1 mg/mL. Accurately weigh 100
mg USP /-menthol into 100 mL vol. flask, add alcohol to dis-
solve, and dil. to vol. with alcohol.
(d) DMAB color reagent. — Dissolve 0.5 g p-dimethylami-
nobenzaldehyde (Sigma Chemical Co.) in 100 mL H 2 S0 4
(1.6+1).
B. Preparation of Calibration Curve
Prep. dil. stds by pipeting aliquots contg 0, 3, 4, 6, 8, and
10 mg menthol into 100 mL vol. flasks and dilg to vol. with
alcohol (1 + 1). Pipet 1 mL each dil. std into 10 mL test tube,
add 5 mL color reagent, mix, and place in boiling H 2 bath
exactly 2 min. Cool in tap H 2 0, and within 15 min det. A at
550 nm against std. Prep, calibration curve by plotting A
against menthol concn (mg/100 mL).
C. Determination
Accurately weigh 2.00-2.15 g cigaret filler and transfer to
distn flask, A. Add 80 ml H 2 and few boiling stones, con-
nect flask to condenser with tube, B, attach adapter, C, to
condenser, and immerse tip in 20 mL alcohol in 100 mL vol.
receiving flask.
Gently heat distn flask until distn begins; then increase heat
and lower receiving flask, D, so tip of adapter is no longer
immersed. Distil until 20 mL distillate collects. Disconnect
condenser from tube, and wash down condenser with alcohol.
Remove receiving flask, dil. distillate to ca 70 mL with al-
cohol, and add H 2 almost to vol. Mix, add alcohol to vol.,
and mix again.
Pipet 1 mL distillate into 10 mL test tube, add 5 mL color
reagent, mix, and place in boiling H 2 bath exactly 2 min.
Cool in tap H 2 0, and within 15 min det. A at 550 nm, using
"color" soln from nonmentholated tobacco carried thru detn
as blank. (If nonmentholated sample corresponding to men-
tholated sample is not available, use reagent blank.) Use non-
mentholated tobacco blank within 15 min after color devel-
opment step. Fresh nonmentholated tobacco blank soln may
be required during multiple sample runs. Det. mg menthol from
calibration curve.
% Menthol = mg menthol /(g original sample x 10)
Ref.: JAOAC51, 650(1968).
CAS-1490-04-6 (menthol)
968.03 Menthol in Cigarette Filler
Gas Chromatographic Method
Final Action 1970
A. Apparatus and Reagents
(a) Gas chromatograph. — Equipped with flame ionization
detector and thermostated injection port and column oven. Use
following conditions for analysis: Column, 1.5 m (5') X ] /s"
od stainless steel packed with 10% (w/w) silicone oil DC-550
on 60-80 mesh Chromosorb W; temps (°): column 150, de-
tector 150, injection port 175; N carrier gas flow ca 35 mL/
min. Adjust H and air flows for max. sensitivity and reason-
able stability.
(b) Mechanical shaker. — Wrist action.
(c) Menthol-anethole std. soln. — 0.250 mg menthol and 0.50
mg anethole/mL. Weigh exactly 0.5000 g tech. grade aneth-
ole and wash into 1 L vol. flask with 200 mL alcohol. Transfer
0.2500 g USP /-menthol to the vol. flask with enough alcohol
to bring to vol. Store soln in dark g-s bottle. Do not use >6
weeks.
(d) Extracting soln. — 0.50 mg anethole/mL. Dissolve 1 .000
g anethole in alcohol in 2 L vol. flask, dil. to vol. with alcohol,
and store in dark.
B. Determination of Ratio Factor
Weigh ca 3 g nonmentholated control filler, contg all usual
humectants but no menthol or anethole, into 125 mL rubber-
stoppered flask. Pipet 50 mL std menthol-anethole soln into
flask, stopper, and shake 1 hron mech. shaker. Let solids set-
tle 15 min and chromatograph 2 pJL aliquot of supernate. Re-
peat twice more to obtain total of 3 replicates of std chro-
matogram. For quant, results, inject both std and unknown
samples by inserting 2" (5 cm) needle to hilt, injecting 2 (jlL
MM QD. TUBING
24/40
500 ML
100 ML
FIG. 968.02— Distillation apparatus; see 968.02C for
explanation of symbols
68
Plants
AOAC Official Methods of Analysis (1990)
rapidly, and withdrawing needle at once. (Menthol elutes in
ca 3 min, anethole in ca 5 min.) After ca 10 min, all other
compds are eluted and new injection can be made.
Draw baselines under menthol and anethole peaks and mea-
sure peak hts in mm. Using mean peak ht of menthol and
anethole from 3 std chromatograms, calc. std ratio factor of
menthol to anethole as follows:
Std ratio factor = peak ht for menthol (0.25 mg/mL)/
peak ht for anethole (0.50 mg/mL)
C. Determination
Accurately weigh 8-8.5 g mentholated cigarette filler and
place in 250 mL rubber-stoppered erlenmeyer. Pipet 100 mL
extg soln into flask, stopper, and mech. shake 2 hr. Let solids
settle 15 min and chromatograph 2 |xL aliquot of supernate.
Draw baselines under menthol and anethole peaks and measure
peak hts in mm. Calc. ratio factor of unknown menthol as
follows:
Ratio factor for unknown = peak ht for unknown menthol/
peak ht for anethole (0.50 mg/mL)
% Menthol = (unknown ratio factor x 0.25 x 10)/
(std ratio factor x g sample)
Ref.: JAOAC 51, 650(1968).
CAS- 1490-04-6 (menthol)
4 a Animal Feed
Joel WL Padmore, Associate Chapter Editor
North Carolina Department of Agriculture
965.16 Sampling of Animal Feed
Procedure
Use slotted single or double tube, or slotted tube and rod,
all with pointed ends.
Take >500 g sample, 1 kg preferred, as follows: Lay bag
horizontally and remove core diagonally from end to end. Det.
number of cores as follows: From lots of 1-10 bags, sample
all bags; from lot of ^11, sample 10 bags. Take 1 core from
each bag sampled, except that for lots of 1-4 bags take enough
diagonal cores from each bag to total >5 cores. For bulk feeds
draw >10 cores from different regions; in sampling small con-
tainers (<10 lb) 1 package is enough. Reduce composite sam-
ple to amt required, preferably by riffling, or by mixing
thoroly on clean oil-cloth or paper and quartering. Place sam-
ple in air-tight container.
A sample from less than these numbers of bags may be de-
clared an official sample if guarantor agrees. For samples that
cannot be representatively taken with probe described, use other
sampling means.
950.02
Animal Feed
Preparation of Sample
Final Action
Grind sample to pass sieve with circular openings 1 mm
(V25") diam. and mix thoroly. If sample cannot be ground, re-
duce to as fine condition as possible. Do not grind molasses
feeds.
Refs.: JAOAC 33, 424(1950); 41, 223(1958); 48, 658(1965);
51, 467(1968).
0.01 mL. Clean tube and condenser with chromic acid clean-
ing mixt., rinse thoroly with H 2 0, then alcohol, and dry in
oven to prevent undue amt H 2 from adhering to inner sur-
faces during detn.
B. Determination
If sample is likely to bump, add dry sand to cover bottom
of flask. Add enough toluene to cover sample completely (ca
75 mL). Weigh and introduce enough sample into toluene to
give 2-5 mL H 2 and connect app. Fill receiving tube with
toluene, pouring it thru top of condenser. Bring to boil and
distil slowly, ca 2 drops /sec, until most of the H 2 passes
over; then increase rate of distn to ca 4 drops/sec.
When all H 2 is apparently over, wash down condenser by
pouring toluene in at top, continuing distn short time to see
whether any more H 2 distils over; if it does, repeat washing-
down process, if any H 2 remains in condenser, remove by
brushing down with tube brush attached to Cu wire and satd
with toluene, washing down condenser at same time. (Entire
process is usually completed within 1 hr.) Let receiving tube
come to room temp. If any drops adhere to sides of tube, force
them down, using Cu wire with end wrapped with rubber band.
Read vol. H 2 and calc. to %.
Refs.: JAOAC 8, 295(1925); 9, 30(1926).
920.36* Moisture in Animal Feed
Drying without Heat over
Sulfuric Acid
Final Action
Surplus 1974
See 7.006-7.007, 12th ed.
934.01 Moisture in Animal Feed
Drying in Vacuo at 95-100°
Final Action
Dry amt sample contg ca 2 g dry material to const wt at 95-
100° under pressure <100 mm Hg (ca 5 hr). For feeds with
high molasses content, use temp. <70° and pressure ^50 mm
Hg. Use covered Al dish >50 mm diam. and <40 mm deep.
Report loss in wt as moisture.
Ref.: JAOAC 17, 68(1934); 51, 467(1968); 60, 322(1977).
925.04 Moisture in Animal Feed
By Distillation with Toluene
Final Action
A. Apparatus
Connect 250 mL flask of Pyrex or other resistant glass by
means of Bidwell-Sterling moisture receiver to 500 mm Liebig
condenser. Calibrate receiver, 5 mL capacity, by distg known
amts H 2 into graduated column, and estg column of H 2 to
69
930.15 Moisture in Animal Feed
Drying at 135°
Final Action
(Not to be used when fat detn is to be made on same sample)
Regulate air oven to 135 ±2°. Using low, covered Al dishes,
934.01, weigh ca 2 g sample into each dish and shake until
contents are evenly distributed. With covers removed, place
dishes and covers in oven as quickly as possible and dry sam-
ples 2 hr. Place covers on dishes and transfer to desiccator to
cool. Weigh, and calc. loss in wt as H 2 0.
Refs.: JAOAC 13, 173(1930); 14, 152(1931); 17, 178(1934);
18, 80(1935).
953.07 Moisture in Animal Feed
In Highly Acid Milk By-products
Final Action
Add ca 2 g ZnO, freshly ignited or oven dried, to flat-bot-
tom dish >5 cm diam. and weigh. Add ca 1 g sample and
70
Animal Feed
AOAC Official Methods of Analysis (1990)
weigh quickly. Add ca 5 mL H 2 and distribute sample evenly
on bottom of dish. Heat on steam bath, exposing max. surface
of dish bottom to live steam until apparently dry. Heat at 98-
100° in air oven 3 hr or to const wt. Cool in desiccator; then
weigh quickly. Det. wt residue. Oil. with twice its vol. C0 2 -
free H 2 0. Add 2 mL phthln, and titr. with 0. IN NaOH to first
persistent pink. Calc. as % lactic acid by wt. (1 mL 0.1N
NaOH = 0.0090 g lactic acid.). To compensate for H 2 formed
when acid is neutzd by ZnO, add 0.1 g to residue wt for each
g acid (as lactic) in weighed sample. Report % residue (cor-
rected) as total solids.
Refs.: JAOAC 36, 213(1953); 37, 253(1954).
942.05
Ash of Animal Feed
Final Action
Weigh 2 g sample into porcelain crucible and place in temp.
controlled furnace preheated to 600°. Hold at this temp. 2 hr.
Transfer crucible directly to desiccator, cool, and weigh im-
mediately, reporting % ash to first decimal place.
Refs.: JAOAC 25, 857(1942); 26, 220(1943),
935.11* Protein in Animal Feed
Qualitative Tests
Final Action
Surplus 1965
A. Biuret Test
See 22.012-22.013, 10th ed.
S. Mitlon Test
See 22.014-22.015, 10th ed.
C. Glyoxylic Acid Test (Hopkins-Cole)
See 22.016-22.017, 10th ed.
D. Adamkiewicz Test
See 22.018, 10th ed.
E. Xanthoproteic Test
See 22.019, 10th ed.
954.01 Protein (Crude) in Animal Feed
Kjeldahl Method
Final Action
Det. N as in 955.04. Multiply result by 6.25, or in case of
wheat grains by 5.70.
Refs.: JAOAC 37, 241(1954); 38, 56(1955).
988.05 Protein (Crude) in Animal Feed
CuS0 4 /Ti0 2 Mixed Catalyst Kjeldahl Method
First Action 1988
(Caution: See safety notes on sulfuric acid and sodium
hydroxide.)
A. Principle
Sample is digested in H 2 S0 4 , using CuS0 4 /Ti0 2 as cata-
lysts, converting N to NH 3 which is distd and titrd.
B. Reagents
(a) Sodium hydroxide soln. — Dissolve ca 450 g NaOH pel-
lets or flakes (low N) in H 2 0, cool, and dil. to 1 L; or use
soln with sp. gr. >1.36.
(b) Boiling stones. — Alundum, 8-14 mesh (No. 1590-D18;
Thomas Scientific Co.).
(c) Methyl red indicator. — Dissolve 1 g Me red (Na salt) in
100 mL MeOH.
(d) Hydrochloric or sulfuric acid std soln. — 0.5N. Prep, as
in 936.15 or 890.01.
(e) Sodium, hydroxide std soln. — 0.1N. Prep, as in 936.16.
After stdzg both acid and base by methods suggested in (d)
and (e), also check one against the other. In addn, check entire
method by analyzing NIST Std Ref. material No. 194,
NH 4 H 2 P0 4 , certified 12.15% N, and a high purity JysineHCl.
C. Apparatus
(a) Digestion.— Kjeldahl flasks with capacity of 500-800
mL.
(b) Distillation. — Digestion flask (e.g.. Corning Glass No.
2020) connected to distn trap by rubber stopper. Distn trap is
connected to condenser with low-S tubing. Outlet of condenser
tube should be <4 mm diam.
D. Determination
Weigh 0.250-1 .000 g sample into digestion flask. Add 16.7
g K 2 S0 4 , 0.01 g anhyd. CuS0 4 , 0.6 g Ti0 2 , 0.3 g pumice,
0.5-1.0 g Alundum granules, and 20 mL H 2 S0 4 . (Add addnli
1.0 mL H 2 S0 4 for each 0.1 g fat or 0.2 g other org. matter if
sample wt is >1 g.)
Include at least 1 sample of high purity lysine -HO in each
day's run as check of correctness of digestion parameters. If
recovery is not complete, make appropriate adjustments.
To digest sample, first adjust heat to bring 250 mL H 2 at
25° to rolling boil in 5 min. Add a few boiling chips to prevent
superheating. Then heat samples at this 5 -min boil rate until
dense white fumes clear bulb of flask (ca 10 min), swirl gently,
and continue heating addn] 40 min. (Note: Reagent propor-
tions, heat input, and digestion time are critical factors — do
not change.) Cool, cautiously add about 250 mL H 2 0, and
cool to room temp. (Note: Add H 2 as soon as possible to
reduce amt of caking. If excessive bumping occurs during distn,
increase diln H 2 from 250 mL to ca 300 mL.)
Prep, titrn beaker by adding appropriate vol. of acid std soln
to amt of H 2 such that condenser tip will be sufficiently im-
mersed to trap all NH 3 evolved. Add 3-4 drops of indicator
soln (c).
Add addnl 0.5- L0 g Alundum granules to cooled digestion
flask. Optionally, 2-3 drops of tributyl citrate may also be
added to reduce foaming. Slowly down side of flask, add suf-
ficient NaOH soln (a) such that mixt. will be strongly alk.
Immediately connect flask to distn app., mix completely, and
distill at ca 7.5-min boil rate until >150 mL distillate is col-
lected in titrn beaker.
Titr. excess std acid in distillate with NaOH std soln (e).
Correct for blank detn on reagents. Calc. % nitrogen:
%N = {[(N acid )(mL acid ) - (mL bk )(N NilOH )
- (mL Na oH)(N Na o H )] x 1400.67}/mg sample
where mL NaOH = mL std base needed to titr. sample; mL acid
~ mL std acid used for that sample; mL bk - mL std base
needed to titr. 1 mL std acid minus mL std base needed to
titr. reagent blank carried thru method and distd into 1 mL std
acid; N acid = normality of std acid; N Nu0 h - normality of std
base. Calc. % crude protein, defined as 6.25 x % nitrogen,
or 5.7 x % nitrogen for wheat grains.
Ref.: JAOAC 70, 907(1987).
AOAC Official Methods of Analysis (1990)
Protein
71
968.06 Protein (Crude) in Animal Feed
Dumas Method
First Action 1968
Final Action 1969
A. Principle
N, freed by pyrolysis and subsequent combustions, is swept
by C0 2 carrier into nitrometer. C0 2 is absorbed in KOH and
vol. residual N is measured and converted to equiv. protein
by numerical factor.
B. Apparatus and Reagents
(a) Nitrogen analyzer and accessories . — Consists of com-
bustion and collection and measuring systems. Suitable instru-
ment, Model 29 A, with following accessories and reagents is
available from Oak Brook Instruments Div. of Perkin Elmer
Corp., 2000 York Rd, Oak Brook, IL 60521 (Perkin Elmer's
current model of nitrogen analyzer is PE2410N nitrogen ana-
lyzer): Al combustion boats, No. 29-412; Vycor combustion
tubes, No. 29-328; CuO-Pt catalyst (CuO wire form with 2.5%
Pt reforming catalyst), No. 29-160; reduced Cu wire, No. 29-
120; Co,0 4 , No. 29-170; CuO powder, fines, No. 29-140;
45% KOH, No. 29-110.
(b) Balance. — Accurate to 0.01 mg.
(c) Barometer. — Hg type, readable to 0.1 mm.
C. Preparation of Samples
Grind to pass No. 30 sieve. Store in capped bottles.
D. Determination
Operate instrument in accordance with instructions of man-
ufacturer. (Following directions apply to Coleman Model 29 A
Nitrogen Analyzer. Consult Operating Directions D-360B,
Coleman Cat. No. 29-904, for addnl details.)
After combustion furnaces have come to thermal equilib-
rium, turn combustion cycle control to START and let proceed
normally thru cycle. Observe indicated temp, on pyrometer of
both upper and lower combustion furnaces at end of combus-
tion portion of cycle. Furnace temps should be 850-900°. If
not, adjust.
Prep, combustion tube by inserting stainless steel screen in
lower end of combustion tube (end farthest from trademark).
In upper end, place enough glass wool to form 6 mm plug
when packed. With 11 mm glass rod, drive glass wool down
to stainless steel plug. Holding tube vertically, pour CuO-Pt
catalyst directly from dispenser bottle into combustion tube until
it reaches upper end of trademark. Tap or vibrate tube on bench
until reagent settles to approx. center of trademark.
Weigh and record wt of empty Al combustion boat. Place
sample in boat. Weigh and record wt of sample and combus-
tion boat. Difference between wts is sample wt. Use following
sample wts (mg) as guides to suitable sample sizes: bermuda
grass 150-300; rice bran, wheat shorts, dehydrated alfalfa 150-
250; range feed 100-200; cottonseed meal 75-150; edible soy
protein 50-150. Weigh sample to nearest 0.01 mg. To avoid
wt changes, record wt within 1 min after sample and boat are
placed on balance. If this is impossible, weigh sample inside
weighing bottle, such as Kimble No. 15165 or 15166.
Turn combustion tube to horizontal, and carefully insert loaded
sample boat into open end of tube. Slide or push boat, without
spilling contents, until it reaches trademark. Raise open end
until tube forms 60-70° angle to horizontal. Tap or vibrate
combustion tube on bench top while rotating tube between thumb
and forefinger. Raise open end of tube and add vol. Co 3 4 and
vol. CuO fines equal to vol. sample. For convenient means of
adding above reagents to samples, place vol. CuO fines and
vol. C03O4, each equal to vol. sample, in addnl combustion
boat; add contents of boat, but not boat itself, to combustion
tube; and rotate partially filled combustion tube between thumb
and forefinger while varying angle of tube 20-45° from hor-
izontal. Continue rotating, tapping, and vibrating until sample
is dispelled from boat and is thoroly mixed with oxidizing agents.
Raise open end until tube forms 60-70° angle to horizontal;
add CuO-Pt catalyst ca 12 mm above sample boat. Tap or
vibrate gently to eliminate voids. Add CuO-Pt catalyst to within
20 mm of top of tube, again tapping or vibrating gently to
eliminate voids.
Install prepd combustion tube in N analyzer. Adjust 45%
KOH soln meniscus to calibrating mark in nitrometer with dig-
ital readout meter. Record counter reading, /?,. (Counter read-
ing should preferably lie between 500 and 1000 uX at this
point. Vent control may be used to assist in arriving at this
counter setting, if necessary.) Record syringe temp., t 1; indi-
cated on special scale thermometer. Add 2 min more to com-
bustion portion of cycle by turning auxiliary timer to setting
3. (Once this is done, addnl 2 min will be automatically pro-
grammed into each subsequent cycle.) Turn combustion cycle
control to START. Let analyzer proceed thru its cycle. After
cycle is complete and combustion cycle control has entered
STAND-BY section, readjust KOH meniscus to calibration mark
with digital readout counter. Record new counter reading, R 2 ,
and syringe temp., t 2 . Det. blank for instrument under same
conditions as actual analysis except omit sample.
E. Calculations
(a) Record observed N vol., V — R 2 ~ R\, where V =
observed N vol. (|JiL), R } = initial counter reading, and R 2 =
final counter reading.
(b) Det. corrected N vol. (in |xL), V c = V - (V b + V t ),
where V b = vol. blank (|ulL), V t = vol. correction for temp.
(|ulL) = C f (t 2 - tO. C r is obtained from Table 968.06A (based
on final counter reading); t 2 and tj are in °K.
(c) Det. corrected barometric pressure, P c = P - (P b +
P v ), where P Q — observed barometric pressure (mm Hg), P b
= barometric temp, correction (from Table 968. 06B), and P v
- pressure correction for vapor pressure of KOH soln (from
Table 968.06C).
Table 968.06A Volume Correction for Temperature Correction
Factor (C f ) (fJtL/°K) a
Final Counter
Reading,
(Nitrometers with
Check Value)
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
12
29
45
62
79
95
112
129
145
162
179
1 Vol. correction, V t = C f (t 2
Table 968.06B Barometric Temperature Correction (P b )
°C
Po
(mm
Hg)
Temperature,
700-749
750-780
10
1.2
1.3
15
1.8
1.9
20
2.3
2.5
25
2.9
3.1
30
3.5
3.7
35
4.1
4.3
72
Animal Feed
AOAC Official Methods of Analysis (1990)
Table 968.06C Pressure Correction (P v ) for Vapor Pressure of
KOH (for Practical Purposes, Temp, of KOH is
Same as Syringe)
Temperature, °K
P v> mm Hg
288
293
298
303
308
313
4.1
5.7
7.4
9.6
12.5
16.5
(Note: Empirical approximation of (P h + P v ) = 11.0 will
be satisfactorily accurate for P Q between 740 and 780 mm Hg
and syringe temp, between 298 and 305°K.)
(d) Calc. % N = (P c x V c x 0.0449)/(r x W), where T
= final syringe temp, in °K and W = sample wt in mg.
Example:
P = 750.1 mm Hg at 25°C; W - 148.91 mg
Start
Finish
Counter readings, blank
500 uX
524 \xL
Counter readings, sample
524
6955
t] = 302. 7°K, h = 303. 0°K, V Q - 6955 - 524 = 6431 \xL
V, - 6431 - [24 + C f (t 2 - t,)] =
6431 - (24 + 35 x 0.3) - 6396 |xL
P Q - 750.1 - (3.1 x 9.6) - 737.4
% N = (737.4 x 6396 x 0.04493)/(303.0 x 148.91) = 4.69%
(e) Calc. % protein = % N x 6.25, or % N x 5.70 in case
of wheat grains.
Ret.: JAOAC 51, 766(1968).
976.05 Protein (Crude) in Animal Feed
Automated Kjeldahl Method
First Action 1976
Final Action 1977
A. Principle
Automation of macro Kjeldahl method is in 6 steps: sample
and reagent addn, initial and final digestion, cooling and diln,
NaOH addn, steam distn and titrn, and automatic pumping of
flask contents to waste. Chemistry is carried out in macro Kjel-
dahl flasks equipped with side arms which are rotated at 3 min
intervals thru each successive step.
B. Apparatus
(a) Kjeldahl {protein/ nitrogen) analyzer. — Kjel-Foss Au-
tomatic, Model 16210 (Foss Food Technology Corp.), orequiv.
(b) Weighing papers. — 120 x 120 mm N-free tissues, Foss
Food Technology Corp., or equiv.
C. Reagents
(a) Kjel-tabs.— Contg 5 g K 2 S0 4 and 0.25 g HgO (Foss
Food Technology Corp.).
(b) Kjeldahl {protein/ nitrogen) analyzer reagents.— Prep.
following according to manufacturer's instructions: (7) Sul-
furic acid. — 96-98%. (2) Hydrogen peroxide.— 30-35%. (3)
Ammonium sulfate std solns. — {a) Std soln I. — Dissolve 30.000
± 0.030 g (NH 4 ) 2 S0 4 in H 2 and dil. to 1 L with H 2 0. (b)
Std soln //.—Dissolve 0.750 ± 0.001 g (NH 4 ) 2 S0 4 in H 2
and dil. to 1 L with H 2 0. (4) Mixed indicator soln. — Dissolve
1.000 g Me red and 0.250 g methylene blue in alcohol and
dil. to 1 L with alcohol. Dil. 10 mL this soln to 1 L with H 2 0.
(5) Sodium hydroxide -sodium thiosulfate soln. — 40% NaOH-
8% Na 2 S 2 3 .5H 2 0. (<5) Dilute sulfuric acid soln.— 0.6%. Dil.
30 mL 96-98% H 2 S0 4 to 5 L with H 2 0.
D. Determination
{Caution: See safety notes on wet oxidation, sulfuric acid,
mercury, and peroxides.)
Place 3 Kjel-tabs in special flask (500 mL of design com-
patible to Foss instrument) in position 1 . Shift dispenser arm
over flask and depress H 2 S0 4 lever, initiating simultaneous addn
of 10 mL 30-35% H 2 2 and 12-15 mL 96-98% H 2 S0 4 (de-
pending on fat content of sample). To flask, add accurately
weighed sample (ca 1.0 g if <45% protein, and ca 0.5 g if
>45% protein) wrapped in weighing paper and close lid. Flask
automatically rotates to position 2 where sample digests 3 min,
and then to position 3 for 3 min addnl digestion. In position
4, flask is cooled by centrifugal blower, lid opens automati-
cally, and 140 mL H 2 is added automatically. Flask rotates
to position 5, where NaOH-Na 2 S 2 3 soln is automatically in-
troduced in excess. Released NH 3 is steam distd quant, into
200 mL tall -form titrn beaker contg 50 mL mixed indicator
soln, and is simultaneously titrd automatically with dil. H 2 S0 4
soln delivered by photometrically regulated syringe. Final po-
sition of syringe is measured by potentiometer, output of which
feeds electronic circuitry for conversion to visual display and/
or printout in % N or % protein with appropriate conversion
factors. In position 6, flask is emptied. Calibrate instrument
initially each day with aliquots of (NH 4 ) 2 S0 4 std solns and
check periodically as stated in operating manual.
Ref.: JAOAC 59, 141(1976).
976.06 Protein (Crude) in Animal Feed
Semiautomated Method
First Action 1976
Final Action 1977
A. Principle
Samples are digested in 250 mL calibrated tubes, using block
digestor. A of NH 3 -salicylate complex is read in flowcell at
660 nm, or NH 3 , is distd into std acid and back-titrd with std
alkali .
B. Apparatus
(a) Block digestor. — Model BD-20 (Technicon Instruments
Corp.) or Model DS-20 (Tecator, Inc., 2875C Towerview Rd,
Herndon, VA 22071). Capable of maintaining 410° and di-
gesting 20 samples at a time in 250 mL calibrated volumetric
tubes constricted at top. Block must be equipped with remov-
able shields to enclose exposed area of tubes completely at or
above ht of constriction.
(b) Automatic analyzer. — AutoAnalyzer with following
modules (Technicon Instruments Corp.), or equiv.: Sampler II
or IV with 40/hr (2: 1) cam (higher ratio cams result in carry-
over and poorer peak sepn); proportioning pump 111; NH 3 anal,
cartridge No. 1 16-D53I-01 (or construct equiv. manifold from
flow diagram); AAII single channel colorimeter with 15 X 1,5-
2.0 mm id tubular flowcell, matched 660 nm interference fil-
ters, and voltage stabilizer; and recorder of appropriate span.
{See Fig. 976.06.)
C. Reagents
(a) Phosphate -tartrate buffer soln. — pH 14.0. Dissolve 50
g NaK tartrate and 26.8 g Na 2 HP0 4 .7H 2 in 600 mL H 2 0.
Add 54 g NaOH and dissolve. Add 1 mL Brij-35 (Technicon
Instruments Corp.), dil. to 1 L with H 2 0, and mix.
AOAC Official Methods of Analysis (1990)
Protein
73
ml/min
To Sampler 1-00 H 2 Q
10 T
—JflStiL.
0.32 Air
1.20 Acid/NaCI
40/H, 2/1 S/W
0.16 Sample
Modified
AO
10 T
Type C Membrane
6" DIALYZER
ftftKL
0.80 Acid/NaCI
0.16 ResamPle
0.80 Acid/NaCI
0.42 Buffer
0.32 Salicylate
0.16 Hypochlorite
120 From F/C
COLORIMETER
660 nm
15 mm F/C
FIG. 976.06 — Flow diagram for semiautomated analysis for crude protein
(b) Sodium chloride -sulfuric acid soln. — Dissolve 200 g
NaCl in H 2 in 2 L vol. flask. Add 15 mL H 2 S0 4 and 2 mL
Brij-35. Oil. to vol. with H 2 and mix.
(c) Sodium hypochlorite soln. — Dil. 6 mL com. bleach soln
contg 5.25% available CI (Clorox, or equiv.) to 100 mL with
H 2 and mix. Prep, fresh daily.
(d) Sodium nitroprussi.de -sodium salicylate soln. — Dis-
solve 150 g NaC 7 H 5 3 and 0.3 g Na 2 Fe(CN) 5 .N0.2H 2 in 600
mL H 2 0. Add 1 mL Brij-35, dil. to 1 L with H 2 0, and mix.
(e) Nitrogen std solns. — Prep. 6 stds by accurately weigh-
ing (±10 mg) 59, 118, 177, 236, 295, and 354 mg (NH 4 ) 2 S0 4
primary std (Fisher Scientific Co. No. A-938, or equiv.; dry
2 hr at 105° before use and assume theoretical value of 21 .20%
N after drying) into individual 250 mL digestion tubes. Pro-
ceed as in 976.06G, beginning "Add 9 g K 2 S0 4 , 0.42 g HgO,
and 15 mL H 2 S0 4 ..." Stds may be stored and reused until
exhausted.
(f) Sodium hydroxide -potassium sulfide soln. — Dissolve 400
g NaOH in H 2 6. While still warm, dissolve 30 g K 2 S in soln,
and dil. to 1 L.
D. Analytical System
If manifold is to be constructed, use clear std pump tubes
for all air and soln flows. All fittings, coils, and glass trans-
mission lines are A All type and size. Use glass transmission
tubing for all connections after pump to colorimeter. Construct
modified AO fitting on sample diln loop using AO fitting, N13
stainless steel nipple connector, and V2" length of 0.035" id
Tygon tubing. Insert N13 nipple approx. halfway into 0.035"
Tygon tubing. Insert tubing into side arm of AO fitting far
enough so resample line will not pump any air. Space pump
tubes equally across pump rollers. Cut 0. 16 mL/min resample
pump tube <1" at entrance before connecting to side arm of
AO fitting. In operation, add buffer and hypochlorite solns thru
metal side arms of A 10 type fittings; add salicylate soln, (d),
thru metal insert to 20T coil. Air, reagents, and sample are
combined immediately after pump thru injection fittings.
E. Start-up
Start automatic system and place all lines except salicylate
line in resp. solns. After >5 min, place salicylate line in resp.
soln and let system equilibrate. If ppt forms after addn of sal-
icylate, pH is too low. Immediately stop proportioning pump
and flush coils with H 2 0, using syringe. Before restarting sys-
tem, check concns of NaCl-H 2 S0 4 soln and phosphate-tartrate
buffer soln.
Pump lowest concn N std soln continuously thru system >5
min and adjust baseline control on colorimeter to read 10%
full scale. Pump highest concn N std soln continuously thru
system until no drift exists (usually ^10 min) and adjust "std.
cal." control to read 85% full scale. Recorder tracings must
be stable and show <0.3 division noise. If noisy conditions
exist, replace dialyzer membrane. When recorder tracing in-
dicates stable condition, immediately start sampling.
F. Shut-Down
Place reagent lines in H 2 0, removing salicylate line first.
Let system wash out £20 min.
G. Colorimetric Determination
(Caution: See safety notes on mercury.)
Weigh samples (See Table 976.06) into dry digestion tubes.
Add 9 g K 2 S0 4 , 0.42 g HgO, and 15 mL H 2 S0 4 to each tube.
Table 976.06 Sample Weight
Protein, %
Sample, g
6-24
1.5±0.1
25-40
1.0±0.1
41-50
0.8±0.1
51-60
0.7±0.1
61-90
0.5±0.01
>90
Weigh sample equiv.
to 50 mg N
74
Animal Feed
AOAC Official Methods of Analysis (1990)
(Calibrated metal scoops may be used for solids.) Insert tubes
into digestor block preheated to 410°, place shields around tubes,
and digest 45 min.
After digestion, remove rack of tubes from block, place in
hood, and let cool 8-10 min, (Time depends upon air flow
around tubes.) Direct rapid spray of H 2 6 (kitchen sink dish
rinsing sprayer works well) to bottom of each tube to dissolve
acid digest completely. If ppt forms, place tube in ultrasonic
bath to aid in redissolving salt. Let cool, dil. to vol., and mix
thoroly, Transfer portion of each sample soln to Auto Analyzer
beaker.
Place stds in tray in increasing order of concn, followed by
group of samples. Analyze lowest concn std in duplicate, dis-
carding first peak. Precede and follow each group of samples
with std ref, curve to correct for possible drift. Analyze stds
and samples at rate of 40/hr, 2/1 sample-to-wash ratio. Prep,
std curve by averaging peak hts of first and second set of stds.
Plot av. peak ht stds against N concn contained in each 250
mL tube.
% Protein = |(mg N/250 mL from graph)
x 6.25 x 100]/mg sample
H. Titrimetric Determination
Digest as in 976. 06G. Cool 5 min and add only enough H 2
to dissolve salts (70—75 mL). Cool and attach digestion tube
to distn head according to manufacturer's directions. Place re-
ceiver flask contg 25 mL std acid, 936.15A or 890.01A, and
5-1 drops Me red indicator on platform. Condenser tip must
be below surface of std acid soln. Add 50 mL NaOH-K 2 S soln
to tube and steam distil vigorously until 125 mL distillate col-
lects. Titr. excess acid with std O.LVNaOH soln, 936.16. Cor-
rect for reagent blank.
%N = [(mL std acid x normality acid) — (mL std NaOH
X normality NaOH)] x 1.4007/g sample
% crude protein ^ % N x 6.25
Refs.: JAOAC59, 134(1976); 62, 290(1979).
984.13 Protein (Crude) in Animal Feed
Copper Catalyst Kjeldahl Method
First Action 1984
(Caution: See safety notes on sulfuric acid and sodium
hydroxide.)
A. Principle
Sample is digested in M 2 S0 4 , using CuS0 4 as catalyst, con-
verting N to NH 3 which is distd and titrd.
B. Reagents
(a) Sodium hydroxide, — Pellets, flakes, or soln witn sp. gr.
>1.36, low N. Dissolve ca 450 g NaOH in H 2 0, cool, dil. to
1 L.
(b) Alundum. — Boiling stones, 8—14 mesh (Thomas Sci-
entific Co., No. 1590-D18).
(c) Methyl red indicator. — Dissolve 1 g Me red (Na salt)
in 100 mLMeOH.
(d) Hydrochloric or sulfuric acid std soln. — 0.5N, Prep, as
in 936.15 or 890.01.
(e) Sodium hydroxide std soln. — 0.1N, Prep, as in 936.16.
After stdzg both acid and base by methods suggested in (d)
and (e), also check one against the other. In addn, check entire
method by analyzing N1ST Std Ref. Material No. 194,
NH 4 H 2 P0 4 , certified 12.15% N, and high purity lysine.HCJ.
C. Apparatus
(a) Digestion.— Uss Kjeldahl flasks with capacity of 500-
800 mL.
(b) Distillation.— -Use digestion flask (Corning Glass Works,
orequiv.) connected to distn trap by rubber stopper. Distn trap
is connected to condenser with Jow-S tubing. Outlet of con-
denser tube should be <4 mm diam.
D. Determination
Weigh 0.250-1.000 g sample into digestion flask. Add 15
g K,S0 4 , 0.04 g anhyd, CuS0 4 , 0.5-1.0 g alundum granules,
and 20 mL H 2 S0 4 . (Add addnl 1.0 mL H 2 S0 4 for each 0.1 g
fat or 0.2 g other org. matter if sample wt is >1 g.)
Include at least one sample of high purity lysine, HC1 in each
day's run as check of correctness of digestion parameters. If
recovery is not complete, make appropriate adjustments.
Heat flask at 5 -min boil rate (burner preheated and adjusted
to bring 250 mL H 2 at 25 D to rolling boil in 5 min) until dense
white fumes clear bulb of flask, swirl gently, continue heating
addnl 90 min. (Note: Reagent proportions, heat input, and
digestion time are critical factors— do not change.) Cool, cau-
tiously add 250 mL H 2 0, and cool to room temp. (Note: If
bumping occurs daring distn, vol. of H 2 may he increased
to ca 275 mL.)
Prep, titrn beaker by adding accurately measured appropri-
ate vol. std acid soln to amt of H 2 such that condenser tip
will be sufficiently immersed. Add 3-4 drops indicator soln
(c).
Add 2-3 drops of tributyl citrate to digestion flask to reduce
foaming; add another 0.5-1 ,0 g alundum granules. Slowly down
side of flask, add sufficient NaOH soln (a) such that mixt. will
be strongly alk. Immediately connect flask to distn app., mix
completely, and distil at ca 7.5-min boil rate until ^.1.50 mL
distillate is collected in titrn beaker.
Titr. excess std acid in distillate with std NaOH soln. Cor-
rect for blank detn on reagents. Calc. % N:
% N = [(N add )(mL, dd ) - (mL w )(N Na0H )
- (mL NaOH )(N NaO M)][1400.67]/mg sample
where mL NaOH ™ mL std base needed, to titr sample; mL acid —
mL std acid used for that sample; mL bk = mL std base needed
to titr. 1 mL std acid minus mL std base needed to titr. reagent
blank carried thru method and distd into 1 mL std acid; N acid
= normality of std acid; N basc = normality of std base. Calc.
% crude protein, defined as 6,25 x % nitrogen, or 5.7 X %
nitrogen for wheat grains.
Ref.: J AOAC 67, 869(1984).
989.03 Fiber (Acid Detergent)
and Protein (Crude)
in Animal Feed and Forages
Near-Infrared Reflectance Spectroscopic Method
First Action 1989
(Generally applicable to detn of acid-detergent fiber and crude
protein in any forage or feed sample)
Method Performance:
Crude protein:
s R = 0.15; RSD R = 0.42%
Ac id -detergent fiber:
s R - 0.34; RSD R - 1.14%
Successful use of method is based on obtaining suitable cal-
ibration for instrument by selecting learning set of samples and
AOAC Official Methods of Analysis (1990)
Protein
75
performing calibration described in Determination (b). Four
rules to be followed in calibration of instrument are stated in
US DA Agriculture Handbook No. 643, p. 45 ("Near Infrared
Reflectance Spectroscopy (NIRS): Analysis of Forage Qual-
ity." U.S. Dep. Agric. Handb. 643, U.S. Government Print-
ing Office, Washington, DC, 96 pp.)- In brief, rules are as
follows:
(7) Be certain calibration samples adequately represent pop-
ulation to be analyzed.
(2) Conduct accurate laboratory analyses on calibration
samples. This step cannot be overemphasized.
(3) Select appropriate data processing technique to extract
pertinent information from spectra.
(4) Select correct wavelengths.
It may be necessary to periodically update a calibration. An-
alyst must monitor results of method to ascertain when this is
necessary. Test sample is usually run each day and its ana-
lytical value is detd periodically by laboratory analysis method.
Accuracy of any chemometric procedure is limited only by
validity of laboratory method used to measure desired quality
parameter for all samples and selection of appropriate learning
set.
A. Principle
Random portions of prepd sample are loaded into sample
holder of NIR spectrometer. Instrument is part of system that
has been calibrated using representative samples from popu-
lation to be tested. Equations selected from calibration statis-
tics, which have been validated, are used to calc. acid-deter-
gent fiber and crude protein content of feed and forage samples.
B. Apparatus
(a) Wavelength-scanning instrument, — Model 6100 or 6350
grating monochromator (Pacific Scientific Corp., Gardner/
Neotec Instrument Div., Silver Spring, MD 20910), or equiv.
Monochromator is described in detail in (I) Landa, I. Rev.
Sci. Instrum. 50, 34-40(1979); and (2) Landa, I., & Norris,
K.H. Appl. Spectrosc. 23, 105-107(1979).
(b) Computer. — PDP 1 1 Series computer equipped with 64
K bytes of main memory; dual RX02 double-density floppy
disks; RL01 5-megabyte or RL02 10-megabyte hard disks. PDP
system software RT-1 1 V5.0 or later (Digital Equipment Corp. ,
Nashua, NH 03061), or equiv.
(c) USD A public software. — Software is described in detail
in USDA Agriculture Handbook No. 643. Software consists
of 14 programs written in FORTRAN IV to collect, store, and
process NIRS data. Repository for software: U.S. Department
of Agriculture, Richard B. Russell Agricultural Research Cen-
ter, Plant Structure and Composition Research Unit, PO Box
5677, Athens, GA 30613. Commercial software is available
from several vendors.
(d) Mill. — Tecator cyclone sample mill with 1 mm screen
(Fisher Scientific Co.), or equiv. Periodically change grinding
ring to ensure consistency of particle size over time.
(e) NIRS sample holder. — Nylon, 2.5 cm diam., 1 cm thick,
with IR trans mittance quartz window. Sample capacity 0.75-
1.75 g. Sample is held in place with sep. back made of rubber
or foam core (Pacific Scientific Corp., Gardner /Neotec In-
strument Div.), or equiv.
(f) Sample storage container . — For maintaining const
moisture concn in samples. For best results, use Poly Kraft
Bags-Mil-B-121 Type II, Grade A, Class I. Place sample in
bag and heat-seal (EDCO Supply Corp., Brooklyn, NY 11232,
or equiv.).
C. instrument Operation
(a) Start-up. — For best results, run instrument continu-
ously. If instrument is cold, warm-up time should be >15 min
and may require 1 h.
(b) Monochromator diagnostic tests. — (/) Instrument
noise. — Scan ceramic ref. to itself. Collect 25 repetitions of
64 scans. Express deviations from zero as av. deviation (bias),
and as root mean sq (RMS), expressed as log (1/R)/10 6 , where
R = reflectance. Bias indicates any systematic change in log
(1/R) level of scans taken over time. Bias values that are all
pos. or all neg. indicate problem with instrument. RMS value
can range from low of 10 to high of 50 without affecting anal-
ysis. Monochromators should have av. noise level below 30
RMS over 100 scans.
(2) Wavelength accuracy. — Use clear polystyrene petri dish
to measure wavelength repeatability and accuracy. Place petri
dish in light beam and then pull out sample drawer to expose
ceramic std. Ref. this scan to measurements without petri dish.
Locate and compare major styrene peaks with known locations
at 1680.3, 2164.9, and 2304.2 nm. Repeatability std dev. should
be <0.05 nm, and accuracy from known location should
be <0.5 nm. Large pos. values for wavelength accuracy and
repeatability usually indicate mech. problems in monochro-
mator.
(c) Maintenance. — Whenever dust accumulates, use vac-
uum, brush, or soft tissue to clean ceramic std, all parts of
drawer assembly, and windows above and below detector. In-
strument operation is described in detail in USDA Agriculture
Handbook No. 643 and in Shenk, J.S., Westerhaus, M.O., &
Hoover, M.R. Proc. Am. Soc. Agric. Eng. (1978), p. 242.
D. Determination
(a) Preparation of sample. — Grind samples for NIRS anal-
ysis in cyclone mill through 1 mm screen. Clean mill between
samples to minimize cross-contamination. Prior to grinding,
dry samples contg >25% moisture in 60° air oven for 24 h.
Mix milled samples well, and place random portion in sample
holder. Continue to add random portions until NIRS sample
holder is 2 / 3 full. Press back into holder until it is tight and
level. As check, invert holder and make certain sample is firmly
pressed against window. If any abnormality is apparent, re-
move back and repeat procedure. Consistency in sample han-
dling and prepn is crucial to successful use of NIRS technique.
(b) Calibration. — To calibrate system for acid -detergent fi-
ber and crude protein detns, randomly select samples that are
representative of population to be analyzed, using either finite
or infinite population. Finite population has defined boundaries
set by analyst which limit population; infinite population has
no such boundaries. Select sufficient number of samples to
represent range of acid-detergent fiber and crude protein concns
and all other variables that may affect chem. and physical
composition of feed or forage (stage of growth, species, pres-
ervation method, etc.). In practical terms, min. of 50 samples
should be considered.
Collect reflectance (R) measurements (log 1 /R) of calibra-
tion samples with program SCAN, Apparatus (c), at 2.0 nm
intervals from 1100 to 2500 nm. Develop multiterm calibra-
tion equations by multiple linear regression of reflectance mea-
surements to acid-detergent fiber and crude protein concns us-
ing program CAL (c). Before regression statistics are evaluated,
examine differences (residuals) between NIRS data and ref.
method data for samples with large /-values. Large pos. or
neg. /-value indicates that residual is 2.5 times std error of
difference between NIRS detn and ref. method detn, and that
laboratory values from ref. method were inaccurate or did not
represent samples at time scan was taken (i.e., subsampling
error). Reanalyze these samples by ref. method.
In addn, evaluate output for samples that have large H-val-
ues. Large H- statistic (>3) indicates that NIRS spectra used
in calibration for that sample differ substantially from NIRS
spectra of other samples. Calcn of H was mathematically de-
rived from co variance matrix according to formula: H =
76
Animal Feed
AOAC Official Methods of Analysis (1990)
X(s 1 x)~ 1 x f [Landa. Apparatus (a)]. High value on diagonal of
H matrix indicates sample that is dissimilar to calibration set
at wavelengths used in equation. Rescan such samples. If 2
scans agree and sample belongs in population, then retain sam-
ple. If scans disagree, then first scan was mistake and should
be discarded.
Examine std error of calibration (SEC) to det. fit of cali-
bration samples: the lower the SEC, the better the fit. Select
equation with SEC about 2 times laboratory repeatability std
dev. for acid-detergent fiber and crude protein ref. method.
Examine coeff. of detn (R 2 ) to det. proportion of variation in
ref. method values among samples explained by NIRS regres-
sion equation. Low R 2 values often indicate that laboratory
data from ref. method are imprecise. If laboratory repeatability
error from ref. method is l U of std dev., select equation with
R 2 >:(X75>
Examine F-statistic of regression coeffs. High F-values in-
dicate that regression coeff. is significantly different from zero;
small ^-values indicate that coeff. contributes little to equation
except to fit random errors. Probability that observed F-value
was obtained solely through chance does not follow std F ta-
bles because F is selected as max. of all wavelength combi-
nations considered. As number of choices increases, large F-
values are needed to signify coeff. fitting more than just ran-
dom errors. Reject equations with F- values <10.
(c) Validation. — Conduct NIRS analysis (program PRE) with
equations selected from calibration statistics on population of
unknown samples. Examine NIRS data for samples with larger
H- values. Large H- value (>3.0) for a few samples indicates
that their NIR spectra are different from spectra of calibration
population. Large lvalue (2.5 times SED) for a few samples
indicates that laboratory values from ref. method were inac-
curate or did not represent samples at time scan was taken. If
many validation samples have large t- and H-values, over- fit-
ting has occurred, and equation is specific to samples in cal-
ibration set. Next, use validation statistics from program STAT
to examine std error of analysis (SEA) by NIRS of chem, com-
position of validation samples. SEA is true indication of per-
formance of equation on unknown samples. Select equation
with lowest bias and SEA. Unlike SEC, which must decrease
with each addnl term, SEA only decreases with addnl terms
until over- fitting becomes important and forces it to increase.
Best equation for routine NIRS analysis is based on both su-
perior calibration and validation statistics.
Ref.: JAOAC71, 1162(1988).
941.04 Urea and Ammoniacal Nitrogen
in Animal Feed
Urease Method
Final Action
A. Reagents
(a) Defoaming soln. — Dow Corning Corp. Antifoam B
Emulsion.
(b) Urease soln. — Prep, fresh soln by dissolving stdzd urease
in H 2 so that each 10 mL neutzd soln will convert N of >0. 1
g pure urea.
Standardization, — To det. alky of com. urease prepn dis-
solve 0.1 g in 50 mL H 2 and titr. with O.LV HC1, using Me
red, 984.13B(c). Add same vol. O.LV HC1 to each 0.1 g urease
in prepg urease soln. To det. enzyme activity, prep, ca 50 mL
neutzd 1% soln. Add different amts of soln to 0.1 g samples
pure urea and follow with enzyme digestion and distn as in
detn. Calc. activity of urease prepn from amt of this urease
soln that completely converted urea, as detd by complete re-
covery of N by distn.
(c) Calcium chloride soln. — Dissolve 25 g CaCl 2 in 100 mL
H 2 0.
B. Determination
Place 2 g sample in Kjeldahl flask with ca 250 mL H 2 0.
Add 10 mL urease soln, stopper tightly, and let stand 1 hr at
room temp, or 20 min at 40°. Cool to room temp, if necessary.
Use addnl urease soln if feed contains >5% urea (ca 12% pro-
tein equiv.). Rinse stopper and neck with few mL H 2 0. Add
>2 g MgO (heavy type), 5 mL CaCl 2 soln, and 3 mL de-
foaming soln, and connect flask with condenser by Kjeldahl
connecting bulb. Distil 100 mL into measured vol. std acid,
936.15 or 890.01, and titr. with std alkali, 936.16, using Me
red, 984.13B(c).
Refs.: JAOAC 24, 867(1941); 25, 874(1942); 27, 494(1944).
967.07 Urea in Animal Feed
Coiorimetric Method
First Action 1967
Final Action 1970
(Applicable to animal feeds and their ingredients)
A. Apparatus
Spectrophotometer . — Instrument with max. band width 2.4
nm at 420 nm, with 1 cm cells.
B. Reagents
(a) p-Dimethylaminobenzaldehyde (DMAB) soln. — Dis-
solve 16.00 g (Eastman Kodak Co. No. 95 only) in 1 L alcohol
and add 100 mL HC1. Stable 1 month. Prep, new std curve
with each new batch of reagent.
(b) Zinc acetate soln. — Dissolve 22. g Zn(OAc) 2 .2H 2 in
H 2 0, add 3 mL HOAc, and dil. to 100 mL.
(c) Potassium ferrocyanide soln. — Dissolve 10.6 g
K 4 Fe(CN) 6 .3H 2 in H 2 and dil. to 100 mL.
(d) Vegetable charcoal. — Darco G-60.
(e) Phosphate buffer soln. — pH 7.0. Dissolve 3.403 g an-
hyd. KH 2 P0 4 and 4.355 g anhyd. K 2 HP0 4 sep. in ca 100 mL
portions freshly distd FLO. Combine solns and diL to 1 L with
H 2 0.
(f) Urea std solns. — (J) Stock soln. — 5 mg/mL. Dissolve
5.000 ± 0.001 g reagent grade urea in H 2 and dil. to 1 L
with H 2 0. (2) Working solns.— -0.2, 0.4, 0.6, 0.8, 1.0, 1.2,
1.4, 1.6, 1.8, and 2.0 mg urea/5 mL. Pipet 2, 4, 6, 8, 10,
12, 14, 16, 18, and 20 mL stock soln into 250 mL vol. flasks
and dil. to vol. with phosphate buffer. (3) Reference soln. —
Use std soln contg 1.0 mg urea/5 mL as ref. std. Store at
<24°. Stable 1 week.
C. Preparation of Standard Curve
Pipet 5 mL aliquots of working std solns into 20 x 150 mm
(25 mL) test tubes and add 5 mL DMAB soln to each. Prep,
reagent blank of 5 mL buffer soln and 5 mL DMAB soln.
Shake tubes thoroly and let stand 10 min in H 2 bath at 25°.
Read A in 1 cm cell at 420 nm with reagent blank at zero A.
Plot A against concn urea. Plot should be straight line; if not,
repeat, using new lot of DMAB .
D. Determination
Weigh LOO g ground sample into 500 mL vol. flask. Add
1 g charcoal, ca 250 mL H 2 0, 5 mL Zn(OAc) 2 soln, and 5
mL K 4 Fe(CN) 6 soln. Shake mech. 30 min and dil. to vol. with
H 2 0. Let stand until ppt settles. Decant thru Whatman No. 40
paper and collect clear filtrate. Pipet 5 mL filtrate into test
tube, add 5 mL DMAB soln, and shake thoroly. Include ref-
erence std (5 mL soln (f)(3) and 5 mL DMAB soln) and re-
AOAC Official Methods of Analysis (1990)
Nitrogen
77
agent blank with each group of samples. Let stand 10 min in
H 2 bath at 25°. Read A at 420 nm against reagent blank.
% Urea = (1.0 x A samp]c X I00)/O4 std X mg sample in aliquot)
Ret.: JAOAC 50, 56(1967).
CAS-57-13-6 (urea)
920.37* Nitrogen (Albuminoid)
in Animal Feed
Final Action
Surplus 1965
See 22.020-22.021, 10th ed.
920.38* Nitrogen (Amido)
in Animal Feed
Final Action
Surplus 1965
% Amido N = % Total N - % albuminoid N
968.07 Nitrogen (Nitrate and Nitrite)
in Animal Feed
Colorimetric Method
First Action 1968
Final Action 1970
A. Principle
Nitrate and nitrite are extd with Cd and Ba chloride soln.
Bulk of sol. proteins are pptd in aJk. soln and clarified soln is
passed thru metallic Cd column, reducing nitrate to nitrite. Ni-
trite is measured colorimetrically.
(Caution: Cd salts are toxic.)
B. Reagents and Apparatus
(a) Nitrate nitrogen std solns. — (I) Stock soln. — 12 |xg ni-
trate N/mL. Dissolve 0.867 g KN0 3 in 1 L H 2 0. Dil. 25 mL
to 250 mL with H 2 0. (2) Working solns. —0.6, 1.2, 1.8, 2.4,
3.0 Ltg N/mL. Dil. 5, 10, 15, 20, and 25 mL stock soln to
100 mL with H 2 0.
(b) Extracting soln. — Dissolve and dil. 50 g CdCl 2 and 50
g BaCl 2 to 1 L with H 2 0. Adjust to pH 1 with HC1.
(c) Ammonium chloride buffer soln. — pH 9.6. Dissolve 50
g NH 4 C1 in 500 mL H 2 and adjust pH with NH 4 OH. Dil. to
1 L with H 2 0.
(d) Sodium hydroxide soln. — 2.5/V. Dissolve 50 g NaOH
in 500 mL H 2 CX
(e) Sulfanilamide soln. — 0.5%. Dissolve 1.25 g sulfanila-
mide in 250 mL HC1 (1 + 1). Soln is stable 1-2 months.
(f) Coupling reagent. — Dissolve 0.5 g 7V-(l-naph-
thyl)ethylenedi amine. HO in 100 mL H 2 0. Store in g-s dark
bottle in refrigerator. Soln is stable several weeks.
(g) Salt soln.— Dissolve 100 g NaCl in 500 mL H 2 0. Add
50 mL buffer soJn, (c), and dil. to 1 L with H 2 0.
(h) Reduction tube. — 25 mL buret or equiv. id chromatgc
tube with stopcock and reservoir (Kontes Glass Co. Cat. No.
K-420280 or Lurex Scientific Cat. No. JC-1506).
C. Preparation of Columns
Prep, supply of metallic Cd by placing Zn rods into 500 mL
20% CdS0 4 soln. After reaction for 3 hr, discard soln and
scrape moss- 1 ike Cd growth from Zn rods. Place Cd in high-
speed blender, add 500 mL H 2 0, and blend 2 sec. Wash fine
metal particles with H 2 onto sieves, collecting only 20™40
mesh size. Fill reduction tube with H 2 and add 2 cm plug of
glass wool. Press any trapped air from glass wool as it is pushed
to bottom of column with glass rod. Add Cd to depth of 10
cm, using min. of very gentle tapping. Wash column with 25
mL 0.107V HC1, two 25 mL portions H 2 0, and finally 25 mL
buffer, (c), dild 1 + 9. Keep column covered with salt soln,
(g), when not in use.
Normally columns can be used repeatedly if kept under salt
soln between analyses. When succession of highly proteina-
ceous or other sol. org. contg samples are treated, flow rate
may decrease gradually. Repeating 25 mL 0.1 0A^ HC1 treat-
ment may restore original flow rate; if not, prep, new column.
Reproducible flow rate is important. Actual rate can be 3-5
mL/min but once established, it must be identical (±0.1 mL)
for samples and stds.
D. Preparation of Standard Curve
Prep, std curve of 3, 6, 9, 12, and 15 \xg nitrate- nitrite N
by pipeting 5.0 mL aliquots of working std solns into 30 mL
beakers. Add 5 mL buffer soln, (c), and 1.5 mL H 2 0, mix
well, and transfer quant, to reduction column, using min. H 2 0.
Adjust flow rate thru column to 3-5 mL/min. Just as reservoir
empties, add 15 mL salt soln, (g). Collect eluate, including
salt wash, in 50 mL vol. flask (total vol. of eluate should be
ca 40 mL). Add 5 mL sulfanilamide soln, (e), mix, and let
stand 3 min. Add 2 mL coupling reagent, (f), mix, dil. to vol.
with H 2 0, mix, and let stand 20 min for max. color devel-
opment. Color is stable ^2 hr. Det. A in 1 cm cells at 540
nm against reagent blank. Plot A against |xg nitrate-nitrite N.
E. Extraction
(a) Low level nitrate samples (grains, meals, supplements,
etc.). — Wash 5.0 g finely ground sample into 250 mL vol.
flask. Add 100 mL extg soln, (b), and 100 mL H 2 0, and mix.
Let stand 1 hr with occasional swirling. Add 20 mL 2.5A NaOH,
dil. to vol. with H 2 0, mix, and filter immediately thru rapid
paper. Pipet 10 mL buffer soln, (c), into 100 mL vol. flask,
dil. to vol. with clear filtrate, and mix.
(b) Dry, high level nitrate products (dried plants, hays,
meals, etc.). — Weigh 5.0 g finely ground sample into 500 mL
vol. flask. Add 100 mL extg soln, (b), and 300 mL H 2 0, and
mix. Let stand 1 hr with occasional swirling, add 40 mL 2.5N
NaOH, dil. to vol. with H 2 0, mix, and filter immediately thru
rapid paper. Pipet 10 mL buffer soln, (c), into 100 mL vol.
flask, dil. to vol. with clear filtrate, and mix.
(c) Grasses, silages, and other wet materials. — Weigh 100
g sample into I gal. capacity high-speed blender. Add 100 mL
extg soln, (b), and 800 mL H 2 0, including vol. H 2 contrib-
uted by sample as detd in 934.01 or 925. 04B. Homogenize 1
min, pour into 2 L beaker, and let stand 1 hr. Add 100 mL
buffer soln, (c) (total vol. 1 L), mix well, and filter thru What-
man No. 42 paper, collecting portion of clear filtrate.
F. Determination
(a) Nitrate plus nitrite nitrogen. — Pipet 25 mL buffered
sample exts, 968.07E(a) or (b), or 5 mL ext, (c), into reduc-
tion column and treat as in 968. 07D, beginning, "Adjust flow
rate thru column ..." Rinse column with 30 mL H 2 be-
tween samples to remove NaCI. Use portion of buffered sam-
ple exts with equiv. diln and pH as ref. soln in detg A at 540
nm. Also det. nitrate- nitrite in reagents and correct for this
blank value. Calc. total nitrate- nitrite N from std curve.
(b) Nitrite nitrogen. — Pipet aliquot clear sample filtrate
(contg <15 fxg nitrite) into 50 mL vol. flask and dil. with H 2
to ca 40 mL. Mix well, add 5 mL sulfanilamide soln, (e), mix,
and let stand 3 min. Add 2 mL coupling reagent, (f), and dil.
78
Animal Feed
AOAC Official Methods of Analysis (1990)
to vol. with H 2 0. Mix we J J and let stand 20 min for max. color
development. Measure A in 1 cm cells against sample ext with
equiv. diln at 540 nm. Correct for nitrite reagent blank.
(c) Nitrate nitrogen. — Calc. by difference between (a) and
(b) above.
G. Calculation
ppra NO r N and/or NO3-N
— fxg N0 3 -N found X diln factor/g sample
Diln factors for exts: 968.07E(a), 11.1; (b), 22.2; (c), 200.
Ref.: JAOAC 51, 763(1968).
971.09 Pepsin Digestibility
of Animal Protein Feeds
Filtration Method
First Action 1971
Final Action 1973
A. Principle
Defatted sample is digested 16 hr with warm soln of pepsin
under const agitation. Insol. residue is isolated by filtering,
washed, dried, and weighed to det. % residue. Residue is ex-
amined microscopically and analyzed for protein. Filtration
method is applicable to all animal proteins. Methods are not
applicable to vegetable proteins or mixed feeds because of
presence of complex carbohydrates and other compds not di-
gested by pepsin.
B. Apparatus
(a) Agitator. — See Fig. 971.09. Continuous, slow speed (1.5
rpm), end-over-end type, to operate inside incubator at 45 ±
2° and carry 8 oz screw-cap prescription bottles, or equiv. Ag-
itator and bottles available from D. E. Sims, 716 Forrest Ave,
Quincy, IL 62301. Stirring or reciprocating (shaking) type ag-
itator cannot be used because solid particles collect on sides
of bottle and do not contact pepsin soln. If heat from agitator
motor raises incubator temp, to >45°, mount motor outside
incubator by drilling hole thru side of incubator and connecting
motor to agitator with extension shaft and coupling (available
from agitator supplier). (Caution: See safety notes on electri-
cal equipment.)
(b) Settling rack. — Wood or metal to hold digestion bottles
at 45° angle. May be made from 2 boards nailed horizontally
FIG. 971. 09— Agitator
into "V" cut into vertical end pieces. Also available from ag-
itator supplier, (a).
(c) Filtering device. — Modified California buchner,
962.09C(d), available from Labconco Corp., 8811 Prospect
Ave, Kansas City, MO 64132, No. 55100. (If edge of screen
is rough, smooth with small -tip soldering iron.) Use with re-
tainer sleeve, 2 X 2.75" od stainless steel tube, available from
agitator supplier, (a).
(d) Glass fiber filter. — 7 cm, Whatman, Inc., No. 934-AH,
or equiv.
(e) Moisture dishes. — Al, 78 mm od X 20 mm, with out-
side cover and vertical sides (Curtin Matheson Scientific, Inc.,
No. 19370-30, or equiv.).
C. Reagent
Pepsin soln. — 0.2% pepsin (activity 1:10,000) in 0.075/V
HC1; do not use pepsin NF or pepsin of activity other than
1 : 10,000. Prep, just before use by dilg 6.1 m'L HCI to 1 L
and heating to 42-45°. Add pepsin and stir gently until dis-
solved. Do not heat pepsin soln on hot plate or overheat.
D. Preparation of Sample
Sieve sample, 965.16, thru No. 20 sieve. Grind portion re-
tained on sieve to pass No. 20 sieve. Combine both portions
and blend by stirring and shaking in pt (500 mL) jar. Thoro
blending is essential. Because of high fat content of many an-
imal products, grinding without sieving may cause sticking in
mill, loss of moisture or fat, or poorly blended sample.
E. Extraction
{Caution: See safety notes on distillation, flammable solvents,
and diethyl ether.)
Prep, extn thimble from 1 1 cm Whatman No. 2 paper, or
equiv., as follows: Fold paper in half; straighten paper and
refold at right angles to first fold; turn paper over and repeat
process with folds at 45° to original fold; while holding creased
paper in one hand, place short test tube (6-8 mm smaller in
diam. than extractor sample holder or cup in which thimble is
to be used) at its center; fold along natural crease lines to form
4-pointed star around tube; and wrap points in same direction
around tube to complete thimble.
Weigh 1.000 g ground sample (0.500 g of poultry byprod-
ucts or hydrolyzed feathers because of gummy nature and amt
of residue) into thimble and ext I hr with ether at condensation
rate of 3-4 drops /sec. (If Soxhlet is used, top of thimble should
extend above siphon tube to avoid loss of solid particles. If
paper contg sample is totally submerged in siphon cup, sample
must be completely wrapped in paper.) Observe ether ext to
det. that no solid particles were carried into sol v. For approx.
fat content detn, evap. ether, and dry and weigh residue. Re-
move paper from sample container or cup and let dry at room
temp. Unfold, and quant, brush defatted sample into digestion
bottle, avoiding contamination by brush bristles or filter paper
fibers. Use of powder funnel is helpful to avoid loss.
F. Pepsin Digestion
To defatted sample in agitator bottle add 150 mL freshly
prepd pepsin soln pre warmed to 42-45°. Be sure sample is
completely wetted by pepsin soln. Stopper bottle, clamp in
agitator, and incubate with const agitation 16 hr at 45°.
G. Treatment of Residue
Dry individual sheets of glass fiber filter, (d), 30 min at 1 10°
in moisture dishes with cover open. Cool in desiccator 30 min
with cover closed, and weigh (Wj).
Remove bottles from agitator. Place in 45° angle settling
rack and loosen caps. Let residue settle > 15 min. Place weighed
filter in California buchner, (c), apply suction, and moisten
AOAC Official Methods of Analysis (1990)
Fat
79
with fLO. Place retainer sleeve on filter and press down gently.
Rinse particles of residue on cap onto filter with small amt
H 2 0. Carry bottle from rack to filter at same angle as settled
and slowly pour contents thru filter as continuous small stream,
avoiding all unnecessary agitation. Liq. passes thru paper as
rapidly as poured, with residue spreading over surface of filter
but not covering it completely until all or practically all of liq.
has passed thru. If filtration rate becomes slow, it may be ac-
celerated by adding acetone washes described below, but only
if no significant amt of digestion mixt. remains on funnel when
acetone is added. (Filtration (passage of aq. mixt. thru filter)
should be complete within 1 min with most proteins.) After
supernate has passed thru filter, quant, transfer residue onto
filter as follows:
Add 15 mL acetone to bottle. Hold thumb over bottle neck
and shake vigorously. Release pressure, replace thumb over
bottle neck, and shake bottle in inverted position over filter.
Remove thumb, letting acetone and residue discharge onto fil-
ter. Repeat rinse with second 15 mL portion acetone, shaking
and releasing pressure as above. Inspect bottle, and rinse fur-
ther with acetone, using policeman, if necessary. If >3 mm
liq. remains on paper when acetone washes are started, it may
be necessary to use three 15 mL acetone washes instead of 2
to increase filtration rate.
After all liq. passes thru funnel, wash residue and inside
surface of retainer sleeve with 2 small portions acetone from
wash bottle or hypodermic syringe, and suck dry. Remove re-
tainer sleeve from funnel. Transfer filter to original moisture
dish. Scrape or brush any residue particles or filter clinging to
retainer sleeve or funnel onto filter in moisture dish. Dry in
oven, cool, and weigh as before (W 2 ). Calc. % indigestible
residue = (W 2 ~~ W,) x 100/g sample.
Det. indigestible protein by transferring filter contg residue
directly to Kjeldahl flask. Proceed as in 954.01. (Caution: Vi-
olent reaction may take place when NaOH is mixed with dild
digestion mixt., caused by large excess H 2 S0 4 due to small
amt org. material from residue and none from glass filter. Avoid
by thoroly mixing and cooling digestion mixt. before addn of
NaOH or by using 20 mL H 2 S0 4 in Kjeldahl digestion instead
of 25 mL.) Make blank detn on 1 sheet of glass filter and
subtract from each sample detn, if necessary. Calc. % protein
based on original sample wt. Result represents % indigestible
protein in sample. Convert to % crude protein content of sam-
ple not digested, "protein indigestible" = % indigestible pro-
tein in sample x 100/% total crude protein in sample.
Refs.: J. Agric. Food Chem. 3, 159(1955). JAOAC 40,
606(1957); 41, 233(1958); 42, 231(1959); 43, 320
(1960); 54, 669(1971); 55, 702(1972).
920.39 Fat (Crude) or Ether Extract
in Animal Feed
Final Action
Use method 920.39A or 920.39C for mixed feeds other than
(/) baked and/or expanded, (2) dried milk products, or (3)
contg urea.
A. Indirect Method
Det. moisture as in 934.01 or 920.36*; then ext dried sub-
stance as in 920. 39C, and dry again. Report loss in wt as ether
ext.
Direct Method
B. Reagent
Anhydrous ether. — Wash com. ether with 2 or 3 portions
fLO, add solid NaOH or KOH, and let stand until most of
H 2 is abstracted from the ether. Decant into dry bottle, add
small pieces of carefully cleaned metallic Na, and let stand
until H evolution ceases. Keep ether, thus dehydrated, over
metallic Na in loosely stoppered bottles. (Caution: See safety
notes on sodium metal and diethyl ether.)
C. Determination
(Large amts H 2 OsoL components such as carbohydrates, urea,
lactic acid, glycerol, and others may interfere with extn of fat;
if present, ext 2 g sample on small paper in funnel with five
20 mL portions H 2 prior to drying for ether extn. Caution:
See safety notes on monitoring equipment, distillation, and
diethyl ether.)
Ext ca 2 g sample, dried as in 934.01 or 920.36*, with an-
hyd. ether. Use thimble with porosity permitting rapid passage
of ether. Extn period may vary from 4 hr at condensation rate
of 5-6 drops /sec to 16 hr at 2-3 drops /sec. Dry ext 30 min
at 100°, cool, and weigh.
Refs.: JAOAC 64, 351(1981); 65, 289(1982).
954.02 Fat (Crude) or Ether Extract
in Pet Food
Gravimetric Method
Final Action 1977
(To be used only on products which have been baked and /or
expanded, and on intermediate moisture pet foods. Not appli-
cable to canned, fresh, or frozen pet food. Such products should
be dried at 70-1 10°, then ground, and drying completed by
934.01 or 920.36* followed by 920.39A or 920.39C.)
(Caution: See safety notes on distillation, diethyl ether, and
petroleum ether.)
Place ca 2 g, accurately weighed, ground, well mixed sam-
ple in Mojonnier fat-extn tube, add 2 mL alcohol to prevent
lumping on addn of acid, and shake to moisten all particles.
Add 10 mL HC1 (25+11), mix well, and set tube 30-40 min
in H 2 bath at 70-80°, shaking frequently. Cool to room temp,
and add alcohol until liq. level rises into constricted portion
of Mojonnier tube.
Add 25 mL ether, stopper with glass, Neoprene, or good
quality rubber stopper thoroly cleaned with alcohol, and shake
vigorously 1 min. Carefully release pressure so that no sol v.
is lost. Wash adhering sol v. and fat from stopper back into
extn tube with few mL redistd pet ether (bp <60°). Add 25
mLredistd pet ether, stopper, and shake vigorously 1 min. Let
stand until upper liq. is practically clear or centrf. 20 min at
ca 600 rpm. Pour as much of ether-fat soln as possible thru
filter consisting of cotton pledget packed just firmly enough
in funnel stem to let ether pass freely into 1 50 mL beaker contg
several glass beads. Rinse lip of tube with few mL pet ether.
Re-ext liq. remaining in tube twice, each time with only 15
mL of each ether, shaking 1 min after addn of each ether. Pour
clear ether soln thru filter into same beaker as before, and wash
tip of tube, stopper, funnel, and end of funnel stem with few
mL of mixt. of 2 ethers (1 + 1). Evap. slowly on steam bath
under gentle stream of air or N. Continue heating on steam
bath 15 min after solv. has evapd; then cool to room temp.
Redissolve dried fat residue in four 10 mL portions Et ether,
filtering each portion thru small pledget of cotton into 100 mL
beaker, contg few glass beads, that has been predried 30 min
at 100°, cooled to room temp, in desiccator, and weighed im-
mediately. Use fifth 10 mL portion ether for rinsing cotton and
funnel. Evap. ether on steam bath, dry 90 min at 100°, cool
80
Animal Feed
AOAC Official Methods of Analysis (1990)
to room temp, in desiccator, and weigh immediately. Correct
this wt by blank detn on reagents used.
Refs.: JAOAC 37, 250(1954); 38, 225(1955); 59, 1218(1976);
60, 322(1977); 65, 456(1982).
932.02 Fat (Crude) or Ether Extract
in Dried Milk Products
Final Action
Proceed as in 932.06A(b) and 932.06B, using 8.5 mL H 2
and 1.5 mLNH 4 OH.
Refs.; JAOAC 15, 524(1932); 17, 190(1934); 18, 351(1935);
28, 80(1945).
948.04 Fat (Crude) or Acetone Extract
in Fish Meal
See 948.16 and 969.24.
962.09 Fiber (Crude) in Animal Feed
Ceramic Fiber Filter Method
Rrst Action 1962
Final Action 1971
Revised First Action 1982
AOCS-AOAC Method
A. Principle
Crude fiber is loss on ignition of dried residue remaining
after digestion of sample with 1.25% H 2 S0 4 and 1.25% NaOH
solns under specific conditions. Method is applicable to grains,
meals, flours, feeds, and fiber-bearing material from which fat
can be extd to leave workable residue.
B. Reagents
(a) Sulfuric acid soln,— 0.255 ± 0.005^. 1.25 g H 2 S0 4 /
100 mL. Concn must be checked by titrn.
(b) Sodium hydroxide soln.— 0,313 ± 0.0057V. 1.25 g
NaOH/ 100 mL, free, or nearly so, from Na 2 C0 3 . Concn must
be checked by titrn.
(c) Prepared ceramic fiber. — Place 60 g ceramic fiber
(Cerafiber, 8 Ib/cu ft, E. J. Bartell Co., 700 Powell Ave, S.W.,
Renton, WA 98055) in blender, add 800 mL H 2 0, and blend
1 min at low speed.
Det. blank by treating ca 2 g (dry wt) of prepd ceramic fiber
with acid and alkali as in detn. Correct crude fiber results for
any blank, which should be negligible (ca 2 mg).
(d) Alcohol.- — 95% or reagent alcohol, MeOH, or isopro-
panol.
(e) Antifoam. — Dow Corning Corp. Antifoam A compd dild
1+4 with mineral spirits or pet ether, or H 2 0-dild Antifoam
B Emulsion (1+4). Do not use Antifoam Spray.
(f) Bumping chips or granules.— -Broken Alundum cruci-
bles or equiv. granules (RR Alundum 90 mesh, Norton Co.,
1 New Bond St, Worcester, MA 01606) are satisfactory.
C. Apparatus
(a) Digestion apparatus, — With condenser to fit 600 mL
beaker, and hot plate adjustable to temp, that will bring 200
mL H 2 at 25° to rolling boil in 15±2 min. (Available from
Labconco Corp., 881 1 Prospect Ave, Kansas City, MO 64132.)
(b) Ashing dishes. — Silica, Vitreosil 70 X 16 mm; or por-
celain, Coors Ceramics Co., 600 9th St, Golden, CO 80401,
No. 60230, or equiv.
(c) Desiccator. — With efficient desiccant such as 4-8 mesh
Drierite (CaCl 2 is not satisfactory).
(d) Filtering device. — With No. 200 type 304 or 316 stain-
less steel screen (C-E Tyler, Inc., 3200 Bessemer City Rd,
Hwy 274, PO Box 8900, Gastonia, NC 28053), easily washed
free of digested residue. Either Oklahoma State filter screen
(see Fig. 962.09A; available from Labconco Corp.) or mod-
ified California plastic buchner. (See Fig. 962. 09B; consists
of 2 piece polypropylene plastic funnel manufactured by Nalge
Co., 75 Panorama Creek Dr, PO Box 20365, Rochester, NY
14602, Cat. No. 4280-0700, 70 mm (without No. 200 screen),
or equiv. (also available from Labconco Corp.). Seal screen
to filtering surface of funnel, using small-tip soldering iron.)
(e) Suction filter. — To accommodate filtering devices. At-
tach suction flask to trap in line with aspirator or other source
of vac. with valve to break vac.
(f) Liquid preheater . — For preheating H 2 0, 1 .25% H 2 S0 4 ,
and 1.25% NaOH solns to bp of H 2 0. Convenient system,
shown in Fig. 962. 09C, consists of sheet Cu tank with 3 coils
of 3 /s" (10 mm) od Cu tubing, 12.5' (3.8 m) long. Solder inlets
and outlets where tubing passes thru tank walls. Connect to
reflux condenser and fill with H 2 0. Keep H 2 boiling with
two 750 watt thermostatically controlled hot plates. Use Tygon
for inlet leads to reservoirs of H 2 0, acid, and alkali; use gum
rubber tubing for outlets. Capacity of preheater is adequate for
60 analyses in 8 hr.
D. Preparation of Sample
Reduce sample (riffle is suitable) to 100 g and place portion
in sealed container for H 2 detn. Immediately det. H 2 0. Grind
remainder to uniform fineness. (Weber mill (Sargent-Welch
Scientific Co. S-60870) with screen 0.033-0.040" (No. 18 or
20), Micro mill (Mikropul, Div. of Hosokawa Micron Inter-
national, Inc., 10 Chatham Rd, Summit, NJ 07901) with screen
V25-V16" (No. 18-No. 12), and Wiley mill with 1 mm (No.
18) screen give comparable fineness.) Since most materials
lose moisture during grinding, det. H 2 on ground sample at
same time sample is taken for crude fiber detn.
E Determination
Ext 2 g ground material with ether or pet ether (initial boil-
ing temp., 35-38°; dry-flask end point, 52-60°; >95% distg
200 mesh st. steel screei
20 go, st. steel back plate'
with 5/64 1 ' holes
FIG. 962.09A— Oklahoma State filter screen
AOAC Official Methods of Analysis (1990)
Fiber
81
FIG. 962.09B— Modified California State buchner funnel, 2-piece
polypropylene plastic, covered with 200-mesh screen, A,
heat-sealed to edge of filtering surface
<54°, and <60% distg <40°; sp gr at 60°F, 0.630-0.660; evapn
residue <0.002% by wt). If fat is <1%, extn may be omitted.
Transfer to 600 mL beaker, avoiding fiber contamination from
paper or brush. Add ca 1.5-2.0 g dry wt of prepd ceramic
fiber, 200 mL boiling 1 .25% H 2 S0 4 , and 1 drop dild antifoam.
(Excess antifoam may give high results; use only if necessary
to control foaming.) Bumping chips or granules may also be
added. Place beaker on digestion app. with preadjusted hot
plate and boil exactly 30 min, rotating beaker periodically to
keep solids from adhering to sides. Remove beaker, and filter
as in (a) or (b).
(a) Using Oklahoma filter screen . —Turn on suction and in-
sert screen (precoated with ceramic fiber if extremely tine ma-
terials are analyzed) into beaker, keeping face of screen just
under surface of liq. until all liq. is removed. Without breaking
suction or raising filter, add 50-75 mL boiling H 2 0. After
FIG. 962.09C— Continuous heater for distilled water, 1.25%
alkali, and 1.25% acid
wash is removed, repeat with three 50 mL washings. (Work
rapidly to keep mat from becoming dry.) Remove filter from
beaker and drain all H 2 from line by raising above trap level.
Return mat and residue to beaker by breaking suction and
blowing back. Add 200 mL boiling 1.25% NaOH and boil
exactly 30 min. Remove beaker, and filter as above. Without
breaking suction, wash with 25 mL boiling 1 .25% H 2 S0 4 and
three 50 mL portions boiling H 2 0. Drain free of excess H 2
by raising filter. Lower filter into beaker and wash with 25 mL
alcohol. Drain line, break suction, and remove mat by blowing
back thru filter screen into ashing dish. Proceed as in (c).
(b) Using California buchner. — Filter contents of beaker
thru buchner (precoated with ceramic fiber if extremely fine
materials are being analyzed), rinse beaker with 50-75 mL
boiling H 2 0, and wash thru buchner. Repeat with three 50 mL
portions H 2 0, and suck dry. Remove mat and residue by snap-
ping bottom of buchner against top while covering stem with
thumb or forefinger and replace in beaker. Add 200 mL boiling
1.25% NaOH and boil exactly 30 min. Remove beaker, and
filter as above. Wash with 25 mL boiling 1.25% H 2 S0 4 , three
50 mL portions H 2 0, and 25 mL alcohol. Remove mat and
residue; transfer to ashing dish.
(c) Treatment of residue. — Dry mat and residue 2 hr at
130 ±2°. Cool in desiccator and weigh. Ignite 30 min at
600±15°. Cool in desiccator and reweigh.
% Crude fiber in ground sample = C
= (Loss in wt on ignition — loss in wt of ceramic fiber blank)
x 100/wt sample
% Crude fiber on desired moisture basis
- C X (100 - % moisture desired)/
(100 — % moisture in ground sample)
Report results to 0,1%.
Refs.: JAOAC 42, 222(1959); 43, 335(1960); 44, 567(1961);
45, 578(1962); 65, 265(1982).
978.10 Fiber (Crude) in Animal Feed
Fritted Glass Crucible Method
First Action 1978
Final Action 1979
A. Principle
Principle is same as in 962. 09A, except sample is exposed
to min. vac. needed to regulate filtration, and heating of sam-
ple solns prevents gelling or pptn of possible satd solns.
B. Apparatus and Reagents
See reagents 962.09B(a), (b), and (f); app. 962.09C(a), (c),
(d), and (f), and in addn:
(a) Filtration apparatus. — System to permit application of
min. vac. necessary for filtration and washing of each sample
within 3-5 min. Each unit consists of reservoir manifold con-
nected to (7) H 2 aspirator thru 120° stopcock, (2) atm. thru
second stopcock with metering device, and (3) receptacle contg
cone-shaped hard rubber gasket which provides vac. seal with
crucible. Vac. gage attached to manifold indicates vac. applied
to crucible. Crucible can be heated before and during filtration
by flow of hot H 2 in surrounding jacket. (For photograph of
app., see JAOAC 56, 1353(1973). Filtration unit is available
as Model 60 1 or 602 (replacement models for Model 1 50) from
Analytical Bio-Chemistry Laboratories, Inc., 7200 ABC Ln,
Columbia, MO 65205).
(b) Crucible. — Fritted glass, 50 mL, coarse porosity. Clean
as follows: Brush, and flow hot tap H 2 into crucible to re-
move as much ash as possible. Submerge crucible in base soln,
82
Animal Feed
AOAC Official Methods of Analysis (1990)
(c)(2), ^5 min, remove, and rinse with hot tap H 2 0. Sub-
merge in HO (1 +1), (c) (1), >5 min, remove, and rinse tho-
roly with hot tap H 2 followed by distd H 2 0. After 3-4 uses,
back wash by inverting crucible on hard rubber gasket in fil-
tration app., and flowing near-boiling H 2 thru crucible under
partial vac.
(c) Cleaning solns.-(J) Acid soln.— HC1 (1 + 1). (2) Base
soln.— Dissolve 5 g Na 2 H 2 EDTA, 50 g Na 2 HP0 4 (tech. grade),
and 200 g KOH in H 2 0, and dil. to 1 L. Storage in sep. wide
mouth containers holding 2-3 L soln into which crucibles can
be placed is convenient.
C. Determination
(Caution: See safety notes on distillation and petroleum
ether.)
Ext 2 g ground material with ether or pet ether (initial boil-
ing temp., 35-38°; dry-flask end point, 52-60°; >:95% distg
<54°, and <60% distg <40°; sp gr at 60°F, 0.630-0.660; evapn
residue <0.002% by wt). If fat is <1%, extn may be omitted.
Transfer to 600 mL reflux beaker, avoiding fiber contamina-
tion from paper or brush. Add 0.25-0.5 g bumping granules,
followed by 200 mL near-boiling 1.25% H 2 S0 4 soln in small
stream directly on sample to aid in complete wetting of sam-
ple. Place beakers on digestion app. at 5 min intervals and boil
exactly 30 min, rotating beakers periodically to keep solids
from adhering to sides. Near end of reflux ing place California
buchner, 962.09C(d), previously fitted with No. 9 rubber
stopper to provide vac. seal, into filtration app., and adjust
vac. to ca 25 mm Hg (735 mm pressure). At end of refluxing,
flow near-boiling H 2 thru funnel to warm it; then decant liq.
thru funnel, washing solids into funnel with min. of near-boil-
ing H 2 0. Filter to dryness, using 25 mm vac, and wash res-
idue with four 40-50 mL portions near-boiling H 2 0, filtering
after each washing. Do not add wash to funnel under vac; lift
funnel from app. when adding wash.
Wash residue from funnel into reflux beaker with near-boil-
ing 1.25% NaOH soln. Place beakers on reflux app. at 5 min
intervals and reflux 30 min. Near end of refluxing, turn on
filtration app., place crucible, (b), in app., and adjust vac. to
ca 25 mm. Flow near-boiling H 2 thru crucible to warm it.
(Keep near-boiling H 2 flowing thru jacket during filtration
and washing.) At end of refluxing, decant liq. thru crucible
and wash solids into crucible with min. of near-boiling H 2 0.
Increase vac as needed to maintain filtration rate. Wash res-
idue once with 25-30 mL near- boiling 1 .25% H 2 S0 4 soln, and
then with two 25-30 mL portions near-boiling H 2 0, filtering
after each washing. (Filtering and washing takes ca 3-5 min/
sample.) Do not add wash to crucible under vac.
Dry crucible with residue 2 hr at 130 ±2° or overnight at
1 10°, cool in desiccator, and weigh. Ash 2 hr at 550 ± 10°, cool
in desiccator, and weigh. Do not remove crucibles from fur-
nace until temp, is <250°, as fritted disk may be damaged if
cooled too rapidly.
% Crude fiber = Loss in wt on ignition x 100/wt sample
Ref.: J AOAC 61, 154(1978).
973.18 Fiber (Acid Detergent) and Lignin
in Animal Feed
First Action 1973
Final Action 1977
(Caution: See safety notes on asbestos.)
A Reagents
(a) Sulfuric acid. — 72% by wt. Stdze reagent grade H 2 S0 4
to sp gr 1.634 at 20° or 24.007V: Add 1200 g H 2 S0 4 to" 440
mL H 2 in 1 L MCA vol. flask with cooling. Stdze to 1634
g/L at 20° by removing soln and adding H 2 or H 2 S0 4 as
required.
(b) Acid-detergent soln. — Add 20 g cety.1 trimethylammon-
ium bromide (tech. grade) to 1 L 1 .Q0N H 2 S0 4 , previously
stdzd. Agitate to aid soln.
(c) Asbestos. — Place 100 g asbestos in 3 L if ask contg 850
mL H 2 0. Add 1.4 L H 2 S0 4 (tech. grade), mix, and let cool
2 hr at room temp. Filter on large buchner and wash with H 2 0.
Resuspend mat in H 2 and pour into bag sewn from rectangle
of fiberglass window screening, 14 x 18 mesh (bag should be
>45 cm wide x 30 cm deep). Wash by immersion and agi-
tation in partly filled sink to remove fine particles. Ash re-
covered asbestos 16 hr in 800° furnace. Store in dry form until
use. Used asbestos may be rewashed, reashed, and reused.
Com. prepd acid-washed asbestos is unsatisfactory unless treated
with 72% H 2 S0 4 and ashed at 800°.
B. Apparatus
(a) Refluxing apparatus. — Any conventional app. suitable
for crude fiber detns. Berzelius beakers (600 mL) and con-
densers made from 500 mL r-b flasks are also satisfactory.
(b) Fritted glass crucibles. — Use coarse porosity, 40-50
mL Pyrex crucible. Wash new crucibles and ash at 500°. Re-
move while still hot and place in 100° forced-draft oven >1
hr. Cool 15 min in desiccator over P 2 5 or Mg(C10 4 ) 2 and
weigh in same order samples are to be weighed. Check balance
after each weighing if crucibles are still warm. Hold length
of time from oven to balance pan as const as possible and
always weigh crucibles in same order.
C. Determination of Acid-Detergent Fiber
Weigh 1 g air-dried sample ground to pass I mm screen, or
approx. equiv. amt wet material, into refluxing container. Add
100 mL acid- detergent soln at room temp.
Heat to boiling in 5-10 min; reduce heat to avoid foaming
as boiling begins. Reflux 60 min from onset of boiling, ad-
justing boiling to slow, even level. Remove container, swirl,
and filter thru weighed (W x ) fritted glass crucible, using min.
suction. Increase vac. only as needed. Shut off vac. Break up
filtered mat with rod and fill crucible 2 / 3 full with hot (90-
100°) H 2 0. Stir and let soak 15-30 sec. Dry with vac. and
repeat H 2 washing, rinsing sides of crucible. Wash twice
similarly with acetone.
Repeat acetone washings until no more color is removed,
breaking up all lumps so that sol v. wets all particles of fiber.
Remove residual acetone with vac. Dry 3 hr or overnight in
100° forced-draft oven and weigh (W 2 ). Calc. % acid-detergent
fiber = 100 (W 2 — W { )/S, where S = g sample x g oven-
dried matter /g air-dried or wet matter, detd on sep. sample.
D. Determination of Lignin
To crucible contg fiber, 973. 18C, add 1 g asbestos. Place
crucible in 50 mL beaker for support or arrange crucibles in
shallow enamel pan. Cover contents of crucible with cooled
(15°) 72% H 2 S0 4 and stir with glass rod to smooth paste,
breaking all lumps. Fill crucible about half-way with acid and
stir. Leave glass rod in crucible; refill with 72% H 2 S0 4 and
stir hourly as acid drains, keeping crucible at 20-23° (cool if
necessary). After 3 hr, filter as completely as possible with
vac. , and wash with hot H 2 until acid-free to pH paper. Rinse
sides of crucible and remove stirring rod. Dry crucible in 100°
forced-draft oven, cool in desiccator over P 2 O s or Mg(C10 4 ) 2 ,
and weigh (W 3 ). Ignite crucible in 500° furnace 2 hr or until
C-free. Place crucible while still hot into 100° forced-draft oven
1 hr. Transfer to desiccator, cool, and weigh (W 4 ).
Det. asbestos blank by weighing 1 g asbestos into tared cru-
cible. Proceed as above, beginning "Cover contents of cru-
cible ..." Record any loss in wt on ashing (W 5 ). Discontinue
AOAC Official Methods of Analysis (1990)
Sugars
83
detn of blank if asbestos blank is <0.0020 g/g asbestos. Calc.
% acid-insol. lignin = (W 3 - W 4 - W 5 )/S.
Refs.: JAOAC 46, 829(1963); 56, 781(1973).
CAS-9005-53-2 (lignin)
974.06 Sugars (Total) in Animal Feed
Modified Fehling Solution Method
First Action 1974
Final Action 1975
A. Reagents
(a) Soxhlet modification of Fehling soln. — Prep, as in
923.09A(a) and (b).
(b) Invert sugar std soln. — 1.0%. Prep, as in 923.09A(c),
but do not neutze. Dil. to 0.5% just before use for analysis of
most products.
(c) Lactose std soln. — 1.0%. Dissolve 5.000 g lactose in
H 2 and dil. to 500 mL. Prep, daily.
B. Apparatus
(a) Lamp. — Fluorescent desk lamp or 150 watt reflector spot
lamp, to illuminate boiling soln.
(b) Heater. — Glas-Col mantle, 250 mL, placed over mag.
stirrer. Adjust heat so that 50 mL H 2 contg stirring bar will
boil in 3 min. Mag. stirring hot plate is also satisfactory.
C. Preparation of Sample and Inversion
(a) Feeds containing molasses .— Weigh appropriate size
sample, prepd as in 950.02 but not ground, to provide final
soln ca 0.5% invert sugar but ^5 g, into 250 mL P flask
(Corning Glass Works No. 5840, or equiv.). Add 150 mL H 2 0,
swirl to wet and mix, and heat just to bp. Let stand to cool,
dil. to vol., mix, and let stand to settle coarse particles. Trans-
fer 50 mL supernate to 100 mL vol. flask and add 2.5 mL HC1
(sp gr 1.18 at 20/4°). Let stand overnight at ^25°, dil. to vol.,
and mix. (If aliquot to be used in detn is >25 mL, it is nec-
essary to neutze inverted soln.)
(b) Feeds containing milk products. — Weigh appropriate
size sample to provide final soln ca 1% lactose into 250 mL
vol. flask. Thoroly moisten sample with H 2 0, swirl to dissolve
lactose, dil. to vol., mix, and let stand to settle coarse parti-
cles. Proceed as in 974.06E(b).
D. Standardization
Fill 50 mL buret, with offset tip, with std sugar soln (invert
sugar for use with 974.06E(a) and lactose with 974.06E(b)).
Proceed as in 968.28D, par. 2, except use same type flask as
used in 974. 06E, do not add H 2 0, and start stirring after addn
of indicator.
E. Determination
(a) Difference method. — Add reagents and stirring bar to
250 mL extn flask (Corning Glass Works No. 5160, or equiv.)
or to erlenmeyer, as in 974.06D. Transfer aliquot inverted soln,
(a), to flask so that >1 but <5 mL std soln will be required
to reach end point, place on preheated mantle or hot plate, heat
to bp, boil 2 min, add ca 1 mL indicator, and begin stirring.
Complete detn by titrg with std sugar soln to same end point
used in stdzn. Color change is not so sharp as in stdzn, but
under suitable light it is definite, discernible, and repeatable.
(b) Alternative method. — Fill buret with sample soln, (b),
or inverted sample soln, (a). As in 974. 06D, place reagents
in flask, place on heater, add sample soln to within 2 mL of
final titrn (detd by trial), bring to bp, boil 2 min, and complete
titrn as in (a).
F. Calculations
% Total sugar (as invert or lactose)
= [(F - M) x / x 1001/IV x (lV/250) X D)]
where F = mL std sugar required to reduce mixed Soxhlet
reagent in stdzn; M — mL std soln required to complete detn
(omit in alternative method); / = concn std soln; V = mL sam-
ple soln in aliquot used; W — g sample; and D = diln factor.
Report total sugars, expressed as invert or as lactose.
Ref.: JAOAC57, 382(1974).
925.05
Sucrose in Animal Feed
Final Action
Place 10 g sample in 250 mL vol. flask. If material is acid,
neutze by adding 1-3 g CaC0 3 . Add 125 mL 50% alcohol by
vol., mix thoroly, and boil on steam bath or by partially im-
mersing flask in H 2 bath 1 hr at 83-87°, using small funnel
in neck of flask to condense vapor. Cool and let mixt. stand
several hr, preferably overnight. Dil. to vol. with neut. 95%
alcohol, mix thoroly, let settle or centrf. 15 min at 1500 rpm,
and decant closely. Pipet 200 mL supernate into beaker and
evap. on steam bath to 20-30 mL. Do not evap. to dryness.
Little alcohol in residue does no harm.
Transfer to 100 mL vol. flask and rinse beaker thoroly with
H 2 0, adding rinsings to flask. Add enough satd neut. Pb(OAc) 2
soln (ca 2 mL) to produce flocculent ppt, shake thoroly, and
let stand 15 min. Dil. to vol. with H 2 0, mix thoroly, and filter
thru dry paper. Add enough anhyd. Na 2 C0 3 or K oxalate to
filtrate to ppt all Pb, again filter thru dry paper, and test filtrate
with little anhyd. Na 2 C0 3 or K oxalate to make sure that all
Pb has been removed.
Place 50 mL prepd soln in 100 mL vol. flask, add piece of
litmus paper, neutze with HC1, add 5 mL HC1, and let inver-
sion proceed at room temp, as in 925.48(c). When inversion
is complete, transfer soln to beaker, neutze with Na 2 C0 3 , re-
turn soln to 100 mL flask, dil. to vol. with H 2 0, filter if nec-
essary, and det. reducing sugars in 50 mL soln (representing
2 g sample) as in 906. 03B. Calc. results as invert sugar.
% Sucrose = [% total sugar after inversion - % reducing
sugars before inversion (both calcd as invert sugar)] x 0.95
Because insol. material of grain or cattle food occupies some
space in flask as originally made up, correct by multiplying
all results by factor 0.97, as results of large number of detns
on various materials show av. vol. of 10 g material to be 7.5
mL.
Refs.: USDA Bur. Chem. Circ. 71. JAOAC 41, 276(1958);
42, 39(1959).
CAS-57-50-1 (sucrose)
920.40* Starch in Animal Feed
Final Action
Surplus
A. Direct Acid Hydrolysis
See 7.080, 13th ed.
B. Diastase Method with Subsequent Acid Hydrolysis
See 7.067, 12th ed.
C. Extraction with Subsequent Enzyme Hydrolysis
See 14.075-14.080, 13th ed.
D. In Presence of Interfering Polysaccharides
See 22.048, 1.0th ed.
84
Animal Feed
AOAC Official Methods of Analysis (1990)
E. In Condensed or Dried Milk Products — Qualitative Test
See 22.049, 10th ed.
920.41* Pentosans in Animal Feed
Final Action
Surplus 1965
See 22.050-22.051, 10th ed.
920.42* Galactan in Animal Feed
Final Action
Surplus 1965
See 22.052, 10th ed.
920.43* Acidity (Water-Soluble)
of Animal Feed
Final Action
Surplus 1965
See 22.053, 10th ed.
925.12* Mineral Salts in Animal Feed
Final Action
Surplus 1974
A. Ferrous Salts
Seel. 074, 1 2th ed.
B. Copper Salts
See 7.075, 12th ed.
C. Potassium Iodide
See 7.076, 12th ed.
968.08 Minerals in Animal Feed
Atomic Absorption Spectrophotometry Method
First Action 1968
Final Action 1969
(Caution: See safety notes on A AS.)
A. Apparatus
Atomic absorption spectrophotometer. — See 965. 09A.
B. Operating Parameters
See Table 965.09, except use fuel-rich air-C 2 H 2 flame for
Ca and Mg, and ranges of operation for jutg element/mL so In
are: Ca 5-20, Cu 2-20, Fe 5-20, Mg 0.5-2.5, Mn 5-20, and
Zn 1-5.
C. Reagents
(See introduction to 965. 09B. Com. prepd std solns may be
used.)
(a) Calcium std solns. — Prep, as in 965.09B(a).
(b) Copper, iron, magnesium, manganese, and zinc std
solns. — Prep, stock solns as in 965.09B(b), (c), (e), (f), and
(g), and dil. aliquots with 0.1-0.5/V HC1 to make >4 std solns
of each element within range of detn.
D. Preparation of Sample Solution
(a) Dry ashing (not applicable to mineral-mix feeds) . — Ash
2—1 g sample in well-glazed porcelain dish. Start in cold fur-
nace, bring to 550°, and hold 4 hr. Cool, add 10 mL 3/V HC1,
cover with watch glass, and boil gently 10 min. Cool, filter
into 100 mL vo\. flask, and dil. to vol. with H 2 0. Subsequent
dilns with 0.1-0.5/V HC1 may be necessary to bring sample
solns into anal, range, except for Ca. Final Ca diln must con-
tain enough La soln, 965.09B(d), to provide 1% La concn
after diln to vol. with H 2 0.
(b) Wet digestion. — Proceed as in 935.13A(a), adding 25
mL HN0 3 for each 2.5 g sample and dilg to 100 mL with FLO.
Digestion can be made at low heat on hot plate, using 600 mL
beaker covered with watch glass. Subsequent dilns with 0.1-
0.5/V HC1 may be necessary to bring sample solns into anal,
range, as in (a).
E. Determination and Calculation
See 965.09D-E.
Refs.: JAOAC 51, 776(1968); 54, 666(1971); 59, 937(1976);
60, 465(1977).
CAS-7440-70-2 (calcium)
CAS-7440-50-8 (copper)
CAS-7439-89-6 (iron)
CAS-7439-96-5 (manganese)
CAS-7440-66-6 (zinc)
927.02 Calcium in Animal Feed
Dry Ash Method
Final Action
(Applicable to mineral feeds only)
Weigh 2 g finely ground sample into Si0 2 or porcelain dish
and ignite in furnace to C-free ash, but avoid fusing. Boil res-
idue in 40 mL HC1 (1+3) and few drops HN0 3 . Transfer to
250 mL vol. flask, cool, dil. to vol., and mix thoroly. Pipet
25 mL clear liq. into beaker, dil. to ca 100 mL, and add 2
drops Me red, 984,13B(c) Add NH 4 O.H (1 + 1) dropwise to
pH 5.6, as shown by intermediate brownish-orange. If over-
stepped, add HC1 (1 +3) with dropper to orange. Add 2 more
drops HCJ (1 + 3). Color should now be pink (pH 2.5-3.0),
not orange. Dil. to ca 150 mL, bring to boil, and slowly add,
with const stirring, 10 mL hot satd (4.2%) soln of (NH 4 ) 2 C 2 04.
If red changes to orange or yellow, add HO (1 + 3) dropwise
until color again changes to pink. Let stand overnight for ppt
to settle. Filter supernate thru quant, paper, gooch, or fritted
glass filter (fine Pyrex is preferable), and wash ppt thoroly with
NH4OH (1 + 50). Place paper or crucible with ppt in original
beaker, and add mixt. of 125 mL LLO and 5 mL H 2 S0 4 . Heat
to >70° and titr. with 0AN KMn6 4 , 940.35, to first slight
pink. Presence of paper may cause color to fade in few sec.
Correct for blank and calc. % Ca.
Refs.: JAOAC 10, 177(1927); 19, 93, 574(1936); 28, 80(1945).
CAS-7440-70-2 (calcium)
935.13 Calcium in Animal Feed
Wet Ash Method
Final Action
A. Preparation of Solution
(Caution: See safety notes on nitric acid and perchloric acid.)
(a) Weigh 2.5 g sample into 500 or 800 mL Kjeldahl flask.
Add 20-30 mL HNO3 and boil gently 30-45 min to oxidize
all easily oxidizable matter. Cool soln somewhat and add 10
mL 70-72% HCIO4. Boil very gently, adjusting flame as nec-
essary, until soln is colorless or nearly so and dense white
fumes appear. Use particular care not to boil to dryness (Dan-
AOAC Official Methods of Analysis (1990)
Minerals
85
ger!) at any time. Cool slightly, add 50 mL H 2 0, and boil to
drive out any remaining N0 2 fumes. Cool, dil., filter into 250
mL vol. flask, dil. to vol., and mix thoroly.
(b) Weigh 2.5 g finely ground sample into Si0 2 or porcelain
dish and ignite as in 942.05. Add 40 mL HC1 (1 +3) and few
drops HN0 3 to residue, boil, transfer to 250 mL vol. flask,
cool, dil. to vol., and mix thoroly.
B. Determination
Pipet suitable aliquot of clear soln, 935.13A(a) or (b), into
beaker, dil. to 100 mL, and add 2 drops Me red, 984.13B(c).
Continue as in 927.02, beginning "Add NH 4 OH (1 + 1) drop-
wise ..." except use 0.05 N KMn0 4 for titrn.
(100 mL is suitable aliquot of sample soln for grain feeds;
for mineral feeds, 25 mL aliquot may be taken and titrd with
0. \N KMn0 4 . For suitable precision, size of sample, aliquot,
and concn of KMn0 4 must be so adjusted that >20 mL std
KMn0 4 soln is used.)
Refs.: Ind. Eng. Chem. Anal. Ed. 7, 116, 167(1935). JAOAC
30, 606(1947); 31, 98(1948); 32, 650(1949); 33,
162(1950); 34, 563(1951).
CAS-7440-70-2 (calcium)
943.01 Chlorine (Soluble)
in Animal Feed
Titrimetric Method
Final Action
A Reagents
(a) Potassium chloride std soln. — 0.001 g Cl/mL. Recrys-
tallize reagent KC1 3 times from H 2 0, dry at 110°, and heat
at ca 500° to const wt. Dissolve 2.1028 g in H 2 and dil. to
1 L.
(b) Silver nitrate soln. — Dissolve 5 g AgN0 3 in 1 L H 2
and adjust soln so that 1 mL — 1 mL std KC1 soln.
(c) Potassium thiocyanate soln. — Dissolve 2.5 g KSCN in
1 L H 2 and adjust so that 1 mL = 1 mL std AgN0 3 soln.
Stdze as in 942.36C.
(d) Ferric sulfate soln.— Dissolve 60 g Fe 2 (S0 4 ) ? -nH 2 in
H 2 and dil. to 1 L.
(e) Ferric sulfate indicator. — To filtered 25% soln of
Fe 2 (S0 4 )ynH 2 0/add equal vol. HN0 3 .
B. Determination
Transfer 3 g sample to 300 mL erlenmeyer. Add 50 mL
Fe 2 (S0 4 ) 3 soln (accurately measured), swirling flask to prevent
caking of sample and to facilitate soln of CI. Add 100 mL (also
accurately measured) NH 4 OH (1 + 19). Swirl flask just enough
to ensure soln of CI and thoro mixing of soln. (Very little
swirling is necessary. If soln is agitated by vigorous vertical
shaking, filtration will be difficult.) Let mixt. settle 10 min.
Filter thru dry 11 cm Whatman No. 41 paper, or equiv. Use
50 mL aliquots (V3 of total) on samples low in CI (0-2% CI)
and 25 mL aliquots ('/e of total) on samples high in CI (>2%).
For mineral and other feeds contg >10% CI, weigh 1 g and
use 15 mL (V10 of total).
If approx. % CI in sample is not known, take 10 mL aliquot
for trial titrn. To this add 10 mL HNO a and 10 mL Fe 2 (S0 4 ) 3
indicator. Dil. to ca 50 mL. Add 0.5 mL KSCN soln and im-
mediately add, with stirring, enough AgN0 3 soln to entirely
eliminate any reddish color. From this titrn calc. vol. AgN0 3
soln necessary to ppt all CI in aliquot to be used, adding excess
equal to ca 10% total vol. necessary, altho somewhat greater
excess will not affect results. Use min. total of 10 mL.
To sample aliquot in 250 mL beaker add 10 mL HN0 3 and
10 mL Fe 2 (S0 4 ) 3 indicator (or 20 mL soln contg equal vols of
these solns). Add, with stirring, calcd vol. AgN0 3 soln. Heat
to boiling and cool to room temp., stirring enough to coagulate
ppt. (Cooling may be hastened by immersion of beakers in
cold H 2 0.) Titr. excess AgN0 3 with KSCN. End point is in-
dicated by first appearance of reddish tint that persists 15 sec.
For accurate work, use ref. soln contg all ingredients except
KSCN. End point is first change in color.
Refs.: JAOAC 26, 87(1943); 28, 80(1945).
CAS-7782-50-5 (chlorine)
969.10 Chlorine (Soluble) in Animal Feed
Potentiometric Method
First Action 1969
Final Action 1970
A Apparatus
Potentiometer. — With Ag-AgCl reference electrode and Ag-
indicating electrode (Fisher Scientific Co. No. 9-313-216 and
13-639-122, or equiv.).
B. Standardization
Weigh 125 mg dry NaCl into 400 mL beaker. Add 200 mL
H 2 and 1 mL HN0 3 .
Null potentiometer and titr. NaCl soln with 0AN AgN0 3
soln. Plot mL AgN0 3 soln against mv or scale readings. Add
titrant in small enough increments so that voltage end point is
obvious. Use same end point for samples.
C. Determination
(a) Samples containing less than 5% sodium chloride. —
Weigh 5.844 g sample into 400 mL beaker. Add ca 200 mL
H 2 and 1 mL HNO3. Swirl mixt. gently and let stand 10 min
for complete soln of chlorides. Titr., while stirring, to same
voltage end point as in stdzn.
% NaCl - mL 0AN AgNO 3 /10
(b) Samples containing more than 5% sodium chloride. —
Weigh 5.844 g sample into 200 mL vol. flask. Add ca 190
mL H 2 and 1 mL HN0 3 , dil. to vol. with H 2 0, mix, and let
stand 10 min. Transfer aliquot contg equiv. of ca 125 mg NaCl
to 400 mL beaker, dil. to ca 200 mL, add 1 mL HN0 3 , and
titr. as in (a).
% NaCl - diln factor x mL 0.1/V AgNO 3 /10
Ref.: JAOAC 52, 607(1969).
CAS-7782-50-5 (chlorine)
952.02
Cobalt in Animal Feed
Colorimetric Method
Final Action
A. Reagents
(a) Cobalt, std soln. — 0.05 mg Co/mL. Dissolve 0.2385 g
CoS0 4 .7H 2 (do not dry; use as received) in H 2 and dil. to
1 L. Dil., if necessary, to suitable concn to prep, std curve.
(b) Nitroso-R salt soln. —Dissolve 1 gC 10 H 4 6H.NO(SO 3 Na)2
in H 2 and dil. to 500 mL.
(c) Spekker acid. — Mix 150 mL 85% H3PO4 and 150 mL
H 2 S0 4 , and dil. to 1 L with H 2 0.
(d) Sodium acetate soln. — Dissolve 500 g NaOAc.3H 2 in
HoO and dil. to 1 L with H,0.
86
Animal Feed
AOAC Official Methods of Analysis (1990)
B. Preparation of Standard Curve
To 1,2, etc., up to 11 mL portions std Co soln in 100 mL
vol. flasks add 2 mL Spekker acid, 10 mL nitroso-R salt soln,
and 10 mL NaOAc soln. Prep, blank by using 2 mL Spekker
acid and 10 mL NaOAc soln, but omitting nitroso-R salt soln.
Bring solns to bp on hot plate. Add 5 mL HN0 3 and boil S:l,
but <2 min. Cool, and dil. to 100 mL.
C. Determination
{Caution: See safety notes on nitric acid and hydrogen
sulfide.)
Ash 2 g sample 2 hr at 600°, transfer to 200 mL vol. flask
with 20 mL HC1 and 50 mL FLO, boil 5 min, cool, and dil.
to vol. Let soln settle. Pipet suitable aliquot into small flask.
For samples contg 0.01-0.2% Co use equiv. of 0.25 g sample.
For other samples, take aliquot contg <0.5 mg Co. Soln no
longer appears to follow Beer's law above this amt.
Pass brisk current of H 2 S thru soln 10 min. Filter directly
into 100 mL vol. flask thru Whatman No. 40 paper. Wash with
ca 50 mL 1% H 2 S0 4 satd with H 2 S. Add 2 small glass beads
and boil off H 2 S. (Flasks must be given individual attention,
as violent bumping may occur.) Shake flasks often. Add 5 mL
HN0 3 and boil until nitrous fumes no longer appear. (Take
care, as vol. of soln will be low and bumping and spattering
may occur. At first indication of this, remove immediately from
hot plate.) Small amt HNO3 remaining will not affect result.
Cool, add 2 drops phthln, and adjust to first faint pink with
ca 30% NaOH soln. Immediately add 2 mL Spekker acid fol-
lowed by 10 mL nitroso-R salt soln and 10 mL NaOAc soln.
Bring to vigorous boil, carefully add 5 mL HNO3, and boil
S:l but ^2 min. Cool, and dil. to vol.
Compare color with std Co solns in photoelec. colorimeter,
using green or No. 54 filter, or in spectrophtr at 540 nm. Read
color within 2 hr. Report % Co to third decimal place.
Ref.: JAOAC 35, 559(1952).
CAS-7440-48-4 (cobalt)
947.03 Copper in Animal Feed
Colorimetric Method
Final Action
A. Preparation of Standard Curve
Dissolve 1 .9645 g CuS0 4 .5H 2 in H 2 and dil. to 500 mL.
(1 mL— 1 mg Cu.) Use from 1 to 10 mL of this soln to prep.
set of stds in 100 mL Pyrex g-s vol. flasks. Add 4 mL HC1,
dil. to 50 mL, add 5 mL tetraethylenepentamine , dil. to vol.
with H 2 0, stopper, and mix thoroly. Prep, blank, using all
reagents except Cu. Filter blank and stds before reading color
as in 947.03B.
B. Determination
Prep, sample soln as in 952. 02C, using 8 g sample. Pipet
50 mL aliquot into 100 mL Pyrex g-s vol. flask, add 5 mL
tetraethylenepentamine, dil. to vol. with H 2 0, and mix
thoroly. Filter, and compare colors within 30 min in photo-
elec. colorimeter (red or No. 66 filter) or read in spectrophtr
at 620 nm. Report % Cu to third decimal place.
Refs.: Anal. Chem. 19, 325(1947). JAOAC 37, 246(1954);
38, 222(1955).
975.08
Fluorine in Animal Feed
First Action 1975
Final Action 1976
Co/orimetric Method
A. Determination
See 944.08, especially 944. 08E.
Ion Seiective Electrode Method
B. Apparatus
(a) Electrodes. — Fluoride ion selective electrode (Model
9409, Orion Research Inc., or equiv.) and single junction cal-
omel ref. electrode, plastic sleeve-type (Model 90-01, Orion
Research Inc., or equiv.).
(b) Magnetic stirrer. — With 4 cm (I 1 //) Teflon-coated stir-
ring bar. Use mat to insulate sample from motor heat.
(c) pH meter. — Corning digital Model 112 (Corning Sci-
entific Instruments, 63 North St, Medfield, MA 02052, or
equiv.).
C. Reagents
(Deionized FLO may be used.)
CAS-7440-50-8 (copper)
(a) Sodium acetate soln. — 3M. Dissolve 408 g NaOAc. 3H 2
with H 2 in 1 L vol. flask. When soln warms to room temp.,
dil. to vol. with H 2 0. Adjust to pH 7.0 with few drops HOAc.
(b) Sodium citrate soln. — 1.32M. Dissolve 222 g Na ci-
trate. 2H 2 with ca 250 mL H 2 in 1 L vol. flask. Add 28 mL
HCIO4, dil. to vol., and mix.
(c) Fluoride std solns. — (J) Stock soln. — 500 ppm. Ac-
curately weigh 1. 105 g NaF (reagent grade, dried 4 hr at 100°)
into 1 L vol. flask. Dissolve and dil. to vol. with H 2 0, and
mix. Store in plastic bottle. (2) Intermediate soln I. — 100 ppm.
Pipet 20 mL stock soln into 100 mL vol. flask, dil. to vol.
with H 2 0, and mix. (3) Intermediate soln II. — 10 ppm. Pipet
2 mL stock soln into 100 mL vol. flask, dil. to vol. with FLO,
and mix. (4) Working solns. — Pipet 3, 5, and 10 mL inter-
mediate soln II and 5 and 10 mL intermediate soln I into five
100 mL vol. flasks to prep. 0.3, 0.5, 1.0, 5.0, and 10 ppm F
working solns, resp. To each add 10.0 mL IN HC1, 25.0 mL
NaOAc. 3H 2 soln, (a), and 25.0 mL Na citrate soln, (b). Dil.
to vol. with H 2 and mix.
D. Preparation of Sample
Accurately weigh well mixed sample contg ca 400 |xg F into
200 mL vol. flask. Pipet in 20 mL IN HC1 and stir 20 min at
high speed on mag. stirrer. Add 50.0 mL NaOAc soln, (a),
and 50.0 mL Na citrate soln, (b), to dissolved sample. Dil. to
vol. with H 2 and mix.
E. Determination
Connect F and ref. electrodes to pH meter, place electrodes
in low concn F soln, and warm up pH meter. Pour 50-70 mL
std and corresponding sample solns into sep. 100 mL beakers.
Place electrodes in each soln and while stirring with mag. stir-
rer at const rate, read mv of std and unknown solns. Rinse and
blot off electrodes and stirring bar between solns. Construct
std curve on 3 cycle semilogarithmic paper. Read ppm F of
sample soln from std curve.
% F = ppm F x mL sample soln x 10~ 6 X 100/g sample
Ref.: JAOAC 58, 477(1975).
CAS-7782-41-4 (fluorine)
AOAC Official Methods of Analysis (1990)
Minerals
87
934.02* Iodine in Mineral Mixed Feeds
Knapheide-Lamb Method
Final Action
Surplus 1965
See 22.084-22.086, 10th ed.
935.14 Iodine in Mineral Mixed Feeds
Elmslie-Caldwell Method
Final Action
(Not applicable to iodized mineral feeds contg little or no org.
matter. Caution: See safety notes on bromine.)
Place sample contg 3-4 mg I in 200-300 mL Ni dish. Add
ca 5 g Na 2 C0 3 , 5 mL NaOH soln (1 + 1), and 10 mL alcohol,
taking care that entire sample is moist. Heat on steam bath to
remove alcohol. Then dry at ca 100° to prevent spattering upon
subsequent heating (30 min is usually enough).
Place dish and contents in furnace heated to 500° and keep
at that temp. 15 min. (Ignition of sample at 500° appears to
be necessary only to carbonize any sol. org. matter that would
be oxidized by Br-H 2 if not so treated. Temp. >500° may
be used if necessary.) Cool, add 25 mL H 2 0, cover dish with
watch glass, and boil gently 10 min. Filter thru 18 cm paper
and wash with boiling H 2 0, catching filtrate and washings in
600 mL beaker (soln should total ca 300 mL). Neutze to Me
orange with 85% H 3 P0 4 and add 1 mL excess.
Add excess Br-H 2 and boil soln gently until colorless, and
then 5 min longer. Add few crystals salicylic acid and cool
soln to ca 20°. Add 1 mL 85% H 3 P0 4 and ca 0.5 g KI, and
titr. I with 0.0057V Na 2 S 2 3 , adding starch soln when liberated
I color is nearly gone, f mL 0.0057V Na 2 S 2 3 = 0.1058 mg I.
Refs.: JAOAC 18, 338(1935); 21, 596(1938); 23, 688(1940);
33, 83(1950).
CAS-7553-56-2 (iodine)
917.04 Manganese (Acid-Soluble)
in Animal Feed
Colorimetric Method
Final Action
A. Reagent
Potassium permanganate std soln. — 500 ppm Mn. Prep, and
stdze as in 940.35, except use 1.4383 g KMn0 4 and 0.12 g
Na oxalate. Transfer aliquot contg 20 mg Mn to beaker. Add
100 mL H 2 0, 15 mL H 3 P0 4 , and 0.3 g Ki0 4 , and heat to bp.
Cool, and dil. to 1 L. Protect from light. Dil. this soln contg
20 ppm Mn with H 2 (previously boiled with 0.3 g KIO4/L)
to make convenient working stds in range of con ens to be
compared.
B. Determination
(Caution: See safety notes on nitric acid and sulfuric acid.)
Ash weighed sample, 5—15 g, at dull red heat (ca 600°) in
porcelain dish. Cool, and add 5 mL H 2 S0 4 and 5 mL HN0 3
to ash in dish or to ash transferred to beaker with 20-30 mL
H 2 0. Evap. to white fumes. If C is not completely destroyed,
add addnl portions HN0 3 , boiling after each addn. Cool slightly,
transfer to 50 or 100 mL vol. flask, and add vol. dil. H 3 P0 4
soln (8 + 92) equal to Vs vol. of flask (25 or 50 mL). Cool,
dil. to vol., mix, and filter or let stand until clear.
If 50 mL flask was used, pipet 25 mL clear soln into beaker
or 50 or 100 mL vol. flask and add 15 mL H 2 0. if 100 mL
flask was used, pipet 50 mL into beaker or 100 mL flask and
add 30 mL H 2 0. Heat nearly to bp, and with stirring or swirl-
ing add 0.3 g KI0 4 for each 15 mg Mn present. Keep 30-60
min at 90-100°, or until color development is complete. Cool,
dil. to measured vol. of 50 or 100 mL, and mix. Compare
with std KMn0 4 soln in photoelec. colorimeter or in spec-
trophtr at 530 nm. Calc. ppm Mn.
Refs.: J. Am. Chem. Soc. 39, 2366(1917). G. Frederick Smith
Chemical Co. Pub. 209, 5th ed. (1950). JAOAC 22,
78, 673(1939); 24, 865(1941); 25, 892(1942).
CAS-7439-96-5 (manganese)
964.06 Phosphorus in Animal Feed
Alkalirnetric Ammonium Molybdophosphate Method
Final Action
A. Reagents
(a) Molybdate soln. — Dissolve 100 g Mo0 3 in mixt. of 144
mL NH 4 OH and 271 mL H 2 0. Cool, and slowly pour soln,
stirring constantly, into cool mixt. of 489 mL HN0 3 and 1 148
mL H 2 0. Keep final mixt. in warm place several days or until
portion heated to 40° deposits no yellow ppt. Decant soln from
any sediment and keep in g-s vessels.
(b) A cidified molybdate soln . — To 1 00 m L mol y bdate soln,
(a), add 5 mL HN0 3 . Filter immediately before use.
(c) Sodium hydroxide std soln. — Dil. 324.03 mL \N alkali,
carbonate-free, 936.16, to 1 L. (100 mL of this soln should
neutze 32.40 mL \N acid; 1 mL = 1 mg or 1% P 2 5 on basis
of 0.1 g sample.) (Since burets in const use may become
so corroded as to increase their capacity, test them at least
annually.)
(d) Std acid soln. — Prep, soln of HC1 or of HN0 3 , corre-
sponding to concn of (c) or to V2 this concn, and stdze by titrn
against (c), using phthln.
0. Determination
Prep, sample soln as in 935.13A(a). Pipet, into beaker or
flask, aliquot corresponding to 0.4 g sample for P 2 5 content
of sample <5%; 0.2 g for 5-20%; 0.1 g for >20%." Add 5-
10 mL HNO3, depending on method of soln (or equiv. in
NH4NO3); then add NH 4 OH until ppt that forms dissolves only
slowly on vigorous stirring, dil. to 75-100 mL, and adjust to
25-30°. If sample does not give ppt with NH 4 OH as test of
neutzn, make soln slightly alk. to litmus paper with NH 4 OH
and then slightly acid with HN0 3 (1 +3). Add 20-25 mL aci-
dified molybdate soln for P 2 5 content <5%; 30-35 mL for
5-20%; and enough acidified molybdate soln to ensure com-
plete pptn for >20%. Shake or stir mech. 30 min at room
temp.; decant at once thru filter and wash ppt twice by de-
canting with 25-30 mL portions H 2 0, agitating thoroly and
allowing to settle. Transfer ppt to filter and wash with cold
H 2 until filtrate from 2 fillings of filter yields pink color on
adding phthln and I drop of the std alkali. Transfer ppt and
filter to beaker or pptg vessel, dissolve ppt in small excess of
the std alkali, add few drops of phthln, and titr. with std acid.
Report as % P.
Ref.: JAOAC 47, 420(1964).
CAS-7723-14-0 (phosphorus)
88
Animal Feed
AOAC Official Methods of Analysis (1990)
965.17 Phosphorus in Animal Feed
Photometric Method
First Action 1965
Final Action 1966
(Not applicable to mineral -mix feeds. Dry ashing procedure is
not applicable to feeds or mineral mixes contg monobasic Ca
phosphate.)
A. Apparatus
Spectrophotometer. — Capable of isolating 400 nm band and
accepting ^15 mm diam. cells.
B. Reagents
(a) Molybdovanadate reagent. — Dissolve 40 g NH 4 molyb-
date.4H 2 in 400 mL hot H 2 and cool. Dissolve 2 g NH 4
metavanadate in 250 mL hot H 2 and cool; add 250 mL 70%
HC10 4 . {Caution: See safety notes on perchloric acid.) Grad-
ually add molybdate soin to vanadate soln with stirring, and
dil. to 2 L.
(b) Phosphorus std solns. — (J) Stock soln. — 2 mg P/mL.
Dissolve 8.788 g KH 2 P0 4 in H 2 and dil. to 1 L. (2)Working
soln. — 0.1 mg P/mL. Dil. 50 mL stock soln to 1 L.
C. Preparation of Standard Curve
Transfer aliquots of working std soln contg 0.5, 0.8, 1.0,
and 1.5 mg P to 100 mL vol. flasks. Treat as in 965. 17D,
beginning "Add 20 mL molybdovanadate reagent, ..." Prep,
std curve by plotting mg P against %T on semilog paper.
D. Determination
Ash 2 g sample, in 150 mL beaker, 4 hr at 600°. Cool, add
40 mL HC1 (1+3) and several drops HN0 3 , and bring to bp.
Cool, transfer to 200 mL vol. flask, and dil. to vol. with H 2 0.
Filter, and place aliquot contg 0.5-1.5 mg P in 100 mL vol.
flask. Add 20 mL molybdovanadate reagent, dil. to vol. with
H 2 0, and mix well. Let stand 10 min; then read %T at 400
nm against 0.5 mg std set at 100% T. (Use ^15 mm diam.
cells.) Det. mg P from std curve.
% P = mg P in aliquot /(g sample in aliquot X 10)
Ref.: JAOAC 48, 654(1965); 59, 937(1976).
CAS-7723-14-0 (phosphorus)
964.07 Microscopy of Animal Feed
Basic Microscopic Examination
First Action 1964
Final Action 1965
4. Apparatus
(a) Magnifier-fluorescent illuminator with desk base, 3x y
or reading glass.
(b) Microscopes and illuminator. — Illuminators: Illumina-
tor for this purpose should have: compactness and flexibility;
transformer or resistor to vary light intensity; focusing adjust-
ment to give uniformly lighted field of view; blue-white color
from cool low-voltage source. (I) Compound microscope . —
For mold counting and other filth and decomposition work,
microscope should have following min. specifications: binoc-
ular body with inclined oculars; 4 parfocal achromatic objec-
tives of ca 4, 10, 20, and 40 X; revolving 4-place nosepiece;
Abbe condenser with N.A. of 1.25; 10x Huygenian or wide-
field eyepieces; fine adjustment; mech. stage. (2) Widefield
stereoscopic microscope recommended for filth examina-
tion. — Microscope should have following min. specifications:
binocular body with inclined oculars; sliding or revolving
nosepiece to accommodate 3 objectives; 3 parfocal objectives
1 X, 3x, and 6 or 7.5 X; paired 10 X and paired I5x widefield
oculars; mounted on base and capable of illumination by trans-
mitted or reflected light. 30 x is ordinarily used for routine
examination of filter papers. Verification at higher magnifi-
cation may be required. Following are preferred:
(7) Widefield stereoscopic microscope . — With arm rests,
flat stage (remove spring holders), optional substage illumi-
nation, inclined eyepiece, and lenses to magnify ca 7-30 x,
15 x optimum.
(2) Compound microscope. — With mech. stage, substage
condenser, inclined binocular eyepiece, 3 position rotating
nosepiece, lenses to magnify ca 36-400X, 120x optimum.
{3) Microscope illuminator. — With iris diaphragm; mova-
ble stand holder with rod to permit adjusting light source as
to ht and angle for substage or direct over-stage lighting; able
to hold 2 blue glass filters or 1 blue and I ground glass; 60-
100 watt bulb.
(c) Sieves.— Nest of 5" No. 10, 20, 40, 60, 80, and bottom
pan.
(d) Stages. — Dark Co glass plates 4 X 4" (Fisher Scientific
Co. No. 13-735); or blue paper and microscope slides.
(e) Spot plates. — Black and white.
(f) Forceps. — Fine pointed, curved. If necessary, bend and
grind on emery wheel for good contact of points.
(g) Dropping bottles. — Amber, 30 mL, as reagent
dispensers.
(h) Microspatula; microstirring rods made by drawing out
glass rods; spoon.
B. Reagents
(a) Chloroform. — Tech. Recover by filtration and distn.
(b) Acetone. — Tech.
(c) Acetone, dilute. — Dil. 75 mL acetone with 25 mL H 2 0.
(d) Dilute hydrochloric acid. — Dil. 1 vol. HC1 with 1 voL
H 2 0.
(e) Dilute sulfuric acid. — Dil. 1 vol. H 2 S0 4 with 1 vol. H 2 0.
(f) Iodine soln. — Dissolve 0.75 g KI and 0.1 g I in 30 mL
H 2 and add 0.5 mL HC1. Store in amber dropping bottle.
(g) Millon reagent. — Dissolve, by gently warming, 1 part
by wt Hg in 2 parts by wt HNO3. Dil. with 2 vols H 2 0. Let
mixt. stand overnight and decant supernate. Soln contains
Hg(N0 3 ) 2 , HgN0 3 , HNO3, and some HN0 2 . Store in g-s bot-
tle. {Caution: See safety notes on mercury salts.)
(h) Molybdate soln.— Add 100 mL 10% NH4NO3 soln to
400 mL molybdate soln, 964.06A(a). Use only clear supernate
to fill 30 mL amber dropping bottle. Discard and refill when
crystn occurs.
(i) Mountant I. — Dissolve 10 g chloral hydrate in 10 mL
FLO and add 10 mL glycerol. Store in amber dropping bottle.
(j) Mountant II. — Dissolve 160 g chloral hydrate in 100
mL H 2 and add 10 mL HC1.
(k) Silver nitrate soln. — 10%. Dissolve 10 g AgN0 3 in 100
mL H 2 0.
C. Standards
(a) Feed ingredients. — Collect ingredients used in grain and
stock feeds known to conform to definitions of Association of
American Feed Control Officials as stds. Store in 4 oz bottles.
To control insects, add ca 1 mL CS 2 , and stopper. Become
thoroly familiar with structural appearance of stds before and
after treatment with org. solvs.
(b) We ed seeds. —Collect common weed seeds occurring in
grains. Most may be found in foreign material obtained after
sieving com. whole grains with U.S. Grain Testing Sieve hav-
ing 5 / G4 " (2.5 mm) triangular holes. Identify from illustration
AOAC Official Methods of Analysis (1990)
Microscopy
89
in "Identification of Crop and Weed Seeds" (USDA Handbook
219 (1963), Government Printing Office, Washington, DC
20402). Store in numbered vials. Become familiar with those
weed seeds designated as prohibited and restricted noxious un-
der state laws of individual concern. (See "State Noxious-Weed
Seed Requirements Recognized in the Administration of the
Federal Seed Act" (USDA, Agricultural Marketing Service,
Grain Div., Hyattsville, MD 20782).)
Ref.: JAOAC47, 504(1964).
970,08 Microscopy of Animal Feed
Identification of Vegetable Tissues
Final Action
A. Principle
Feeds are fractionated according to particle size and cleared
where necessary for clear observation; conglomerates are dis-
integrated into constituents and fractions arranged on stage
suitable for microscopic examination at lowest magnification
that permits identification of components when compared to
std feed ingredients.
B. General Methods
(a) Scratch feeds. — Spread representative portion of sam-
ple on white paper and examine under magnifier-fluorescent
illuminator at 3X or with reading glass. Identify grains and
weed seeds; note other foreign material, heat- and insect-dam-
aged particles, live insects, and rodent excreta; examine for
smut, ergot, and mold ("Grain Inspection Manual," USDA).
(b) Mashes comparatively free from adhering fine parti-
cles.— (1) Low power microscopy. — Arrange in nest form 3
sieves that will adequately fractionate feed according to par-
ticle size. Generally, for cattle feeds use No. 10, 20, and 40;
for poultry feeds, No. 20, 40, and 60. Include bottom pan.
Add ca 10 g unground feed (plastic tablespoon makes con-
venient scoop) to nest, and sieve thoroly. With spatula, spread
portion from each sieve on 4 x 4" Co glass stage and place
under stereoscopic microscope. (Blue paper may also be used
as stage.) Arrange illuminator above and near stage so light
strikes sample at angle of ca 45° for shadow contrast. Adjust
magnification (ca 15 X optimum), illumination, and light filters
to individual preference for clear observation. Blue light or
northern daylight is preferred. Examine each fraction on stage
sep. and systematically. Observe feed particles, continually
probing, turning, and testing resistance to pressure with for-
ceps. Note particle size, shape, color, resistance to pressure,
texture, odor, and major structural features. Compare with stds.
If desired, transfer individual particles with forceps to second
glass plate for direct comparison with corresponding tissues
from stds. Likewise transfer and break up conglomerates by
gentle pressure with flat end of forceps. Make list of observed
ingredients. Neglect trace grains which may be normal inpur-
ities in major grains. (Consult "Official Grain Standards of the
United States," USDA, for amts of "other grains" permissible
as impurities in whole grains.)
(2) High power microscopy. — Lower illuminator and select
filters so adequate blue light is reflected thru substage con-
denser of high power microscope. With microspatula, transfer
little of fine sievings from bottom sieve and pan to slide, add
2 drops mountant I, stir, and disperse with microstirring rod.
Examine microscopically (120X optimum). Compare histo-
logically with stds. Remove slide, add 1 drop I soln, stir, and
re-examine. Starch cells are stained pale blue to black; yeasts
and other protein cells, pale yellow to brown. If further tissue
clarification is desired, boil little of same fine sievings 1 min
with ca 5 mL mountant II. Cool, transfer drop or 2 of bottom
settlings to slide, cover, and examine microscopically.
(c) Oily feeds or those containing large particles obscured
by adhering fine particles, — (Most poultry feeds and un-
knowns are best examined by this technic.) Place ca 10 g un-
ground feed in 100 mL tall -form beaker and nearly fill with
CHC1 3 (hood). Stir briefly and let settle ca 1 min. With spoon,
transfer floating (org.) material to 3.5" (9 cm) watch glass,
drain, and dry on steam bath. Sieve, and proceed as in (b). If
desired, filter, dry, suspend fine particles in CHC1 3 , and ex-
amine microscopically (rarely necessary).
(d) Feeds in which molasses has caused lumpiness and oth-
erwise obscured vision. — Place ca 10 g unground feed in 100
mL tall-form beaker. Add 75 mL 75% acetone, stir few min
to dissolve molasses, and let settle. Carefully decant and re-
peat extn. Wash residue twice with acetone by decantation,
dry on steam bath, sieve, and proceed as in (b).
(e) Pellets or crumbles. — Gently grind few pellets at time
in mortar with pestle with enough pressure to sep. pellet into
its constituents, but not to break up constituents themselves.
Sieve first grind thin No. 20 sieve and return particles re-
maining on sieve to mortar for further grinding. Depending on
nature of pellet, proceed with ground material as in (b), (c),
or (d).
970.09 Microscopy of Animal Feed
Identification of Animal Tissues
and Mineral Constituents
Final Action
A. Principle
Feeds contg animal tissues and minerals when suspended in
CHCI3 readily sep. into 2 fractions: (/) Org. fraction which
floats, consisting of muscle fibers, connective tissue, dried
ground organs, feather remains, hoof and horn particles, etc.
from either animal or marine products, plus all vegetable tis-
sues. (2) Mineral fraction which sinks, consisting of bones,
fish scales, teeth, and minerals.
B. Preparation of Sample
Perform CHC1 3 flotation sepn as in 970.08B(c). Collect
floating material and dry on steam bath. Decant CHCI 3 , collect
mineral fraction, and dry on steam bath.
C. Identification of Animal Tissue
Examine dried floating material as in 970.08B(b).
D. Identification of Major Mineral Constituents
Place dried mineral fraction on nest of No. 40, 60, and 80
sieves and bottom pan. Sieve and place the 4 fractions in sep.
groups on same Co glass plate or blue paper stage. Examine
under stereoscopic microscope at ca 1 5 x . Animal and fish
bones, fish scales, and mollusc shells are generally recogniz-
able. Salt usually occurs in cubes which may be dyed. Calcite
form of limestone occurs as rhombohedrons.
£ Confirmatory Tests
With forceps, place unknown particle on glass plate and break
up by applying gentle pressure with flat surface. Working un-
der stereoscopic microscope, sep. particles ca 2.5 cm and place
beside each a fractional drop of reagent solns listed by touch-
ing end of dropper to plate. Push particle into liq. with mi-
crostirring rod and observe what occurs at interface. Follow
order given until pos. identification is obtained. If preferred,
perform tests in black spot plate.
(a) Silver nitrate soln. — (/) Crystal immediately turns chalk
white and slowly expands: chloride, probably salt. (2) Crystal
90
Animal Feed
AOAC Official Methods of Analysis (1990)
turns yellow and yellow needles begin to grow; mono- or di-
basic phosphate, generally dicalcium phosphate. (3) Sparingly
sol. white needles form (Ag 2 S0 4 ): sulfate, Mn-MgS0 4 . (4)
Particles slowly darken: bone.
(b) Dilute hydrochloric acid, — (/) Vigorous effervescence:
CaC0 3 . (2) Mild effervescence or none: make following tests.
(c) Molybdate soln. — Formation of minute yellow crystals
at some distance from particle: tricalcium phosphate, either bone
or rock phosphate. (All phosphates react, but mono- and di-
basic phosphates have been identified with AgN0 3 .)
(d) Milton reagent. — (/) Disintegrated particles mostly float,
turn pink to red (protein), and fade in ca 5 min: bone phos-
phate. (2) Particles appear to swell and disintegrate but remain
on bottom: defluorinated rock phosphate. (3) Particles merely
disintegrate slowly: rock phosphate.
(e) Dilute sulfuric acid. — Long, thin white needles slowly
form on addn of drop of H 2 S0 4 (J + 1) to HC1 (1 + 1) soln of
particle: confirms Ca.
975.09 Identification of Furazolidone,
Tylosin, and Zoalene
See 973.80.
963.07 Ethoxyquin in Animal Feed
Fluorometric Method
First Action 1963
Final Action 1964
A. Reagents and Apparatus
(a) Quinine sulfate reference soln. — 1 p,g/mL 0.1 TV H 2 S0 4 .
Dissolve 0.100 g quinine sulfate USP (dried at 120° for 3
hr before using) in 1 L 0.LV H 2 S0 4 . Dil. 10 mL aliquot of
this soln to 1 L with 0.17V H 2 S0 4 . Use to calibrate photoflu-
orometer.
(b) Ethoxyquin std solns. — Add 100.0 mg liq. ethoxyquin
to 100 mL vol. flask and dil. to vol. with pet ether (Soln A).
Dil. 5 mL Soln A to 100 mL with pet ether (Soln B, 50 \xg/
mL). Dil. 5 mL Soln B to 100 mL with pet ether (Soln C, 2.5
pg/mL). Dil. 10 mL Soln C to 20 mL with pet ether (1.25
|jLg/mL) and 5 mL to 25 mL (0.50 |xg/mL).
(c) Photofluorometer.— Equipped with primary filter pass-
ing 365 nm Hg line (Corning Glass Works No. 5874 (CS7-
39), or equiv.) and secondary filter passing 420-500 nm
(Corning Glass Works 3389 + 5543 + 4784, half stock thick-
ness, or equiv.).
B. Preparation of Standard Curve
Adjust photofluorometer to read with pet ether and 100
with quinine sulfate ref. soln. Obtain fluorescence readings for
ethoxyquin std solns contg 0-2.5 (xg/mL. Plot readings against
(Jig ethoxyquin/mL on linear paper.
C. Determination
Place 10 ± 0.1 g finely ground sample in 100 mL beaker
and slurry with 50 mL MeOH. Stir and let stand 10 min. De-
cant thru plug of glass wool into 250 mL vol. flask. Reslurry
residue with two 50 mL portions MeOH, decant, and filter,
combining all filtrates. Dil. to vol. with MeOH. Transfer 25
mL aliquot to 250 mL separator, add 100 mL H 2 0, and mix
well. Add 50 mL pet ether, stopper, and shake moderately 1
min. Let stand few min to sep. (If emulsion forms, add ca 100
mg NaCl crystals. After emulsion breaks, drain aq. lower layer
into 250 mL beaker.) Transfer pet ether layer to second 250
mL separator, return aq. layer to first separator, and re-ext
with two 25 mL portions pet ether.
Add 50 mL H 2 to combined pet ether exts in separator,
stopper, and shake moderately. Let sep., drain lower aq. layer,
and discard. Transfer pet ether layer to 100 mL vol. flask, and
dil. to vol. with pet ether. Adjust photofluorometer as above
and det. fluorescence readings. Obtain pg ethoxyquin/mL from
std curve. Ppm ethoxyquin = 100 X jjig/mL.
Add 50 mL H 2 to combined pet ether exts in separator,
stopper, and shake moderately. Let sep., drain lower aq. layer,
and discard, Transfer pet ether layer to 100 mL vol. flask, and
dil. to vol. with pet ether. Adjust photofluorometer as above
and det. fluorescence readings. Obtain pg ethoxyquin /mL from
std curve. Ppm ethoxyquin = 100 X pg/mL.
If untreated feed is available, prep, std curve from series of
samples contg 0-250 pg ethoxyquin/ 10 g and carried thru detn.
Refs.: JAOAC 44, 560(1961); 46, 306(1963); 47, 512(1964).
CAS-9 1-53-2 (ethoxyquin)
970.10
See 936.11.
Cyanogenetic Glycosides
In Animal Feed
970.11 Hydrocyanic Acid in Animal Feed
See 915.03.
Drugs in Animal Feed
See chapter on drugs in animal feed.
Molasses and Molasses Products in Animal Feed
See chapter on sugars and sugar products.
5. Drugs in Feeds
Hussein S. Ragheb and Robert L Small idge, Associate Chapter Editors
Purdue University
(Medicated feeds may deteriorate under improper storage con-
ditions. When possible, use reasonably fresh samples, store
them in the cold, and grind just before analysis.)
965.16 Sampling of Animal Feed
Procedure
Use slotted single or double tube, or slotted tube and rod,
all with pointed ends.
Take ^500 g sample, 1 kg preferred, as follows: Lay bag
horizontally and remove core diagonally from end to end. Det.
number of cores as follows: From lots of 1 — 10 bags, sample
all bags; from lot of >: 1 1 , sample 10 bags. Take I core from
each bag sampled, except that for lots of 1-4 bags take enough
diagonal cores from each bag to total >5 cores. For bulk feeds
draw >10 cores from different regions; in sampling small con-
tainers (<J0 lb) 1 package is enough. Reduce composite sam-
ple to amt required, preferably by riffling, or by mixing tho-
roly on clean oil-cloth or paper and quartering. Place sample
in air-tight container.
A sample from less than these numbers of bags may be de-
clared an official sample if guarantor agrees. For samples that
cannot be representatively taken with probe described, use other
sampling means.
950.02
Animal Feed
Preparation of Sample
Final Action
Grind sample to pass sieve with circular openings 1 mm
(V25") diam. and mix thoroly. If sample cannot be ground,
reduce to as fine condition as possible. Do not grind molasses
feeds.
Refs.: JAOAC 33, 424(1950); 41, 223(1958); 48, 658(1965).
963.32* 2-Acetylamino-5-Nitrothiazole
in Feeds
Spectrophotometric Method
First Action 1963
Final Action 1964
Surplus 1977
See 42.011-42.016, 13th ed.
969.53 Aklomide in Feeds
Spectrophotometric Method
First Action 1969
Final Action 1971
(Applicable in presence of sulfanitran or roxarsone)
A. Reagents
(a) Titanous chloride soln. — 4% aq. Prep, fresh on day of
use from 20% soln or solid TiCl 3 .
(b) Sodium nitrite soln. — 0.1% aq. Prep, fresh on day of
use.
(c) Ammonium suljamate soln. — 0.5%. Dissolve 500 mg
NH4SO3NH2 in H 2 and dil. to 100 111L. Prep, fresh weekly.
(d) Coupling reagent. — 0.1% aq. N-naphthylethylenedi-
amine.2HCl. Prep, fresh weekly and store in dark glass bottle
in refrigerator.
(e) Aklomide std solns. — 2-Chloro-4-nitrobenzamide, pu-
rified for std use, available from Salsbury Laboratories, 2000
Rockford Rd, Charles City, 1A 50616. (1) Stock soln. — I mg/
mL. Transfer 100 mg aklomide to 100 mL vol. flask, dissolve
in ca 75 mL MeOH, dil. to vol. with MeOH, and mix well.
(2) Intermediate soln. — 10 |xg/mL. Pipet 10 mL stock soln
into 100 mL vol. flask, dil. to vol. with MeOH, and mix. Pipet
5 mL into 50 mL vol. flask. Evap. to dryness on H 2 bath
with aid of gentle air stream and cool to room temp. Add ca
30 mL 0.X5N HC1, shake 10 min intermittently, dil. to vol.
with 0.157V HC1, and mix well. (3) Working solns.— 0, 0.4,
0.8, 1.2, 1.6, and 2.0 M-g/rnL. Transfer 0, 1,2, 3, 4, and 5
mL intermediate soln to sep. 25 mL vol. flasks and dil. to vol.
with 0.157V HC1.
B. Preparation of Standard Curve
Transfer 4 mL aliquot from each working std soln to sep.
colorimetric tubes and proceed with reduction, color devel-
opment, and measurement as in 969. 53C. Tubes contain 0,
1.6, 3.2, 4.8, 6.4, and 8.0 |xg aklomide/tube, equiv. to 0,
0.008, 0.016, 0.024, 0.032, and 0.040% aklomide in feed when
5 g sample is taken. Plot A against % aklomide.
C. Determination
Weigh 5 g sample contg ca 0.025% aklomide into 100 mL
vol. flask, add 75 mL MeOH, and heat 30 min in 60° H 2
bath, shaking occasionally. Remove flask, cool to room temp.,
and dil. to vol. with MeOH. Mix thoroly and Jet stand 40 min
to settle feed particles.
Pipet 5 mL clear supernate into 50 mL vol. flask and dil.
to vol. with 0.157V HCI. Mix well and filter thru Whatman
No. 4 paper into 125 mL erlenmeyer. (If filtrate is cloudy,
re filter.) Pipet 4 mL filtrate into each of 2 tubes, add 2 drops
4% TiCl 3 from dropper, mix, and let stand 2 min. Add 2 drops
107V NaOH from dropper, mix until white ppt persists, and
acidify with 2.0 mL HCI. Mix and let stand until soln clears.
Add 0.5 mL NaN0 2 to one tube and 0.5 mL H 2 to second
tube as blank; mix. After 3 min, add 0.5 mL 0.5% NH 4 sul-
famate to each tube and mix. After 2 min, add 0.5 mL cou-
pling reagent to each, mix, and let color develop 15 min. Read
A of soln at 545 nm in colorimeter or spectrophtr. Subtract
reading of feed blank. Det. % aklomide in feed directly from
std curve.
Ref.: JAOAC 52, 438(1969).
CAS-301 1-89-0 (aklomide)
969.54 Aklomide in Feeds
Thin Layer Chromatographic Qualitative Test
Final Action 1971
A. Reagents
(a) Spray reagent. — Dissolve and dil. 0.1 g p-(dimethyl-
amino)cinnamaldehyde (DMC) (No. J436, J.T. Baker, Inc.)
91
92
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
to 100 mL with 1.07V HCL (Soln is stable >1 month.) Just
before use, add 1 mL 20% T\C\ 3 to 25 mL DMC soln and
mix. Discard after 1 hr.
(b) Aklomide reference std. — 1 mg/mL. See 969.53A(e)(y).
B. Test
Ext 10 g sample with 25 mL MeOH, shaking occasionally
during 15 min. Filter thru Whatman No. 4 paper into 50 mL
beaker. Cone, filtrate to ca 2 mL on steam bath. Spot ca 10
jxL on 250 |xm silica gel G TLC plate along with ref. std and
develop ca 30 min, in ether. Remove from tank and air dry
ca 15 min. Spray plate with DMC-TiCl 3 reagent. (Caution:
See safety notes on spraying chromatograms.) Aklomide forms
reddish pink spot. Compare R f value to that of ref. std.
Ref.: JAOAC 52, 438(1969).
CAS-301 1-89-0 (aklomide)
964.28 p-Aminobenzoic Acid In Feeds
Spectrophotometric Method
Final Action 1965
A. Preparation of Standard Solution
Transfer 0.100 g /?-aminobenzoic acid (99 + % purity, avail-
able from ICN Pharmaceuticals inc., Life Sciences Group) to
100 mL vol. flask, dissolve in 5 mL LV NaOH, and dil. to
vol. with H 2 0. Dil. 5 mL aliquot to 200 mL with H 2 (1 mL
= 25 jjug). Place 2, 4, and 6 mL aliquots dild soln (50, 100,
150 (xg) in 100 mL vol. flasks, add 3 mL HC1 to each, dil.
to vol. with H 2 0, and mix.
B. Determination
Transfer 5 g freshly ground feed to 250 mL vol. flask, add
135 mL H 2 0, making slurry of first 10 mL to wet sample com-
pletely, and then add 15 mL HC1. Mix, and place on steam
bath 25 min, swirling occasionally until soln darkens. Cool,
dil. to 250 mL with H 2 0, and let feed particles settle. Pipet
50 mL into 100 mL vol. flask, dil. to vol. with H 2 0, and mix
thoroly. Pour soln into 250 mL beaker, add filter-aid, and filter
thru 18.5 cm Whatman No. 2 paper, or equiv., discarding first
10-15 mL, if turbid.
Pipet two 10 mL aliquots into 50 mL beakers, add 5 mL
H 2 and 2 mL fresh 0.10% NaN0 2 soln, mix, and let stand
3 min. Add 2 mL 0.50% NH 4 sulfamate soln, mix, and let
stand 2 min. Then add, to one beaker only, 1 mL coupling
reagent, 969.53A(d), and to other 1 mL H 2 0. Mix solns and
wait 10 min. Det. A against H 2 at 545 nm in spectrophtr.
(Avoid false readings due to N bubbles on cell walls.) Subtract
blank A from sample A and calc. |xg found by ref. to std curve.
% /?-aminobenzoic acid in feed = fxg found/ 1000
K /?-aminobenzoate = p-aminobenzoic acid x 1.278
Prep, stds by treating 10 mL aliquots of 3 final std solns,
representing 5, 10, and 15 fig, as in detn, beginning ". . .
add 5 mL H 2 ..." Plot A at 5, 10, and \5 juug and draw
straight line.
C. Qualitative Tests
(To differentiate p-aminobenzoic acid, arsanilic acid, and
sulfaquinoxaline)
Place 10 mL prepd sample filtrate in separator. Ext with 10
mL peroxide-free ether by vigorous shaking 30 sec. Let layers
sep., and drain aq. layer into another separator. Re-ext with
10 mL ether and drain aq. layer into third separator for third
extn with same vol. ether. After final extn, drain aq. layer into
fourth separator, add 5 mL H 2 0, mix, and couple soln as in
964.28B, second par. Wait 10 min, add 5 drops HC1 and 10
mL isoamyl alcohol, and ext gently ca 30 sec. Let stand until
layers sep. Red color in sol v. is due to p-aminobenzoic acid;
that in lower layer, to arsanilic acid. Drain as much aq. layer
as possible and again ext with 10 mL sol v. Arsanilic acid re-
mains as distinct color in aq. layer, not as mere trace due to
incomplete removal of jP-aminobenzoic acid. Combine ether
exts, wash with 5 mL H 2 ? discard, and ext with 10 mL 1%
Na 2 CO ? soln; acidify, and couple again to prove presence of
sulfaquinoxaline.
Ref.: JAOAC 47, 214(1964).
CAS- 150- 13-0 (p-aminobenzoic acid)
CAS-98-50-0 (arsanilic acid)
CAS-59-40-5 (sulfaquinoxaline)
953.1 9* 2-Amino-5-Nitrothiazole
In Feeds
Spectrophotometric Method
Final Action
Surplus 1980
See 42.025-42.027, 13th ed.
961.24 Amprolium in Feeds
Spectrophotometric Method
First Action 1961
Final Action 1962
A. Principle
Amprolium is extd from feed with aq. MeOH. Ext is pu-
rified by chromatgy on alumina, and amprolium reacts with
2,7-naphthalenediol, K 3 Fe(CN) 6 , KCN, and NaOH in MeOH
to form colored compd with absorption max. at 530 nm. There
is no interference from usual components of com. feeds, vi-
tamins, antibiotics, picolines, or pyrimidines. Nithiazide, En-
heptin A, and nitrofurazone show some interference.
B. Reagents
(Caution: See safety notes on cyanides.)
(a) Alcoholic sodium hydroxide soln. — Dil. 15.0 mL aq.
NaOH soln, (i), with anhyd. MeOH to 200 mL. Stopper, and
mix well.
(b) Alumina. — Reagent grade suitable for chromatgy. Should
pass following test: Vigorously shake 10 g alumina with 100
mL H 2 in 250 mL g-s flask >2 min. Let settle, decant, and
det. pH potentiometrically. pH should be 9.5-10.5. Recovery
of amprolium may vary among different brands of alumina.
Test column recovery by spiking ext from nonmedicated feed.
(c) Amprolium std soln. — 25 p,g/mL. Weigh 25.0 mg Am-
prolium Ref. Std (available from Merck & Co.) into 50 mL
vol. flask, dissolve in dil. MeOH, (e), dil. to vol., and mix.
Dil. 5 mL to 100 mL in vol. flask with dil. MeOH. Soln is
stable 1 week.
(d) Color developing reagent. — Add 5 mL K 3 Fe(CN) 6 soln
to 90 mL naphthalenediol soln, (f ), in 250 mL g-s flask, and
mix well. Add 5 mL KCN soln, (g), stopper, mix well, and
let stand 30-35 min. Add 100 mL ale. NaOH soln, (a), and
mix. Use within 75 min, filtering thru medium porosity fritted
glass filter just before use.
(e) Dilute methyl alcohol, — Mix 2 vols anhyd. MeOH with
1 vol. H 2 0. Cool to room temp, before use.
(f) Naphthalenediol soln. — Dissolve 25 mg 2,7-naphtha-
lenediol (Eastman Kodak Co.) in 1 L anhyd. MeOH.
(g) Potassium cyanide soln. — Dissolve 1.0 g KCN in 100
mL H 2 0. Kept tightly stoppered, soln is stable 2 weeks.
(h) Potassium ferricyanide soln. —Dissolve 200 mg
AOAC Official Methods of Analysis (1990)
Amprolium
93
K 3 Fe(CN) 6 in 100 mL H 2 0. Kept tightly stoppered, soln is
stable 2 weeks.
(i) Sodium hydroxide soln. — Dissolve 2.25 g NaOH in 200
mL H 2 0.
C. Extraction
Accurately weigh amt ground feed (<15 g) contg 1.5-2.5
mg amprolium and transfer to 250 mL g-s flask. Add 100.0
mL dil. MeOH, stopper, and stir mag. or shake mech. 60 min.
Filter thru Whatman No. 42, or equiv., paper and collect 25-
40 mL clear filtrate, rejecting first 10-15 mL. Filtrate should
be clear. Refilter, if necessary, thru fresh paper or centrf. until
clear.
D. Chromatography
(a) Preparation of alumina. — To 200 g Al 2 3 , (b), add 1
L H 2 0. Stir mixt. 30 min. Filter slurry thru fast paper on buch-
ner funnel. Wash A1 2 3 on filter with three 100 mL portions
of anhyd. MeOH. Air-dry under vac. until A1 2 3 reaches room
temp. Prepd A1 2 3 should be free-flowing. Store in g-s bottle.
(b) Preparation of column. — Constrict end of 40 cm length
of 9- 10 mm id glass tubing by rotating in hot flame until open-
ing is 4—5 mm. Insert small plug of Pyrex glass wool in lower
end of tube and compress with glass rod to thickness ca 2-3
mm. Transfer 5.0 g prepd alumina to dry tube and pack by
gentle tapping of tube. Prep. sep. column for each sample.
Note: Column recovery of amprolium may vary among dif-
ferent brands of basic alumina. Test column recovery by spik-
ing ext from nonmedicated feed.
(c) Chromatography of feed extract.— Pipet 25 mL clear
ext onto column and let pass thru column by gravity. Reject
first 3 mL eluate and collect next 5 mL for color development.
E. Determination
Mark 3 sep. 15 mL centrf. tubes as X, S, and B. To X add
4.00 mL clear eluate from column; to S add 4.00 mL ampro-
lium std soln, and to B add 4.00 mL dil. MeOH as blank. Add
10.0 mL color developing reagent to each tube, stopper, mix,
and let stand 20 min. Centrf. 2-3 min, decant into 1 cm cells,
and cover. (If solns are not clear and free from suspended par-
ticles, decant into cells thru small plug of Pyrex glass wool.)
Det. A of solns X and S in spectrophtr or colorimeter at 530
nm against soln B as ref. within 20-25 min after adding color
developing reagent.
% Amprolium in feed - (2.5A x C)/(A' x W)
where A and A' refer to sample and std, resp., C = mg am-
prolium in final aliquot of std soln (0.100 mg), and W = g
original sample.
Refs.: JAOAC 44, 5(1961); 62, 399(1979); 72, 105(1989).
CAS- 121-25-5 (amprolium)
965.47 Amprolium in Feeds
Fluorometric Method
First Action 1965
Final Action 1967
(Applicable in absence of antibiotics except procaine penicillin
and chlortetracycline)
A. Reagents
(a) Amprolium std solns. — (7) Stock soln. — 0.20 mg/mL.
Weigh 20.0 mg Amprolium Ref. Std (available from Merck
& Co.) and dissolve in enough TCA soln, (d), to make 100,0
mL. (2) Working soln. — 1 ^g/mL. Dil. 5.00 mL stock soln
to 100 mL with TCA soln and mix well. Further dil. 10 mL
of this soln to 100 mL with H 2 0, and mix well.
(b) Potassium ferricyanide soln. — Dissolve 2 g K 3 Fe(CN) 6
in 100 mL H 2 0.'
(c) Silver nitrate soln. — Dissolve 5 g AgN0 3 in 100 mL
H 2 0.
(d) Trichloroacetic acid (TCA) soln. — Dissolve 5 g
CCI3COOH in 100 mL H 2 0.
B. Extraction
Grind feed sample to pass No. 20 sieve and mix thoroly.
(High-speed blender grinds most feeds to desired fineness in
ca 3 min.) Weigh sample contg ca 750 jmg amprolium and
transfer to 250 mL g-s flask. Add 100.0 mL TCA soln, stop-
per, and agitate 30 min on mag. stirrer or mech. shaker.
Filter by gravity thru Whatman No. 42 paper, rejecting first
5 mL. Collect ^10 mL clear filtrate. Transfer 5.00 mL clear
ext to 50 mL vol. flask, dil. to vol. with H 2 0, and mix well.
This is dild sample ext.
C. Development of Fluorophor
Mark three 50 mL centrf. tubes X, Y> and Z. To tube X add
15.00 mL dild sample ext; to tube Y add 1.50 mL TCA soln
and 13.50 mL H 2 as blank soln; and to tube Z add 15.00 mL
amprolium working std soln. To all tubes add 5.00 mL NaOH
soln (3 + 10), stopper with polyethylene stoppers, and mix
well. Immediately add 0.50 mL AgN0 3 soln to all tubes, stop-
per, and mix well. Let all tubes stand 2 min. Then to all tubes
add 3.0 mL K 3 Fe(CN) 6 soln, stopper, mix, and let stand 3.0
min.
During this 3 min wait, add 15 mL /7-BuOH to all tubes, as
overlay, and stopper. After 3 min, vigorously shake all tubes
1 .0 min, and centrf. 1 min. Transfer 10.0 mL aliquots of upper
BuOH layer from all tubes to test tubes. Add 1 .00 mL absolute
alcohol to each tube and mix well.
D. Measurement of Fluorescence
(Caution: See safety notes on photofluoro meters.)
(a) For instruments designed to accommodate 10 X 10 mm
cells and using monochromatic light for excitation. — Set ac-
tivation wavelength at 400 nm (uncorrected) and emission
wavelength at 455 nm (uncorrected). Transfer ca 2.0 mL fluo-
rophor BuOH ext to cell and read.
(b) For instruments designed to accommodate JO x 40 mm
cells and using filters to adjust wavelengths for excitation and
emission. — Use Kopp Glass Co. No. C5840 filter placed after
light source to adjust excitation wavelength and Kopp No. C3385
filter placed behind cell to adjust emission wavelength. Trans-
fer entire contents of test tube contg extd fluorophor to cell
and read.
E. Calculations
% Amprolium in feed
- (X - Y) x C/[150 x (Z - Y) x W]
where X, Y, and Z are fluorescence readings of sample, re-
agent blank, and std, resp.; C = \xg in 15 mL std soln (15.0);
and W - g sample.
Ref.: JAOAC48, 285(1965).
CAS-121-25-5 (amprolium)
981.27* Arprinocid in Feeds
Liquid Chromatographic Method
First Action 1981
Surplus 1988
See 42.021-42.026, 14th ed.
94
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
982.42* Arprinocid in Premixes
Spectrophotometric Method
First Action
Surplus 1988
See 42.027-42.032, 14th ed.
954.17 Arsanilic Acid in Feeds
Spectrophotometric Method
Final Action 1960
(Applicable in absence of sulfonamides)
A. Determination
Transfer 4.0 g freshly ground sample to 200 mL vol. flask,
and add ca 80 mL H 2 and 4 mL 0.5N NaOH. Place flask on
steam bath ca 5 min, swirling occasionally. Carefully add 20
mL HC.I, mix, and cool to room temp. Dil. to vol. with H 2 0,
mix, pour into 250 mL beaker, add some Filter-Cel, or equiv. ,
and filter thru Whatman No. 42, or equiv., paper, discarding
first 5 mL.
Pipet 5 mL aliquots of clear filtrate into each of two 20 X
175 mm test tubes. To each tube add 2 mL 0.1% NaN0 2 soln,
mix, and let stand 5 min. Add 2 mL 0.5% NH 4 sulfamate soln
and let stand 2 min. Then add, to 1 tube only, 1 mL coupling
reagent, 969.53A(d), mix, and let stand 10 min before dilg
both solns to vol. of 15 mL. Mix well (if bubbles appear, filter
thru glass wool), and det. A against H 2 at 538 nm in spec-
trophtr or with 540 nm filter in photometer. Subtract A of blank
from sample A. Det. jxg arsanilic acid in aliquot (equiv. to 100
mg sample) from std curve.
0. Preparation of Standard Curve
Transfer 0.100 g pure arsanilic acid to 100 mL vol. flask,
add ca 20 mL H 2 and 2 mL 0.5N NaOH, and dissolve. Dil.
to vol. with H 2 and mix well. Transfer 10 mL to 100 mL
vol. flask, dil. to vol. with H 2 0, and mix well. Dil. 5 mL of
this soln to 250 mL with H 2 in vol. flask, and mix well (1
mL — 2 |xg arsanilic acid). Pipet aliquots of 0, 2, 3, 5, and
8 mL of this std soln into 20 x 175 mm test tubes, add 1 mL
HO (1 + 1) to each tube, and continue as in 954. 17A, be-
ginning "To each tube add 2 mL 0.1% NaN0 2 ..." Subtract
blank A from A of stds and plot differences against 4, 6, 10,
and 16 |xg arsanilic acid in aliquots.
Refs.: JAOAC 37, 257(1954); 40, 452(1957).
CAS-98-50-0 (arsanilic acid)
957.22 Arsenic (Total) in Feeds
Colorimetric Test
First Action 1957
Final Action 1960
A. Reagents
(a) Arsenic trioxide. — NIST As 2 3 SRM 83, or equiv.
(b) Magnesium oxide-magnesium nitrate slurry. — Suspend
75 g MgO and 105 g Mg(N0 3 ) 2 .6H 2 in enough H 2 to make
1 L. Agitate vigorously before addn to sample. (Freshly prepd
slurry gives ash which is easily disturbed by air currents.)
(c) Stannous chloride soln. — Dissolve 40 g As-free SnCl 2 -
H 2 in HC1 and dil. to 100 mL with HC1. Effective as long
as it discharges yellow color in sample ext.
(d) Absorbing soln. — Transfer with graduate 25 mL 1.5%
HgCl 2 soln, and with pipet 3.75 mL 6/V H 2 S0 4 and 3.75 mL
0.03/V KMn0 4 , into 250 mL graduate. Dil. to 250 mL with
H 2 and mix. Prep, fresh daily.
(e) Ammonium molybdate reagent. — Dissolve 1 g
(NH4) 2 Mo0 4 in 100 mL 5 AN H 2 S0 4 . Soln keeps several weeks.
(Prep. 5.4V H 2 S0 4 by dilg 6N (9 + 1).)
(f) Hydrazine sulfate reagent. — 0.15%. Dissolve 0.15 g
N2H4.H2SO4 in 100 mL H 2 0. Soln keeps several weeks.
B. Apparatus
(Do not clean app. and glassware with detergents, as they in-
terfere with color development. Haemo-Sol, available from
Scientific Products, Inc., or equiv., is satisfactory.)
(a) Evaporating dishes. — 70 mL; Coors No. 430, size 00A,
or equiv.
(b) Arsine evolution apparatus .—Bend 6 mm id glass tub-
ing at 120 3 angle ca 10 cm from one end and at 60° angle ca
15 cm from other end. Plug shorter end with glass wool im-
pregnated with satd Pb(OAc) 2 soln and insert in rubber stop-
per, placed in top of 125 mL erlenmeyer, so that end of tube
projects just below stopper. Plug other end with un impreg-
nated glass wool and connect thru rubber tubing to glass tube,
constricted at lower end, that reaches to bottom of 50 mL large
neck vol. flask, or if preferred, 50 mL centrf. tube, marked
exactly at 50 mL and approx. at 20 mL.
C. Preparation of Sample Solution
Weigh ground sample contg <50 |xg As (unless aliquot is
to be taken from digested soln) into 70 mL ashing dish. If
>2.5 g sample is used, increase amt of slurry and size of ash-
ing dish. Add ca 10 mL well mixed slurry, (b), and enough
H 2 to permit thoro mixing with stirring rod. Rinse stirring
rod, and dry sample at 100°. Ash 2-4 hr at 550-600°. (Slight
C residue does not interfere. Use care to avoid loss of ash.)
Cool, and moisten residue with H 2 0. Cover dish with watch
glass and add ca 15 mL HCl (1 + 1). Let stand overnight, or
heat on H 2 bath with agitation until ash dissolves. Filter thru
Whatman No. 30 paper into 125 mL erlenmeyer. Rinse filter
with enough hot H 2 0, in several portions, to obtain ca 60 mL
filtrate.
D. Preparation of Standard Curve
Dissolve 0.660 g As 2 3 in 25 mL 10% NaOH soln, dil. to
1 L with H 2 0, and mix. Dil. 10 mL aliquot to 1 L with H 2
(1 mL = 5 |xg As). Transfer 0, 2, 4, 6, 8, 10, 12, and 14 mL
aliquots from buret into 125 mL erlenmeyers. Dil. each to ca
60 mL with H 2 and proceed as in 957. 22E. Plot A against
jxg As.
E. Arsine Evolution
Add ca 10 mL HCl, 2 mL Kl soln, (15%: keep in dark;
discard when soln turns yellow), and 0.5 mL SnCL soln, (c).
Swirl, heat in H 2 bath 5 min, and cool. Have all parts of
evolution app. ready for immediate assembly, with ca 20 mL
absorbing soln, (d), in 50 mL vol. flask or centrf. tube marked
at 50 mL. Add 5-6 g Zn, 30 mesh, to digested soln; quickly
insert stopper contg glass tubing into erlenmeyer and place de-
livery tube against bottom of vol. flask or centrf. tube so that
bubbles will be small. Use few drops of H 2 to test for leaks
between rubber stopper and erlenmeyer. Connecting glass tube
must be large enough so bubbles will not carry over Pb compds
from impregnated glass wool plug into absorption flask.
F. Color Development
After 30 min, disconnect rubber tubing, leaving delivery tube
in receiving vessel so that any Hg arsenide on tube will be
exposed to color-developing reagents. Add 1.0 mL NH 4
molybdate reagent, (e), and mix by forcing air thru delivery
AOAC Official Methods of Analysis (1990)
BUQUINOLATE
95
tube. Add 1.0 mL hydrazine sulfate reagent, (f), and again
mix. Heat in boiling H 2 bath 20 min. Rinse delivery tube
with H 2 and remove. Cool to room temp., dil. to 50 mL,
and mix. Filter thru tight glass wool plug in funnel or centrf.
(Do not use filter paper, as color will be adsorbed.) Read A
against H 2 at 5:750 nm. Max. A is at 840 nm. Det. As con-
tent from std curve.
As x 2.90 = arsanilic acid; As X 2.24 - arsenosobenzene;
As X 3.51 = 3-nitro-4-hydroxyphenyIarsonic acid; As x 3.3
= 4-nitrophenyl arson ic acid; As X 3.47 = /?-ureidobenzenear-
sonic acid.
Refs.: Ind. Eng. Chem. Anal. Ed. 15, 408(1943); 24,
1821(1952). Sandell, "Colorimetric Determination of
Traces of Metals," 3rd ed., 1959. JAOAC 40,
455(1957).
CAS-7440-38-2 (arsenic)
960.62* Bithionol in Feeds
Spectrophotometric Method
First Action 1960
Final Action 1961
Surplus 1970
See 38.035-38.037, 11th ed.
967.34 Buquinolate in Feeds
Fluorometric Method
First Action 1967
Final Action 1972
A. Principle
Buquinolate is extd from feed with CHC1 3 , coned to small
vol., and sepd from interfering substances by TLC utilizing 2
solv. systems. Buquinolate is eluted from substrate and detd
fluorometrically.
B. Reagents
(a) Alcohol, 80%.— Dil 84.3 mL alcohol to 100 mL with
H 2 0.
(b) Developing solvent. — Mix CHC.l 3 with alcohol (10 +
1). Prep, fresh daily.
(c) Buquinolate std solns. — (/) Stock soln. — 0.5 mg/mL.
Dissolve 50.0 mg Buquinolate Ref. Std (available from Nor-
wich Eaton Pharmaceuticals, Inc., 17 Eaton Ave, Norwich,
NY 13815) in CHC1 3 to make 100 mL. Warm mixt. on steam
bath as necessary. Soln is stable 1 month if protected from
evapn. (2) Working soln. — 100 jmg/mL. Pipet 5 mL stock soln
into 25 mL vol. flask, dil. to vol. with CHC1 3 , and mix well.
Prep, fresh daily.
C. Apparatus
(a) Developing tanks. — Line developing tanks (for plates
<20 x 20 cm) with Whatman 3 MM paper. Add 100 mL
CHC1 3 to one tank; add 100 mL developing solv., (b), to sec-
ond tank. Prep, each tank fresh daily.
(b) Plates for TLC. — Clean plates thoroly with alkyl ben-
zene sulfonate- type detergent (Ajax, or equiv.) and brush; rinse
plates with H 2 and then with acetone. Let plates air dry. Slurry
60 g silica gel G (Brinkmann No. 68-00-261-3) with 120 mL
H 2 0. Pour into suitable applicator and spread 0.500 mm layer
on 20 x 20 cm plates. Air dry 15-30 min; then dry 2 hr at
110°. Cool and store plates in desiccator until used.
(c) Fluorometer. — Either spectrophotofluorometer or filter
fluorometer may be used. (Suitable filters are: excitation, PTR
Optics type UV-7E (UV Spectrum Filter, No. 59-07-9, PTR
Optics Corp., 145 Newton St, Waltham MA 02154); emis-
sion, Kopp Glass Co. filters C7380 and C5840.) (Caution: See
safety notes on photo fluoro meters.)
D. Determination
(Caution: See safety notes on hazardous radiations.)
Grind ca 100 g sample to pass No. 30 sieve and mix thoroly.
Accurately weigh sample contg 1.25 mg buquinolate into 250
mL g-s erlenmeyer. Pipet 100 mL CHCl 3 into sample flask.
Shake mech. 1 hr. Filter ext thru Whatman No. 54 paper on
buchner with mild vac. (Take care to prevent solv. loss by
evapn.) Transfer exactly 80 mL ext to 150 mL beaker and
evap. almost to dryness on steam bath. Take up residue in
small portion CHC1 3 and transfer to 10 mL vol. flask with
small portions CHC1 3 . Dil. to vol. with CHC1 3 and mix well.
Apply 250 fxL sample ext and 250 |xL working std soln to
TLC plate. Place spots ca 25 mm from bottom of plate and
40 mm apart. (Do not touch pipet to plate.) Develop plate in
CHCI3 developing tank, (a), until solv. front nearly reaches
top of plate (ca 1 hr). Observe plate under short wavelength
UV light: Buquinolate remains at origin; feed background mi-
grates. Transfer air-dried (5-10 min) plate to tank contg de-
veloping solv., (b). Let plate develop until solv. front ad-
vances 12 cm. Air dry 5-10 min. Examine plates under short
wavelength UV light. Buquinolate migrates from origin (R { ,
0.4-0.6). With spatula, outline each buquinolate spot plus blank
spot of equiv. area and R { . Remove adsorbent from around
buquinolate spots and discard. Quant, transfer each spot to sep.
g-s 25 mL erlenmeyers. Pipet 10 mL 80% alcohol, (a), into
each flask, shake mech. 20 min, and centrf.
Det. intensity of fluorescent radiation (7) of sample, std, and
blank in 10 X 10 mm silica cells, at excitation and emission
wavelengths of 265 and 375 nm, resp.
% Buquinolate
1A * sample
4iank)/(^sl
k )j X (0. 1 25 /g sample)
Ref.: JAOAC 50, 264(1967).
CAS-5486-03-3 (buquinolate)
963.33* Cadmium Anthranilate
in Feeds
Spectrophotometric Method
First Action 1963
Final Action 1964
Surplus 1974
See 42.046-42.047, 12th ed.
977.35 Carbadox in Feeds
Spectrophotometric Method
Final Action 1981
(Applicable to levels >0.0055%. Carbadox solns are light sen-
sitive. Exts must be protected from direct sunlight or artificial
light.)
A. Apparatus
(a) Filter aid. — Celite 545 (Manville Filtration and Min-
erals) or Millipore prefilter pad (No. AP2504700, Millipore
Corp., Ashby Rd, Bedford, MA 01730), or equiv.
(b) Spectrophotometer. — For use at 520 nm.
96
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
B. Reagents
(a) Carbadox std solns. — (J) Stock soln. — 1.10 mg/mL.
Weigh 110.0 mg Carbadox Ref. Std (available from Pfizer,
Inc., Quality Control, Agricultural Div., 1 107 S Rt 291, Lee's
Summit, MO 64048) into 100 mL vol. flask, dissolve in CHC1 3 -
MeOH (3 + 1), and dil. to vol. with same sol v. Ultrasonic
bath speeds dissoln. Prep, fresh daily. (2) Working soln. —
0. 1 10 mg/mL. Pipet 10 mL stock soln into 100 mL vol. flask,
dil. to vol. with CHCI 3 -MeOH (3 + 1), and mix well. Prep.
fresh daily.
(b) Methanolic hydrochloric acid: soln. — IN. Dil. 85 mL
HCl'to 1 L with MeOH.
(c) Methanolic sodium hydroxide soln. — 0.057V. Dissolve
2.0 g NaOH in MeOH and dil. to 1 L with MeOH. Prep, fresh
weekly or sooner if ppt forms.
(d) Potassium phosphate soln. — \M. Dissolve 136gKH 2 P0 4
in H 2 and dil. to 1 L.
(e) Sodium hydroxide -sodium chloride soln. — Dissolve 100
g NaCl in 0.17V NaOH and dil. to 1 L with OAN NaOH.
(f) Stannous chloride soln. — Prep, immediately before use.
Add 8.0 g SnCl 2 .2H 2 to 100 mL methanolic IN HC1. Place
in 55-60° H 2 bath and swirl intermittently until soln is clear
(ca 20 min). Stopper and cool to room temp. Use within 2 hr.
C. Preparation of Samples
(Caution: See safety notes on chloroform and methanol.)
Weigh duplicate portions ground feed into 250 mL erlen-
meyers: 2.000 g for 0.0330-0.0606% carbadox; 5.000 g,
0.0110-0.0330%; and 20.00 g, 0.0055-0.0110%. Wet each
portion with 10 mL H 2 0, let stand 5 min, and add 140 mL
CHCl 3 -MeOH (3 + 1), Add 15.0 mL working std soln to 1
portion. Stopper both flasks loosely or with polyethylene stop-
per with pinhole, and boil gently 1 hr. Cool to room temp.
Using three 25 mL portions CHCl 3 -MeOH (3 + 1), quant,
transfer mixt. to buchner precoated with Celite or contg pre-
f ilter pad, collecting filtrate under vac. in 250 mL vol. flask.
Dil. to vol. with CHCl 3 -M.eOH (3 + 1), and mix well. Pipet
100 mL aliquot into 250 mL separator contg 50 mL NaOH-
NaCl soln. Shake 10 sec and discard lower CHC1 3 layer. Add
50 mL CHCI 3 , shake 10 sec, and discard CHC1 3 layer. Add
10 mL KH 2 P0 4 soln, and ext with three 50 mL portions CHC1 3 ,
combining exts in r-b flask. Do not let any solids at interface
drain into flask. Evap. to dryness, using rotary evaporator and
60° H,0 bath.
Conduct reagent blank of H 2 and CHCl r MeOH (3 + 1)
thru boiling, filtration, extns, and evapn, omitting addn of feed
and carbadox.
Alternatively, weigh samples as above and ext as follows:
Wet each portion with L0 mL H 2 0, let stand 5 min, and add
140 mL CHCla-MeOH (3 + 1) to one flask. Prep, spiked sam-
ple by adding 130 mL CHCl 3 -MeOH (3 + 1) and 10 mL car-
badox working std soln to other flask. Break up any clumps
with spatula. Stopper tightly and let stand overnight in dark at
room temp. Using three 25 mL portions of CHCl 3 -MeOH (3
+ 1) quant, transfer mixt. to buchner precoated with Celite or
contg pref ilter pad, collecting filtrate under vac. in 250 mL
vol. flask for unspiked sample and in 500 mL vol. flask for
spiked sample. Proceed as above, beginning "Dil. to vol. with
CHCl 3 -MeOH ..."
D. Determination
Dissolve residue in flask from sample, sample plus std, and
blank in 5.00 mL 0.05/V methanolic NaOH. Add 20.0 mL SnCl 2
soln, swirl gently, and let stand 10 min for complete color
development. If necessary, clarify soln by filtration thru small
glass wool plug. If alternative overnight leach was used, clar-
ify soln by centrifg 10 min at 100 rpm. Within 15 min after
completion of color development, det. A of clear solns at 520
nm against MeOH as ref. sol v. Subtract A of blank from A of
sample and A' of sample plus std,
% Carbadox = (A/g sample) x [\/(A' -A)]
X (mg carbadox /mL working std soln)
x (l g/1000 mg) x 15 mL aliquot X 100
When alternative overnight leach was used, change calcn to:
% Carbadox = (A/g sample) x [1/(2 A' - A)]
x (mg carbadox/mL working std soln)
X (1 g/1000 mg) X 10 mL aliquot X 100
Refs.: JAOAC 60, 1059(1977); 62, 982(1979).
CAS-6804-07-5 (carbadox)
969.55 Decoquinate in Feeds
Fluorometric Method
First Action 1969
Final Action 1972
A. Principle
Decoquinate is extd from feed with 1% CaCL-MeOH soln.
After addn of H 2 and acid, drug is extd into CHC1 3 , then
sepd from interfering materials by chromatgy on Florisil. De-
coquinate is eluted from column with 1% CaCL-MeOH and
detd by fluorometry against std treated similarly.
B. Reagents
(a) Calcium chloride-methanol soln. — 1%. Dissolve 10 g
anhyd. CaCl 2 , reagent grade, in 1 L MeOH, spectral grade
(EM Science OmniSolv No. MX0488), or equiv. redistd, re-
agent grade, anhyd. MeOH. Filter thru Whatman No. 2 paper.
(b) Decoquinate std solns. — (1) Stock soln. — 300 |mg/mL.
Weigh 30 mg Decoquinate Ref. Std (available from Hess &
Clark Inc.). Dissolve and dil. to 100 mL with .1% CaCU-MeOH
soln. Prep, fresh monthly. (2) Working soln. — 6 [xg/mL. Pi-
pet 5 mL stock soln into 250 mL vol. flask and dil. to vol.
with 1% CaCl 2 -MeOH soln. Checks of this soln in 1 cm quartz
cells at 265 nm against spectral grade MeOH (ca 0.660). Prep,
fresh std when A is outside range 0.620-0.700. Soln is stable
>1 week. (3) Fluorescence reference soln. — 1.5 [xg/mL. Pi-
pet 25 mL working std soln into 100 mL vol. flask and dil.
to vol. with spectral grade MeOH. Check A at 265 nm as above.
Prep, fresh std when A is outside range 150-0.190.
(c) Florisil. — 100-200 mesh (Fisher No. F-101).
C. Apparatus
(a) Chromatographic columns. — Draw 30 cm length of 9
mm tubing (7 mm id) to drip tip. Insert small glass wool plug
to support adsorbent. Close drip end with short piece of tubing
and pinch clamp. Add 5 mL CHC1 3 to column, then 0.5 ±
0.01 g Florisil. Add 2 mL addni CHC1 3 and stir with thin glass
rod to settle adsorbent. Remove tubing and wash down sides
of tube with CHC1 3 . Prep, just before use.
(b) Separators. — 125 or 250 mL with Teflon stopcocks.
(c) Fluorometer. — (Caution: See safety notes on photo-
fluorometers.) Either spectrofluorometer or filter fluorometer
may be used. Excitation filter: UV-2 (UV Spectrum Filters
No. 14-16-8, 325 nm); emission filter: S/UV (UV Spectrum
Filters No. 14-01-4, 390 nm) (PTR Optics Corp., 145 Newton
St, Waltham, MA 02154).
D. Determination
Weigh 10 g sample into 125 mL erienmeyer, add exactly
50 mL 1% CaCL-MeOH soln, stopper, and shake mech. 20
AOAC Official Methods of Analysis (1990)
DlBUTYLTIN DlLAURATE
97
min. Decant soln into centrf. tube and centrf. 5 min at mod-
erate speed. Pipet 10 mL clear supernate into 125 mL sepa-
rator. Prep, std by pipetting 10 mL working std soln into an-
other separator. Add exactly 10 mL CHC1 3 to each funnel by
pipet and swirl to mix. Add 100 mL dil. HC1 (1 + 19) to each
funnel. Shake gently by inverting 25 times; then allow 15 min
for phases to sep. Drain CHC1 3 layer into centrf. tube and centrf.
5 min. Remove by aspiration any droplets of floating H 2 phase
that seps.
Pipet 5 mL CHC1 3 sample soln onto Florisil column. Pipet
5 mL CHC1 3 std soln onto another column. Pipet 5 mL CHC1 3
onto third column (reagent blank). Pass two 10 mL portions
anhyd. MeOH thru each column. Let MeOH drain to surface
of Florisil and discard column effluent. Elute with 15 mL 1%
CaCl 2 -.M.eOH soln, collecting in tube marked at 15 mL. Mix
well, centrf. if not clear, and transfer to fluorometer cells. Set
activation wavelength of fluorometer at 325 nm and emission
wavelength at 390 nm. Set fluorometer sensitivity with fluo-
rescence ref. std to give convenient scale reading (e.g., 100).
Det. fluorescence of samples, std, and reagent blank. Subtract
reagent blank correction, if any, from reading of std and sam-
ples.
% Decoquinate in feed - (0.003 x corrected fluorescence
of sample) /corrected fluorescence of std.
Ref.: JAOAC51, 1279(1968).
CAS- 18507-89-6 (decoquinate)
977.36 Dibutyitin Difaurate in Feeds
Atomic Absorption Spectrophotometry Method
First Action 1977
Final Action 1979
A. Principle
DBTD is extd from feed with CHC1 3 , ext is filtered to re-
move feed particles, and aliquot is coned in presence of MeOH
until CHCI3 is removed. MeOH soln is dild and filtered to
remove feed interference, and Sn is detd by AA using air-C 2 H 2
flame.
B. Apparatus and Reagents
(a) Atomic absorption spectrophotometer. — Double beam,
operated at 286.3 nm with air-C 2 H 2 flame and direct readout
using 10 mv recorder. Optimize instrument according to man-
ufacturer's instructions.
(b) Hot plate. — Regulated to ±3°.
(c) Mechanical shaker. — Wrist-action type (Burrell Corp.,
or equiv.).
(d) Tin std solns. — (/) Stock soln. — 500 (xg/mL. Accu-
rately weigh ca 0.217 g dibutylin bis(2-ethy1hexanoate) (NIST
SRM No. 1057 or Eastman Kodak No. 10427, % Sn certified)
into 100 mL vol. flask, and dissolve and dil. to vol. with MeOH.
(2) Working soln. — 10 jxg/mL. Pipet 2 mL stock soln into
100 mL vol. flask, add 1.0 mL HC1, and dil. to vol. with
MeOH.
C. Preparation of Sample and Extraction
Grind sample in high-speed blender to pass No. 20 sieve (ca
3 min), and mix thoroly. Accurately weigh aliquot contg ca
10 \ig Sn/mL in final soln (see Table 977.36), and transfer
to 125 mL erlenmeyer. Add 50 mL CHC1 3 , mix, and place
flask in 55-60° H 2 bath. Let sample reach bath temp.; then
stopper tightly. Continue heating addnl 30 min, swirling oc-
casionally. Remove from H 2 bath and shake mech. 20 min.
Filter thru Whatman No. 4 paper, and collect >30 mL filtrate
in 50 mL erlenmeyer.
Table 977.36 Sample Weights for DBTD-Containing Feeds
%DBTD
Feed Sample, g
0.020 (Polystat)
0.0375 (Tinostat)
0.0700 (Wormal)
0.1400 (Wormal)
13.00
7.00
3.75
2.00
Pipet 25 mL filtrate into 100 mL graduated beaker, add 2
boiling chips and 0.25 mL HO, and cone, to ca 10 mL at
gentle boil on hot plate. Add 20 mL MeOH and cone, sample
to ca 10 mL again; then repeat with addnl 20 mL and 30 mL
portions MeOH. (Raise temp, of hot plate to maintain gentle
boiling as ratio of MeOH to CHCI3 increases.) Remove from
heat and let cool to room temp. Transfer MeOH soln to 25
mL vol. flask, washing beaker and funnel with 2-3 five mL
portions MeOH, dil. to vol. with MeOH, and mix thoroly.
Filter thru Whatman No. 42 paper and collect filtrate in an-
other 25 mL vol. flask.
Prep, blank by dilg 1 mL HC1 to 100 mL with MeOH.
D. Determination
(Caution: See safety notes on AAS.)
Let spectrophtr warm up thoroly and equilibrate by aspir-
ating MeOH 15 min, using air-C 2 H 2 flame and triple slot burner
head. Zero spectrophtr by aspirating blank; then aspirate sam-
ple and std solns, using conditions given in (a). Repeat se-
quence for each sample.
% DBTD - A x C X 50 x 5.32 x 10" 6 x (lOO/A') x W
= (A/A') x (0.266/W)
where A and A' refer to sample and std, resp.; C — g std/mL;
and W - g sample.
Ref.: J AOAC 60, 1054(1977).
CAS-77-58-7 (dibutyitin dilaurate)
956.10* Diethylstilbestrol in Feeds
Spectrophotometric Method
First Action
Surplus 1988
See 42.059-42.062, I4th ed.
970.85 Dimetridazole in Feeds
Spectrophotometric Method
First Action 1970
Final Action 1988
A. Principle
Dimetridazole is extd from feeds with MeOH, sepd from
interfering substances by two alumina chromatgc steps, and
detd spectrophtric at its UV wavelength max. Nihydrazone,
furazolidone, zoalene, 2-chIoro-4-nitrobenzamide, tylosin, and
large amts procaine (from procaine penicillin) interfere.
B. Apparatus and Reagents
(a) Spectrophotometer. — For use in UV.
(b) Chromatographic tubes. — 13 x 150 mm and 15 x 250
mm, constricted at bottom to hold glass wool plug and 6 mm
od delivery tube.
(c) Aluminum oxide. — Suitable for chromatgy, 961.24B(b).
To det. suitability of alumina, perform detn on feed that does
not contain dimetridazole or other imidazole drugs. If feed ap-
98
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
pears to contain >0.004% dimetridazole, use another batch of
alumina.
(d) / ,2 -Dimethyls -nitroimidazole {dimetridazole) std
solns. — (/) Stock soln. — 0.1 mg/mL. Weigh 100 mg dime-
tridazole std into 100 mL vol. flask. Dissolve in H 2 by shak-
ing frequently ca 20 min. Dil. to vol. with H 2 and mix. Pipet
20 mL into 200 mL vol. flask, dil. to vol.. with H 2 0, and mix.
(2) Working solns.— Pipet 5, 10, 20, 30, and 40 mL stock
soln into sep. 100 mL vol. flasks. Add 5.0 mL 3iV HC1 to
each, immediately dil. to vol. with H 2 0, and mix. Pipet 5 mL
each soln and 5 mL 0.10/V NaOH into sep. 50 mL erlenmey-
ers. Stopper and mix. These solns contain 2.5, 5, 10, 15, and
20 |JLg dimetridazole/mL.
C. Preparation of Standard Curve
Proceed as in 970. 85F, using working std solns and blank
prepd by mixing 5 mL 0.\5N HC1 with 5 mL 0.107V NaOH.
Read A against blank for recording or manual spectrophtrs.
Construct std curve by plotting A against jxg dimetridazole/
mL.
D. Preparation of Sample
Weigh portion finely ground feed contg 0.5-2.0 mg di-
metridazole (usually 5 g) into 100 mL vol. flask. Add 70-75
mL MeOH and place in 60° const temp, bath 30 min. Make
certain that H 2 level covers flask to ca 3 mm below MeOH
level. Swirl flask 2 or 3 times during first 5 min to heat evenly.
Cool to room temp., dil. to vol. with MeOH, and mix. Let
stand 5-10 min to let coarse feed particles settle.
E. Chromatography
Place small glass wool plug in bottom of 250 x 15 mm
chromatgc tube and add 8 cm layer alumina; pack column tightly
to prevent streaking. (If streaks enter effluent, pos. bias is in-
troduced.) Decant methanolic ext onto column so that settled
feed particles are not disturbed. Collect ca 30 mL eluate in 50
mL vol. flask. Stopper until ready for use.
(Note: Dimetridazole sublimes at temps >70°; manner of
sol v. removal is critical.) For feed contg 0.015% dimetrida-
zole, pipet 15 mL effluent (4 mL if feed contains 0.06%; 3
mL if feed contains 0.10%) into 125 mL suction or r-b flask
and evap. under reduced pressure from H 2 aspirator. If 15
mL is taken, use hot plate (low heat) or H 2 0-bath to reduce
to 3-4 mL. Shake to prevent bumping. When vol. approaches
3-4 mL remove flask from heat and remove last 3-4 mL only
with heat from palm of hand. Continue shaking to prevent
bumping. Do not attempt to attain complete dryness because
part of the 2-3 drops of oily residue is dimetridazole.
Wash down walls of flask, beginning at base of neck, with
5.0 mL 0.10AT NaOH. Swirl to wash walls. Let stand 5 min
and add 5.0 mL 0.157V HO. Swirl to mix and wash flask walls.
Stopper until ready for chromatgy.
F. Determination
Prep, second alumina column by inserting small glass wool
plug into bottom of 150 x 13 mm chromatgc tube, add 4 cm
layer alumina, and tap gently to pack column lightly. Pour
entire 10 mL soln onto column and let pass thru by gravity.
Collect effluent in 50 mL erlenmeyer. Force out liq. adhering
to column by applying air pressure with rubber bulb. Swirl
flask to mix. Stopper until ready to read. Pass blank soln of
5 mL 0.157V HC1 and 5 mL 0.107V NaOH thru sep. 4 cm alu-
mina column as above.
(a) Using recording spectrophotometer . — Fill matched pair
silica cells with reagent blank and with sample soln (always
use same cell for blank) and scan from 330 to 310 nm. Read
A at peak and obtain concn of soln in [xg/mL from std curve.
% Dimetridazole = [((xg/mL from std curve)
x diln factor x 100]/(g sample X 10 6 )
where diln factor = 66.67 for feeds contg 0.015%; 250.0,
0.06%; and 333.3, 0.1%.
(b) Using manual spectrophotometer . — Locate peak A of
sample soln (ca 318 nm), using matched pair silica cells, and
set wavelength at peak. Read A of sample and blank solns and
correct sample for blank. Obtain concn of soln in |ag/mL from
std curve, and calc. % in feed as above.
Refs.: JAOAC 48, 301(1965); 53, 646(1970).
CAS-55 1-92-8 (dimetridazole)
964.29 Ethopabate in Feeds
Colorimetric Method
First Action 1964
Final Action 1967
A. Principie
Ethopabate is extd from feed by 50% MeOH at room temp.
Clear filtrate is acidified with dil. HC1 and extd with CHC1 3 .
Most interfering substances (amines, p-aminobenzoic acid,
procaine) are sepd. CHCI3 ext is washed with Na 2 C0 3 soln to
remove sulfaquinoxaline, acetyl-(/;>-nitrophenyl) sulfanila-
mide, and chlortetracycline. Ethopabate is converted to free
amine by controlled acid hydrolysis. Free amine is diazotized
and coupled; colored complex is extd with w-BuOH and read
at 550 nm.
B. Reagents
(a) Dilute hydrochloric acid.—0.3N. Dil. 25 mL HC1 with
H 2 to 1 L.
(b) Sodium carbonate soln. — Dissolve 40 g anhyd. Na 2 C0 3
in H 2 and dil. to 1 L.
(c) Coupling reagent (NED). — Dissolve 50 mg iV-(l-naph-
thyl)ethylenediamine.2HCl in 25 mL H 2 0. Prep, fresh as
needed.
(d) Ethopabate std solns. — (J) Stock soln. — 0.400 mg/mL.
Weigh 40.0 mg Ethopabate Ref. Std (available from Merck &
Co.) into 100 mL vol. flask, dissolve in MeOH, and dil. to
vol. (2) Intermediate soln. — 40 fig/mL. Pipet 10 mL stock
soln into 100 mL vol. flask, dil. to vol. with aq. MeOH (1 +
1), and mix well. Stored in tightly stoppered flasks, solns are
stable >1 month. (3) Working soln. — 16.0 (xg/20.0 mL. Pipet
5 mL intermediate soln into 250 mL vol. flask, dil. to vol.
with aq. MeOH (1 + 1), and mix well.
C. Extraction
Grind feed sample to pass No. 20 sieve and mix thoroly.
(High-speed blender grinds most feeds to desired fineness in
ca 3 min.) Accurately weigh sample contg ca 80 jutg ethopabate
(do not exceed 20 g). Transfer to 250 mL g-s flask. Add 100.0
mL aq. MeOH (1 + 1) and mag. stirrer bar, stopper tightly,
and stir 1 hr. (Mech. shaker that provides vigorous agitation
may be used.) Centrf. , or filter portion of ext thru fast paper.
Collect only enough filtrate to supply aliquot for test. If nec-
essary, store exts overnight at room temp, in tightly stoppered
flasks.
D. Removal of Interferences
Pipet 20 mL clear ext into 50 mL centrf. tube. Add 5.0 mL
dil. HC1 (1 + 9) and 10 mL CHC1 3 , stopper with polyethylene
stopper, and shake vigorously 3 min on mech. shaker. Centrf.,
and carefully transfer bottom CHC.I3 layer into clean 50 mL
centrf. tube, using syringe with long needle. Repeat extn with
two more 10 mL portions CHC1 3 . Add 10 mL Na 2 C0 3 soln to
AOAC Official Methods of Analysis (1990)
Furazolidone
99
combined CHC1 3 exts, stopper, and shake 3 min. Centrf. , and
without disturbing interface, draw off most of top aq. layer,
using syringe, and discard. Repeat washing with another 10
mL Na 2 C0 3 soln, discarding washing. Add 10 mL H 2 to CHC1 3
ext, stopper, shake vigorously ca 1 min, and centrf. Draw off
aq. layer and discard. Repeat with another 10 mL H 2 0. (To
avoid loss of drug and low results, do not disturb interface on
CHC1 3 , and complete extn and washings in shortest time pos-
sible. Prolonged contact with HC1 and Na 2 C0 3 may cause par-
tial hydrolysis of ethopabate.)
Pipet 20 mL aq. MeOH (1 + 1) into 50 mL centrf. tube,
add 5.0 mL dil. HC1 (1 + 9), and proceed as for sample (re-
agent blank).
Pipet 20 mL ethopabate working std soln (16.0 jxg) into 50
mL centrf. tube, add 5.0 mL dil. HO (1 +9), and proceed
as for sample (std).
E. Conversion of Ethopabate to Free Amine
Quant, transfer washed CHC1 3 exts to sep. 100 mL beakers.
Rinse each centrf. tube with two 3 mL portions aq. MeOH
(I 4- 1), adding rinsings to beaker. Place beaker on steam bath
and evap. CHC1 3 to vol. of ca 2 mL. Add 5.0 mL aq. MeOH
(1 + 1) and swirl beaker to dissolve completely.
Quant, transfer soln to r-b centrf. tube. Rinse beaker with
10, 1.0, and 5 mL portions 0.3N HO. Immerse tube in boiling
H 2 bath so that level of liq. in tube is just below level of
H 2 bath. Heat 45 min. Remove tube from hot H 2 bath and
cool to 10-15° in cold H 2 bath.
F. Development and Measurement of Color
Remove tubes from cold H 2 bath. Add 1.0 mL freshly prepd
0.2% NaN0 2 soln to each tube, mix, and let stand 2 min. Add
1.0 mL 1.0% NH 4 sulfamate soln, mix, and let stand 2 min.
Add 1.0 mL NED soln, mix, and let stand 10 min. Add 5.0
g NaCI and 5.00 mL n-BuOH, stopper, and shake vigorously
until NaCI dissolves. Centrf., carefully transfer portion of clear,
colored ale. layer to 1 cm cell, and read A at 555 nm against
/7-BuOH. Correct for reagent blank.
% Ethopabate in feed
= 0.008 x (A - A B )/[(A' ~ A B ) x W]
where A, A B , and A' refer to sample, reagent blank, and std,
resp., and W = g original sample.
Refs.: JAOAC 47, 221(1964); 48, 280(1965).
CAS-59-06-3 (ethopabate)
985.51 Furazolidone in Feeds and Premixes
Liquid Chromatographic Method
First Action 1985
Final Action 1988
(Applicable to premixes contg 2-22% furazolidone and to feeds
contg 0.005-0.05% furazolidone. Note: Furazolidone solns are
light-sensitive. Protect exts and stds from direct sun and
artificial light.)
(Caution: See safety notes on centrifuges, distillation, pipets,
acetonitrile, acetone, and dimethylformamide.)
A. Principle
Unground premix is extd with DMF, and concn of ext is
adjusted with 5% tetraethyl ammonium bromide (TEAB) to ca
55 jmg furazolidone/mL for LC. Complete feed is extd with
H 2 0~acetone on continuous extn app., solv. is evapd, and res-
idue is dissolved in DMF. 5% TEAB is added to sep. fat. Ext
is cooled and clarified for LC.
B. Apparatus
(a) Liquid chromatograph. — Instrument capable of main-
taining const pulseless flow of mobile phase at 0.5-1.5 mL/
min. Operating conditions: flow rate 1.5 mL/min; loop injec-
tion vol. 20 julL; detector sensitivity 0.32 AUFS or adjusted to
produce working std peak response 60-80% full scale; detec-
tor wavelength 365 nm (settings from 390 to 405 nm may be
used to improve selectivity for very low level samples if chro-
matgc column does not adequately resolve interfering peaks).
(b) Chromatographic column. — Any reverse phase col-
umn, Cig or C 8 , with particle size <10 |mm that will produce
single, sharp furazolidone peak with peak skew <\A. Guard
column may be used.
(c) Continuous extraction apparatus. — Goldfisch (Lab-
conco Corp., 8811 Prospect Ave, Kansas City, MO 64132,
No. 3001), or equiv.
(d) Extraction thimbles. — Whatman, single thickness, 19
x 90 mm (Scientific Products,' Inc., No. E6480-4).
(e) Sample clarification filter . — 13 mm glass fiber pre-filter
pads (Gelman No. 66073, available from Fisher Scientific Co.,
No. 09-731 A) inserted in 0.5 in. id 5 mL syringe barrel
(Pharmseal, Scientific Products, No. S9504-5) or any filtration
device designed for clarification of aq. chromatgc samples.
C. Reagents
(a) Extractants . — DMF, reagent grade, for premixes. Ace-
tone (reagent grade)-H 2 (93 + 7) for complete feeds.
(b) Diluent. — 5% tetraethylammonium bromide (5% TEAB,
w/v) (Eastman Kodak Co., No. 1516) in distd, deionized H 2 0.
Keep in refrigerator. For correct final conens, warm to room
temp, before pipetting.
(c) Furazolidone std solns.— (Y) Stock soln. — Ca 1.1 mg/
mL. Accurately weigh 0. 1 10 ± 0.005 g furazolidone std (Hess
and Clark, Inc., 7th & Orange Sts, Ashland, OH 44805) and
record exact wt to nearest 0.1 mg (WJ. Transfer into 100 mL
vol. flask, dissolve and dil. to vol. with DMF (sonication aids
dissolution). Soln is stable if stored in dark. (2) Intermediate
soln.—Ca 110 |ULg/mL. Dil. 10.0 mL stock soln to 100 mL
with DMF. Soln is stable if stored in dark. (3) Working std
soln. — Ca 55 (xg/mL. Mix 10.0 mL intermediate soln with
equal vol. 5% TEAB. (Mix equal vols. Do not dil. to vol.)
Let soln cool to room temp. Prep, daily. Diln of std (D s ) =
2000.
(d) Mobile phase.— CH 3 CN (LC quality)-2% HOAc in distd,
deionized H 2 (20 + 80), or as adjusted to give capacity factor
(k) of ca 2.5 for furazolidone.
D. Extraction
(a) Complete feeds. — Det. approx. sample wt to contain ca
550 |jLg furazolidone by using formula, sample wt, g = 0.555/
% guarantee. Accurately weigh (to nearest 0.01 g) calcd wt
of ground, mixed sample (±5%) into extn thimble (W u = ac-
tual sample wt). Press cotton plug down onto top of feed to
prevent channeling. Add 45-50 mL acetone-H 2 (93 + 7)
extractant and 2 or 3 boiling chips to extn beaker. Ext 8-18
h on extn app. at "Hi" setting. Evap. solv. on steam bath (stream
of air directed into beakers or blowing across beakers, as with
partially closed hood door, hastens evapn). if any H 2 is ev-
ident in beaker after initial evapn, add ca 25 mL acetone, swirl
to mix, and re-evaporate (repeat as necessary to remove all
H 2 0). Remove from steam bath as soon as evapn is complete.
Add 5.00 mL DMF, heat on steam bath just until bottom of
beaker is hot (15-30 s) and swirl, washing sides, to dissolve
residue (generally all residue dissolves in warm DMF). Add
5.00 mL 5% TEAB, mix, pour soln into 15 mL centrifuge
tube, and let cool. Centrifuge 5-10 min at 2000 rpm (2500 X
g). Using disposable pipet attached to aspirator and trap, re-
100
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
Table 985.51 Sample Sizes, Dilutions, and Total Sample
Dilutions for Assay of Furazolidone in Feeds
Total
Label
Sample
Claim,
Sample Wt,
Dilns with
Diln (D u ),
%
g
DMF, mL
mL
2.2
1.00
none
400
3.3
1.00
30/50
666.7
11.0
1.00
20/100
2000
22.0
1.00
10/100
4000
move fat layer floating on supernate. Diln for complete feed
samples (£> u ) = 10.
(b) P remixes. — Accurately weigh (±5%) amt unground
sample indicated in Table 985.51 into 500 mL g-s erlenmeyer
(W u = actual sample wt). Add by pipet 200.0 mL DMF, stop-
per, and shake flask 30 min. Either let suspended material set-
tle or centrf. or filter portion of ext. Dil. with DMF to ca 1 10
jjig furazolidone/mL. To 5.00 mL 5% TEAB soJn add 5.00
mL dild ext, mix, and let soln cool to room temp. Clarify as
for complete feeds, (a). (Total sample diln = D u , Table 985.51.)
E Determination
(a) Complete feeds, — Make several injections of furazoli-
done working std soln, adjusting mobile phase strength to give
k ca 2.5 and peak ht 60-80% full scale (k = (A, - t )/t , where
t = distance from injection to first perturbation of std chro-
matogram). Make 2 or more injections of std to ensure 1-2%
repeatability of peak responses. Bracket each 2 sample injec-
tions by std injections. Use av. peak ht P s (or av. peak area)
of stds bracketing each pair of samples to calc. furazolidone
concn in samples (sample peak response = P u ). If no drift in
std peak hts is evident thruout run, then use av. for all std
injections in calcns.
(b) P remixes. — Det. furazolidone as for complete feeds (a).
Diln (£> u ) is shown in Table 985.51.
F. Caicuiation
% Furazolidone
= (Pu x W s x D u x ]00)/(P s x W u x D s )
where P u and P s = peak response of sample (unknow r n) and
std, resp.; W u and W s - g sample and std, resp,; and D u and
D & - mL total dilns of sample and stds, resp. Det. total dilns
as in following example: If extn of 1 g sample in 200 mL solv.
is followed by serial dilns of 20/100 and 5/10, then total diln
is 1 g/200 mL X 20/100 X 5/10 - 1 g/2000 mL, and D u
= 2000 mL.
Ref.: JAOAC68, 1033(1985).
CAS-67-45-8 (furazolidone)
960.63 Furazolidone, Nitrofurazone,
or Bifuran in Feeds
Colorimetric Method
First Action 1960
Final Action 1961
A. Reagents
(a) P he ny [hydrazine hydrochloride soln. — Dissolve 0.5 g
phenylhydrazine.HCl in 50 mL H 2 0. Prep, fresh daily. Mix
equal vol. of this soln with HC1.
(b) Furazolidone std solns . — (I ) Stock soln . — . 55 mg/mL .
Weigh 55 mg furazolidone std (available from Hess & Clark,
Inc.) into 100 mL vol. flask, dil. to vol. with dimethylform-
amide (DMF), and mix. Soln is stable several months when
protected from light. (2) Working soln. — Prep, working std
corresponding to label declaration. For feeds contg 0.011%
furazolidone, pipet 2 mL stock soln into 100 mL vol. flask,
add 48 mL DMF, and dil. to vol. with H 2 0. For feeds contg
0.00275% furazolidone, pipet 0.5 mL stock soln into 100 mL
vol. flask, add 49.5 mL DMF, and dil. to vol. with FLO.
(c) Nitrofurazone std solns.— (J) Stock soln. — 0.56 mg/mL.
Weigh 56 mg nitrofurazone std (available from Hess & Clark,
Inc.) into 100 mL vol. flask, dil. to vol. with DMF, and mix.
Soln is stable several months when protected from light. (2)
Working soln. — Prep, working std corresponding to label dec-
laration. For feeds contg 0.0056% nitrofurazone, pipet 1 mL
stock soln into 100 mL vol. flask, add 49 mL DMF, and dil.
to vol. with H 2 0. For feeds contg 0.01 12% nitrofurazone, pi-
pet 2 mL stock soln into 100 mL vol. flask, add 48 mL DMF,
and dil. to vol. with H 2 0.
(d) Bifuran std solns. — (J) Stock soln. — 0.1285 mg/mL.
Pipet 20 mL nitrofurazone stock soln and 3 mL furazolidone
stock soln into 100 mL vol. flask and dil. to vol. with DMF.
(2) Working soln. — For feeds contg 0.0064% total nitrofur-
ans, prep, working std by pipetting 5 mL bifuran stock soln
into 100 mL vol. flask, adding 45 mL DMF, and dilg to vol.
with H 2 0.
(e) Adsorbent. — To 100 parts alumina, 961.24B(b), in screw
cap bottle, add 4 parts Mg(OH) 2 , shake until thoroly mixed,
then add 5 parts H 2 0, and mix until all lumps disappear. Store
in tightly sealed container.
B. Determination
(Caution: See safety notes on pipets, toxic solvents, and di-
methylformamide.)
Grind coarse or pelleted feeds to "20 mesh" thru cutting-
type mill such as Wiley Intermediate. Finer feeds need not be
ground. Weigh 10 g sample into 125 mL erlenmeyer, add ex-
actly 50 mL DMF, stopper loosely, and place in boiling H 2
bath 5 min. Mech. shake 10 min and filter thru rapid paper.
To 25 mL filtrate add 25 mL H 2 and mix.
Prep, ca 20 mm diam. adsorption column, contg adsorbent,
to ht of 5 cm. Pass the 50% DMF sample soln thru column,
discarding first 3 mL eluate. (If column flow stops, break up
gummy film at top of adsorbent, using Jong thin glass rod.)
Pipet 5 mL aliquots of eluate into each of 2 numbered test
tubes. Protect one tube from light. To other tube, add 3 drops
freshly prepd 2% soln ofNa hydrosulfite and let stand 20 min,
shaking at ca 5 min intervals. Treat 5 mL aliquots of working
std soln in exactly same manner.
Pipet 5 mL phenylhydrazine.HCl soln into each of the num-
bered test tubes contg samples and stds. Mix and place tubes
in 70° H 2 bath 25 min; cool in 15° H 2 bath 5 min. Add
exactly 10 mL toluene to each tube, stopper, and shake vig-
orously 40 times. Centrf. or filter toluene soln directly into
absorption cell thru cotton wad inserted in stem of small fun-
nel. Read A of solns at 440 nm.
% Furazolidone
Lv*samp. ^"* red. samp.
% Total nitrofurans (bifuran)
.) x 0.011 (or0.00275)]/(A nd - A icdslc] )
I, v* samp. ^ red. samp
% Nitrofurazone
t\/*samp. ^red.samp.
) x 0.0064]/(/\ s
) X 0.0056 (or 0.011)]/(A std - A rcd , td )
Refs.: JAOAC 40, 463(1957); 41, 333(1958); 43, 310(1960);
44, 30(1961); 52, 233(1969).
CAS-67-45-8 (furazolidone)
CAS-59-87-0 (nitrofurazone)
AOAC Official Methods of Analysis (1990)
Ipronidazole
101
973.80
Furazolidone
and ZoaBene in Feeds
Qualitative Tests
First Action 1973
Final Action 1988
A. Apparatus
See 964.07A.
B. Reagents
(a) Dimethylformami.de {DMF). — Reagent grade.
(b) Alcoholic potassium hydroxide soln. — 4%. Dissolve 4
g KOH in 100 mL alcohol. If premixed with DMF (1 + 9),
prep, fresh daily.
(c) Ethylene diamine . — Use in hood.
C. Preparation of Sample
Gently grind pellet, cube, or crumble forms with mortar and
pestle. Sieve thru nest of Nos. 10, 20, and 30 sieves with pan.
Drugs usually are coned in portion in pan.
D. Identification
(a) DMF test. — Place 9 drops DMF and 1 drop ale. KOH
soln in each of 3 depressions of white spot plate. Sprinkle ca
0.01 g fine feed material into each soln from tip of spatula
while observing reaction under microscope. Furazolidone pro-
duces intense blue color, easily detected at > 0.0025%. Zo-
alene gives bright green, easily detected at >0.0025%. Color
of minute particles of zoalene fades rapidly; color of larger
particles persists 3-5 min.
(b) Ethylenedi amine test. — Place dry filter paper at bottom
of petri dish and sprinkle ca 0.5 g fine feed evenly over paper.
Dispense 2-4 mL ethylenediamine under edge of paper so as
to wet entire paper and sample. Examine under stereoscopic
microscope at 10x for particles developing bright purple, in-
dicating zoalene, or deep red, indicating furazolidone. (Blood
meal, frequently used in livestock feed, also gives deep red
color with reagent.)
Refs.: JAOAC 56, 762(1973); 60, 389(1970).
CAS-67-45-8 (furazolidone)
CAS- 148-0 1-6 (zoalene)
960.64* Glycarbylamide in Feeds
Colorimetric Method
First Action 1960
Final Action 1961
Surplus 1970
&?<? 38.066-38.071 , 11th ed.
971.44 Ipronidazole in Feeds
Gas Chromatographic Method
First Action 1971
A. Principle
Ipronidazole is extd from feed with warm 0.2JV HCl, trans-
ferred to benzene after alkalinization, and measured by GC
with electron capture detector.
B. Apparatus
(a) Gas chromatography — With electron capture detector.
Conditions: temps (°) — column 180 ± 2 (isothermal), injec-
tion 240 ± 2, detector 210 ± 2; N How 60 mL/min (install
3' (0.9 m) x V/ molecular sieve 5 A trap in N line).
(b) Recorder. — 0- I mv input and l.O sec full scale de-
flection, chart speed 0.75"/min, or equiv.
(c) Gas chromatographic column. — 0.9 m (3') X l /s" od
stainless steel tubing packed with 5% Carbowax 20M-tere-
phthalic acid (TPA) on 100™ 120 mesh Chromosorb G, acid-
washed and dichlorodimethylsilane-treated (Applied Science).
(d) Column preparation. — Dissolve 1.0 g Carbowax 20M-
TPA in 30 mL CHC1 3 and let stand over 19.0 g Chromosorb
G 3 days with occasional swirling. After standing, evap. CHC1 3
under N and dry 1 hr in 70° oven. After packing, condition
column 2 days at 200° in slow stream of N before use.
C. Reagents
(a) Benzene. — Redistil from all-glass app., discarding first
and last 10%. (Caution: See safety notes on distillation, flam-
mable solvents, toxic solvents, and benzene.)
(b) Ipronidazole. — Available from Hoffmann-La Roche,
Inc.; prep, std soln contg 0.3 fxg ipronidazole/mL benzene.
D. Chromatography of Standard
Inject 5 )ulL std ipronidazole soln into gas chromatograph
and adjust conditions to obtain ca 5 min retention time. Det.
area under ipronidazole peak as product of peak ht (cm) and
width (cm) at half ht, using slope baseline technic. After sat-
isfactory performance of column is established, inject at least
duplicate 5 |xL aliquots of std soln (1.5 ng/5 |xL) at beginning
of work day and periodically thereafter (e.g., after each 5-6
sample injections). Measure ipronidazole peak area in each case.
E. Preparation of Sample
(Caution: See safety notes on pi pets.)
Ext mash-type feeds without prior grinding. Grind pelleted
feeds to pass No. 30 sieve before extn. (Method for feeds is
described for 0.0060% level of ipronidazole with modifica-
tions indicated for 0.0030 and 0.0090% levels.)
Transfer 10 g sample into 500 mL vol. flask (for 0.0030%
level, use 20 g sample) and add exactly 200 mL 0.27V HCl
previously warmed to 40 ± 3°. Stopper flask and shake mech.
20 min. Let settle few min. Centrf. portion of supernate and
pipet 10 mL (for 0.0090% level, transfer 5.0 mL and add 5
mL 0.2/V HCl) to g-s centrf. tube. Place tube in cold H 2 bath
and add 0.5 mL 5N NaOH to make alk. (pH - 10-12.5; check
with Accutint pH paper; do not hold el u ate at high pH for
prolonged time). Pipet 20 mL benzene into tube, stopper, shake
mech. 5 min, and centrf. 10 min at 2500 rpm. Pipet 5 mL
clear benzene ext into 25 mL vol. flask, dil. to vol. with ben-
zene, and mix. This is sample soln.
F. Determination
Inject 5 uX sample soln into chromatograph. Measure area
of ipronidazole peak obtained from chromatogram.
% Ipronidazole = (B x C x D)/(B' x 10,000)
where B and B' = areas under ipronidazole peak for sample
and std solns, resp.; C = p,g ipronidazole/mL in std soln (0.3);
and D = diln factor (0.0030% level, D - 100; 0.0060%, 200;
and 0.0090%, 400).
Ref.: JAOAC 54, 72(1971); 57, 29(1974).
CAS- 14885-29- 1 (ipronidazole)
980.36 Melengestrol Acetate
in Feed Supplements
Gas Chromatographic Method
First Action 1980
Final Action 1988
(Caution: See safety notes on chloroform, hexane, and
methanol.)
102
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
A. Principle
MGA is extd from aq. slurry of supplement sample with
hexane, partitioned from hexane into aq. MeOH, and then from
aq. MeOH into CH 2 C1 2 . After evapn of CH 2 CI 2 , dried ext is
transferred to alumina column with hexane, and eluted with
CHC1 3 -hexane soln. MGA in el u ate is detd by GC. MGA can
be assayed in cattle feed supplements contg 0.125 mg/lb to
1.00 mg MGA/lb supplement (0.28-2.2 mg/kg).
B. Apparatus
(a) Extractor. — Liq.-liq. (Ace Glass No. 6840-96 or equiv.).
(b) Gas chromato graph.— Tracor Model MT-220 (replace-
ment Model 540, Tracor Instruments Inc.), or equiv., with
63 Ni pulsed electron affinity detector, 0.6 m (2') x 3 mm id
glass column packed with 1% OV-17 on 100-200 mesh Gas-
Chrom Q (Applied Science), and Tracor 1.0 mv recorder with
chart speed of 0.5"/m.in. Operating conditions. — Linde
99.996% high purity N, or equiv., carrier gas 50 mL/min;
purge, off; temps (°) — injector 235, detector 300, column 225;
detector pulse ht 60 V, pulse interval 300 jxsec, sensitivity 80
x 10"" AFS.
(c) Rotary-evaporator. — Valley Electromagnetics, One
Wolfer Park, Spring Valley, IL 61362, or equiv., 976.36B(m).
(d) Chromatographic tube. — 18 x 500 mm, fitted with
coarse porosity fritted glass disc and Teflon stopcock (Fischer
and Porter Co., or equiv.).
C. Reagents
(a) Aluminum oxide. — Woelm acid, anionotropic, activity
grade 1 for column chromatography (ICN Pharmaceuticals or
equiv.).
(b) Cholesteryl chloroacetate (CCA) internal std soln. — (I)
Stock soln. — 500 jig/mL. Dissolve 125 mg (Aldrich Chemi-
cal Co., C7680-9) in 250 mL absolute ethanol-hexane (5 +
95). (2) Working soln I. — 50 fig/mL. Pipet 10 mL stock soln
and dil. to 100 mL with absolute ethanol-hexane. (3) Working
soln II. — 5 (xg/mL. Pipet 10 mL stock soln and dil. to 1 L
with absolute ethanol-hexane (5 + 95).
(c) Medroxyprogesterone acetate {MAP) extraction std
soln. — (/) Stock soln. — 2 mg/mL. Dissolve 200 mg (The Up-
john Co., 7000 Portage Rd, Kalamazoo, Ml 49001) in 100
mL absolute alcohol (requires overnight shaking). (2) Working
solns. — ie, 20, 30 and 60 |xg/mL. Pipet 8, 10, 15 and 30
mL aliquots stock soln and dil. to I L with absolute alcohol
for use with 0.125-0.250, 0.250-0.450, 0.450-0.750, 0.750-
1.00 mg MGA/lb samples, resp.
(d) Melengestrol acetate (MGA). — (I) Stock soln. — 25 |jLg/
mL. Dissolve 100 mg Ref. Std (The Upjohn Co.) in 100 mL
absolute alcohol. Dil. 5.0 mL to 200 mL with hexane. (2)
Working soln. — 1.25 jxg/mL. Dil. 10 mL stock soln to 200
mL with absolute alcohol-hexane (5 + 95).
(e) Gas chromatography reference soln. — CCA, 5 |xg/mL;
MAP 2.4 fxg/mL; MGA, 0.125 u.g/mL. Pipet 10 mL each
CCA (50 u.g/mL) and MGA (1.25 fig/mL) and 4 mL MAP
(60 |mg/mL) and dil. to 100 mL with hexane.
(f) Solvents. — Distd-in -glass hexane and CH 2 C1 2 , pesticide
and gas chromatgy grade (Burdick & Jackson Laboratories, or
equiv.).
(g) Solvent partition soln. — Mix 0.25% aq. Na?S0 4 soln
with MeOH (30 + 70).
D. Extraction
Grind dry samples to pass 1 mm screen. Thoroly mix sample
and place ca 15 g sample, weighed to nearest 0.01 g, into
extractor, and pipet 10 mL appropriate extn std soln, (c), for
level MGA being assayed. Rinse sample to bottom of extractor
with 30 mL H 2 0. Place mag. stirring bar in extractor. Fill to
side arm with hexane and insert solv. return tube in position.
Attach extractor to condenser and 500 mL receiving flask contg
100 mL hexane and several SiC boiling chips. Ext sample 3
hr by heating receiving flask enough to have rapid reflux (^20
mL/min) at condenser while stirring sample vigorously, ft is
imperative that reflux rate be fast enough to completely ext
sample in 3 hr. 250 watt Glas-Col heating mantle operated at
120 v will do this. Control emulsions by regulating stirring
rate. Sample must be stirred vigorously and continuously. Up
to 30 mL addnl H 2 may be added thru condenser to aid stir-
ring action, if necessary. Let apparatus cool, set receiving flask
aside, and transfer extractor contents to 1 or 2 L separator. Let
phases sep. clearly; discard aq. (lower) layer and transfer hex-
ane layer to 2 L r-b flask and roto-evap. just to dryness. Dis-
solve residue in 100 mL solv. partition soln, (g).
E. Solvent Partition
Quant, transfer ext in 500 mL receiving flask to 500 mL
separator. Rinse 2 L flask with 100 mL solv. partition soln,
(g), into receiving flask and then quant, transfer receiving flask
contents into same separator. Gently shake funnel by inverting
15 times, let solv. layers sep. clearly, and drain lower layer
into 1 L separator contg 200 mL CH 2 C1 2 . Repeat rinsing flasks,
transfers, and partitioning with 3 or more 100 mL portions
solv. partition soln. Vigorously shake combined exts in 1 L
separator 15 sec, let phases sep, clearly, and drain lower
(CH 2 C1 2 ) layer into original 500 mL receiving flask. Repeat
partition into CH 2 CI 2 2 more times, using 40 mL CH 2 C1 2 each
time. Roto-evap. combined exts to near dryness. Remove re-
sidual H 2 with >2 sep. addns of 10 mL absolute alcohol and
roto-evap. solv. Remove residual alcohol with two 10 mL por-
tions hexane, roto-evapg each portion. Dissolve residue in 10
mL hexane.
F. Alumina Column Chromatography
Prep, column by slurrying 20 g aluminum oxide in CHC1 3
and transfer with CHC1 3 wash bottle to tube, (d), contg ca 100
mL CHC1 3 . While column drains, let alumina settle and add
5 g Na 2 S0 4 to column while letting CHC1 3 slowly drain. Use
ca 100-150 mL total CHC1 3 in this step. In this and following
steps, it is important to let each portion of solv. added drain
to top qfNa 2 S0 4 layer before adding next portion. CHC1 3 must
contain 0.5-1.0% alcohol. Rinse column with 10-15 mL hex-
ane, followed by three 50 mL hexane washes. While column
drains, quant, transfer ext to column with four 10 mL portions
and one 75 mL portion hexane. Discard all washings collected
to this point. Elute MGA with five 50 mL portions CHCI3-
hexane (33 + 67) solv., using each portion to rinse flask contg
ext; collect 250 mL eluate in 250 mL vol. flask.
G. Determination
Thoroly mix column eluate and pipet aliquot for analysis as
follows: 0.125-0.250 mg MGA/lb, 40 mL; 0.250-0.450 mg
MGA/lb, 30 mL; 0.450-0.750 mg MGA/lb, 20 mL; 0.750-
1.00 mg MGA/lb, 10 mL, and evap. to dryness in 50 mL g-
s r-b flask, using warm (45-60°) hot plate or H 2 bath and
stream of N or air. When aliquot approaches dryness, add ca
5 mL absolute alcohol and evap. to remove residual H 2 0. Re-
peat addn of alcohol if necessary until aliquot dries com-
pletely. Dissolve in 10.0 mL 5 juig/mL CCA working soln II,
(b)(3). Inject 3 julL each of sample and std into gas chromato-
graph operated as in 980.36B(b). Order of elution, min: MAP,
4-6; MGA, 6-8: CCA, 8-1 1 . Make several injections to achieve
reproducible (< ± 5%) peak hts or areas depending on type
and condition of gas chromatograph. // is imperative that GC
response be linear. Check linearity using MGA cones 0.5 and
2.0 times GC ref. soln, (e), concn (0.0625, 0.125, and 0.25
(mg/mL). Keep CCA concn const.
AOAC Official Methods of Analysis (1990)
NlCARBAZIN
103
H. Calculations
Calc. mg MGA/lb sample:
mg MGA/lb
= R X R' X 1.25 X (250/mL aliquot) X (0.4536/W)
where R and R' — peak ht ratios of MG A/CCA in sample and
CCA/MGA in std, resp.; 1.25 - |mg MGA in 10 ml std soln
(e); 250 = mL eluate collected; mL aliquot = 10-40 mL ali-
quot taken; 0.4536 - conversion factor to obtain mg/lb; W =
g sample extd. MAP is added to sample prior to extn to in-
dicate magnitude of extn efficiency and cleanup losses. Reas-
say samples showing <85% MAP recovery after reasons for
low MAP recovery have been detd.
% MAP recovered
= R { X R 2 X 24 x (100/Af) X (250/mL aliquot)
where /? t and R 2 = peak ht ratios of MAP/CCA in sample
and CCA/MAP in std, resp.; 24 - fxg MAP in 10 mL std soln
(e); M. = [Jig MAP added to sample; and (250/mL aliquot) are
defined above.
Ref.: J AOAC 63, 425(1980).
CAS-29 19-66-6 (melengestrol acetate)
971.45* Nequinate in Feeds
Spectrophotofluorometric Method
First Action 1971
Final Action 1974
Surplus 1975
See 42.084-42.087, 1 3th ed.
956.11 Nicarbazin in Feeds
Spectrophotometric Method
Final Action
(Presence of furazolidone, nitrofurazone, or nihydrazone may
cause high results. Confirm presence of nicarbazin by
Identification Test, 956. 11F.)
A. Reagents
(a) Dimethylformamide (DMF). — Reagent grade.
(b) Alumina. —See 961.24B(b).
(c) Alcohol. — Formulas SDA Nos. 2B, 3A, or 30 may be
used.
(d) Alcoholic sodium hydroxide soln. — Dil. 2.0 mL clear
50% NaOH soln, 936.16B(b), to 100 mL with alcohol. Centrf.
in stoppered tube. Prep, fresh daily.
(e) Nicarbazin std solns. — (7) Stock soln. — Weigh 25.0 mg
Nicarbazin Ref. Std (available from Merck & Co.) into 500
mL vol. flask, and dissolve in ca 150 mL DMF with aid of
gentle heat. Cool, dil. to vol. with DMF, and mix well. Store
protected from light. (2) Working soln. — 12.5 |ULg/mL. Trans-
fer 25.0 mL stock soln to 100 mL vol. flask and dil. to vol.
with DMF. Mix well.
B. Preparation of Column
Use glass tube 22 mm id, ca 50 cm long, constricted at
lower end. Place plug of glass wool in constricted end and add
30 g alumina in 3 portions. Tamp each portion with glass rod
while applying gentle suction. Wash column with 25 mL DMF,
draining to point 1-2 cm above bed level before adding sample
to column. Prep, column for each sample and std.
Never let column run dry; keep head of liq. at all times.
C. Preparation of Sample
(Caution: See safety notes on distillation, toxic solvents, and
dimethylformamide.)
Weigh 10.0 g sample into 250 mL erlenmeyer and add 100.0
mL DMF. Heat just to bp on hot plate in hood with intermit-
tent stirring. Cool to room temp, by immersing in H 2 bath.
Decant supernate into centrf. tubes and centrf. 3 min.
D. Determination
(Caution: See safety notes on pipets.)
Pipet 25.0 mL clear ext onto column and let pass thru col-
umn with aid of gentle suction. Wash column with three 10
mL portions DMF and reject washings. Elute with nine 5 mL
portions alcohol, discarding first 15 mL eluate and collecting
next 25 mL eluate in 25 x 200 mm tube. Quant, transfer eluate
into 50 mL vol. flask and dil. to vol. with alcohol. Mix well.
Pipet 25.0 mL working std soln onto another column and
proceed as for sample.
Pipet two 15.0 mL portions sample soln into sep. 25 mL
vol. flasks. To one add 5.0 mL ale. NaOH soln and adjust
vol. of both solns to 25 mL with alcohol. Read A of yellow
soln formed in first flask within 5 min in spectrophtr or col-
orimeter at 430 nm against second soln as blank. Calc. wt
nicarbazin from std curve.
E. Preparation of Standard Curve
Pipet 10, 15, and 20 mL aliquots of chromatgd working std
soln into sep. 25 mL vol. flasks, add 5 mL ale. NaOH, and
dil. to vol. with alcohol. Mix well. Measure A within 5 min
at 430 nm against alcohol.
Prep, std curve by plotting A against jxg nicarbazin.
F. Identification Test
Place alcohol in 1 cm quartz cell and clear chromatgd sam-
ple soln in matched cell. Det. A at 2 nm intervals from 340
to 349 nm with Beckman Model DU spectrophtr, or equiv.,
at min. slit width. Absorption max. at 344 ± 4 nm confirms
presence of nicarbazin.
Refs.: JAOAC 39, 321(1956); 40, 469(1957); 41, 326(1958).
CAS-330-95-0 (nicarbazin)
967.35 Nicotine in Feeds
Spectrophotometric Method
First Action 1965
Final Action 1967
(Applicable in the presence of phenothiazine, dibutyltin di-
laurate, and 2,2'-dihydroxy-5,5'-dichlorodiphenylmethane)
A. Principle
Nicotine is extd with alkali, steam distd, extd with CHCI 3 ,
and detd spectrophtric in acidic soln.
B. Reagents
(a) Dilute hydrochloric acid. — 0.05JV. Dil. 4.1 mL HCI to
1 L with H 2 0.
(b) Nicotine std soln. — 0.012 mg/mL. Accurately weigh
ca 100 mg nicotine and dil. to 100 mL in vol. flask with 0.05/V
HCI. Transfer 3.0 mL aliquot to 250 mL vol. flask and dil.
to vol. with 0.05N HCI. (Caution: Nicotine is very toxic.)
(c) Antifoam. — Antifoam A (Dow Corning Corp.), or equiv.
104
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
C. Apparatus
(a) Distillation flask. — 250 mL r-b flask and CJaisen distg
head or 250 mL Claisen flask.
(b) Condenser. — Graham coil type with 300 mm jacket
(Corning No. 2500, or equiv.). Must be used in vertical po-
sition.
(c) Ultraviolet recording spectrophotometer. — Double beam,
capable of scanning UV spectrum from 220 to 300 nm, with
1 cm cells.
D. Determination
(Detn can be interrupted at any step where soln is acidic.)
Accurately weigh representative portion of feed, ground thru
No. 20 sieve, contg ca 3 mg nicotine and transfer to 250 mL
centrf. bottle. Add 100 mL 0.5% NaOH soln, stopper (Neo-
prene or rubber), and shake vigorously 1 min. Centrf. ca 5
min at 1500 rpm. Decant free flowing and viscous liq. into
400-600 mL beaker. Rinse lip and centrf. bottle into beaker
with few mL H 2 0, being careful not to dislodge solid material.
Repeat extn, centrf g, and rinsing twice, combining supernates
in beaker. Adjust soln to pH 2-3 with HO and evap. on hot
plate to ca 100 mL. Cool, adjust to pH 10-14 with NaOH (1
+ 1), and transfer quant, to distn flask, using min. of H 2 0.
Vol. must be <125 mL. Add 10 drops antifoam to flask. Place
tip of condenser below surface of 7 mL H 2 S0 4 (1 +5) in 500
mL flask or beaker (container must be tilted at first to obtain
sufficient depth). Steam distil at rate of >:8 mL/min. (It will
be necessary to heat Claisen flask as distn proceeds to avoid
condensation of steam in flask.) Collect ca 300 mL distillate.
When distn is complete, rinse condenser into receiver with ca
5 mL H 2 0.
Transfer distillate to 500 mL separator, rinse with H 2 0, make
distinctly alk. (pH 10-14) with NaOH (1 + 1), and ext with
five 20 mL portions CHC1 3 . Combine CHC1 3 exts in 250 mL
separator. Ext nicotine from CHC1 3 with 20, 20, 15, 15, and
15 mL portions 0.05W HC1. Combine HC1 exts in 125 mL
separator and shake gently few sec with 15 mL pet ether to
remove any remaining CHC1 3 . Drain clear HO layer into 250
mL vol. flask and wash pet ether with addnl 10 mL 0.05./V
HO. Drain acid layer into vol. flask, dil. to vol. with 0.05W
HO, and record UV spectrum from 220 to 300 nm in 1 cm
cell against 0.05/V HO in recording spectrophtr. Draw line
tangent to 2 min. obtained (ca 226 and 280 nm), drop per-
pendicular from point of max. A (ca 259 nm) to tangent line,
and det. net A. Similarly det. net A' of std soln.
% Nicotine in feed
= (Net A x mg nicotine in final std soln
x 100)/(Net A' x g sample x 1000)
ReL: JAOAC 47, 226(1964).
CAS-54-11-5 (nicotine)
971.46 Nifursol in Feeds
Spectrophotometry Method
First Action 1971
Final Action 1984
A. Principle
Nifursol is extd from feed with dimethyl formamide (DMF).
Feed interferences are removed from DMF soln by column
chromatgy on alumina. Drug is reacted with phenylhydrazine
to form 5 -nitro furfural phenylhydrazone, which is extd into
toluene and detd spectrophtric at 555 nm immediately after
addn of methylbenzethonium hydroxide.
B. Apparatus
(a) Chromatographic tubes. — 400 X 20 mm id, with glass
wool plug.
(b) Spectrophotometer . — For use at 555 nm.
C. Reagents
(a) N ,N-Dimethy Iformamide (DMF) solns. — (1) 95%
DMF.— Dil. 95 parts DMF (No. DX1730, EM Science or
equiv.) with 5 parts H 2 0. (2) 50% DMF.— Dil. 50 parts DMF
with 50 parts H 2 0. (Caution: See safety notes on dimethyl-
formamide.)
(b) Alumina. — To 100 parts 80-200 mesh alumina (Alcoa
F-20, Fisher Scientific Co., A-540), add 6 parts powd Mg(OH) 2 .
Shake mixt. in screw-cap bottle until thoroly mixed. Add 8
parts H 2 and immediately mix until all lumps disappear.
(c) Phenylhydrazine soln. — Prep, immediately before use
by dissolving 0.25 g phenylhydrazine. HO crystals in 25 mL
H 2 0. Add 25 mL HCJ.
(d) Methylbenzethonium hydroxide. —\M soln in MeOH (No.
23, 394-3, Aldrich Chemical Co., Inc.).
(e) Nifursol std solns. — (1) Stock soln. — 0.25 mg/mL.
Weigh 25 mg nifursol (mp 224-226°) into 100 mL vol. flask.
Add 5 mL DMF, mix until all material is dissolved, and dil.
to vol. with MeOH. (2) Working soln. — 6.25 |xg/mL. Pi pet
5.0 mL stock soln into 200 mL vol. flask, add 100 mL 95%
DMF, and dil. to vol. with H 2 0. (Nifursol solns are not stable
for long periods of time; prep, fresh as needed.)
D. Extraction
Weigh 5.00 g finely ground feed into 250 mL erlenmeyer.
Add exactly 50 mL 95% DMF, stopper, and shake gently 5
min on mech. shaker. Place sample 30 min in 60° H 2 bath,
remove, and shake vigorously 30 min on mech. shaker. Filter
thru rapid paper in buchner, using vac. Transfer 40.0 mL ali-
quot of filtrate to beaker, add 40.0 mL H 2 0, stir, and let stand
30 min in dark.
E. Chromatography
Add 7 cm specially prepd alumina to chromatgc tube contg
glass wool plug. Tap column walls to settle alumina; then add
1.5 cm Ottawa sand (No. S-15195, Sargent-Welch Scientific
Co.). Prewash column with 50 mL 50% DMF just before use.
Add DMF sample soln to column, discard first 60 mL eluate,
and collect next 12 mL. (Decrease in flow rate may occur with
some feed exts due to accumulation of fine ppt that settles on
surface of alumina. Increase flow rate by stirring top of alu-
mina and sand to break up ppt layer or by applying gentle air
pressure to top of column.)
F. Determination
Pipet 5 mL aliquot into 20 mL test tube. Add 5 mL freshly
prepd phenylhydrazine soln to tube, mix, and place 20 min in
40° H 2 bath. Cool tube under cold tap H 2 (<20°) 5 min,
add exactly 5 mL toluene, stopper tube, and shake vigorously.
(Caution: Do not use black rubber stoppers.) Centrf. 5 min to
clear toluene layer and transfer 3.0 mL toluene to 1 cm cell.
Add 0.1 mL methylbenzethonium hydroxide to cell and mix
immediately. Read sample within 1 min at 555 nm on spec-
trophtr. Det. concn of nifursol from std curve.
G. Preparation of Standard Curve
Pipet 0, 2, 3, 4, and 5 mL aliquots working soln (equiv. to
0.000, 0.0050, 0.0075, 0.0100, and 0.0125% in feed) into
sep, 20 mL test tubes. Dil. to 5 mL with 50% DMF. Develop
color as in 971. 46F and plot A against % drug in feed.
Ref.: JAOAC 54, 66(197.1).
CAS-16915-70-1 (nifursol)
AOAC Official Methods of Analysis (1990)
Nitarsone
105
961.25 Nihydrazone in Feeds
Colorimetric Method
First Action 1961
Final Action 1962
A. Reagents
(a) 95% Dimethylformamide (DMF).— Dil. 95 parts DMF
(Eastman Kodak Co. No. 5870, or equiv.) with 5 parts H 2 0.
(Caution: See safety notes on dimethylformamide.)
(b) Nihydrazone std solns. — (/) Stock soln. — Weigh 110
mg cryst. nihydrazone (available from Norwich Eaton Phar-
maceuticals Inc., 17 Eaton Ave, Norwich, NY 13815) into
100 mL vol. flask, dissolve in DMF, and dil. to vol. with
DMF. Protected from light, soln is stable several months. (2)
Working soln.— For feeds contg 0.011% nihydrazone, pipet 1
mL aliquot stock soln into 100 mL vol. flask, add 50 mL DMF,
and dil. to vol. with H 2 0.
B. Determination
Weigh 10 g sample into 125 mL erlenmeyer, add exactly
50 mL 95% DMF, stopper loosely, and place in boiling H 2
bath 5 min (or until temp, of solv. reaches 90°). Shake mech.
10 min and filter thru rapid paper. To 25 mL filtrate add 25
mL H 2 and mix. Let stand, protected from light, >30 min.
(Some solids may sep.; standing for longer time is permissi-
ble.)
Prep, ca 20 mm diam. adsorption column contg adsorbent,
960.63A(e), to ht of 5 cm. (With highly colored feeds, use
somewhat longer column.) Use plug of cotton or glass wool
to support column, and similar plug or layer of washed sea
sand on top. Pass sample soln thru column, collecting ca 15
mL eluate. Pipet 5 mL aliquots into each of 2 tubes. Protect
1 tube from light; to other add 3 drops freshly prepd 2% No.
hydrosulfite soln, mix, and let stand 5 min. Treat 5 mL ali-
quots dild std soln similarly.
Pipet 5 mL phenylhydrazine soln, 960.63A(a), into all test
tubes, mix, and heat 20 min in 40° H 2 bath. Cool by placing
tubes in 15° H 2 bath 5 min. Add exactly 10 mL toluene to
each tube, stopper, and shake vigorously 40 times. Sep. and
centrf. toluene layer, and read A at 440 nm.
% Nihydrazone
L v* samp. ** red. samp./
X 0.011j/(A std - A redskl )
Ref.: J AOAC 44, 2(1.961).
CAS-67-28-7 (nihydrazone)
970.86 Nitarsone in Feeds
Spectrophotometric Method
First Action 1970
Final Action 1973
A. Principle
Nitarsone is extd from feed with 50% dimethyls ulf oxide
(DMSO) and sepd from interferences by alumina chromatgy.
Nitro group is reduced with TiCl^ and resulting amine assayed
colorimetrically at 530 nm with Bratton-Marshal! reaction.
Arsanilic acid and carbarsone interfere.
B. Reagents
(a) Nitarsone std solns. — (7) Stock soln. — 1 mg/mL. Weigh
100 mg nitarsone std (available from Salsbury Laboratories,
Inc.) into 100 mL vol. flask and dil. to vol. with 4% NaOH.
(2) Working soln. — 50 jjug/mL. Dil. 10 mL stock soln to 200
mL with 4% NaOH.
(b) Activated alumina. — Alcoa grade F-20, 80™ 200 mesh
(available from Fisher Scientific Co. as Alumina, adsorption,
Fisher No. A-540). To det. suitability of alumina, perform en-
tire detn on 100 u,g nitarsone. Recovery should be >95%.
(c) Dimethy [sulfoxide (DMSO) soln. — 50%. Dil. with equal
vol. H 2 0. (Caution: DMSO can be harmful. Avoid skin con-
tact by wearing heavy rubber gloves. Use effective fume re-
moval device.)
(d) Titanous chloride soln. — 4% aq. Prep, fresh daily from
20% soln open <3 months and kept refrigerated, or from solid
TiCl 3 . If >1 min is required for color disappearance in detn,
use fresh source of TiCl 3 . (Caution: TiCl 3 is corrosive. Wear
disposable plastic or rubber gloves. Avoid contact with eyes.)
(e) Sodium nitrite soln. — 0.1% aq. Prep, weekly.
C. Preparation of Standard Curve
Pipet 0, 2, 5, 10, 15, 20, and 25 mL working soln into sep.
100 mL vol. flasks and dil. to vol. with 4% NaOH. Pipet 10
mL from each flask into sep. 50 mL vol. flasks, add \5 mL
4% NaOH, and dil. to vol. with H 2 0. Pipet 4 mL from each
flask into sep. test tubes and develop color as in 970. 86E,
beginning "... add 2 drops 4% TiCl 3 ..." Std concns cor-
respond to 0, 0.004, 0.010, 0.020, 0.030, 0.040, and 0.050%
nitarsone in feeds. Plot std curve of A against % drug in feed.
D. Preparation of Sample
Accurately weigh 5 g finely ground feed into 100 mL vol.
flask. Add 75 mL 50% DMSO, place sample on wrist action
mech. shaker, and shake at room temp. 30 min. Dil. to vol.
with 50% DMSO and mix. Transfer 30-40 mL to 50 mL centrf.
tube and centrf. 10 min at 2000 rpm.
E. Determination
(Caution: See safety notes on pi pets.)
Add alumina to 20 x 400 mm chromatgc tube with fritted
glass disk to depth of 7 cm. Tap tube wall to settle alumina;
then add 1 cm layer of sand. Prewash column with 50 mL 50%
DMSO before use.
Pipet 10 mL supernate from prepn of sample onto pre-
washed column. For feeds contg >0.04% nitarsone, use smaller
aliquot. Let sample enter column and then wash into column
with several 5 mL portions H 2 0. Wash column with 75 mL
H 2 and discard el u ate.
Elute nitarsone with 65 mL 4% NaOH, discarding first 15
mL. Collect remaining eluate in 100 mL vol. flask, letting
column run dry. Nitarsone is eluted with ca 25-30 mL eluant.
Dil. eluate to vol. with H 2 and mix.
Pipet 4 mL dild eluate into 2 test tubes, add 2 drops 4%
TiCl 3 to each with mixing, and shake or mix on Vortex mixer
until black color disappears. Add 2 mL HC1 to each and mix
thoroly. Add 0.5 mL 0.1% NaN0 2 , (e), and mix. After 5 min,
add 0.5 mL 0.5% NH 4 sulfamate, 969.53A(c), and mix. After
2 min, add 0.5 mL 0.1% coupling reagent, 969.53A(d), to
one tube and 0.5 mL H 2 to second tube for blank. Let color
develop 1 5 min; then read A of sample and blank at 530 nm
on spectrophtr. Correct sample A for blank A and det. amt
nitarsone in sample from std curve.
Ref.: JAOAC 53, 641(1970).
CAS-98-72-6 (nitarsone)
960.65* Nithiazide in Feeds
Spectrophotometric Method
First Action 1960
Final Action 1961
Surplus 1989
See 42.122-42.125, 14th ed.
106
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
967.36* Nitrodan in Feeds
Colorimetric Method
First Action 1967
Final Action 1968
Surplus 1989
(Not applicable in presence of interfering nitro compels)
See 42.126-42.129, 14th ed.
959.18 Nitromide in Feeds
Spectrophotometric Method
Final Action
A. Reagents
(a) Diethylamine reagent (DEA), aged, — (1 year or older.)
Fresh DEA may be artifically aged as follows: Place 1 L DEA
in dry 2 L flask with 40 g Na or K fluosilicate. Connect flask
to 60 cm (24") bulb reflux condenser and reflux on sand bath
2-3 days in hood. When reagent is sufficiently "aged," 2 mL
clear DEA added to 8 mL dimethyl sulfoxide contg 50 fig 3,5-
DNBA should develop max. color in ca 40 min. A as read on
Beckman DU spectrophtr at 560 nm should be ca 0.375; on
Klett-Summerson photoelec. colorimeter with No. 56 filter, ca
200. Reagent must be free from turbidity. Prep, new std curve
for each batch of DEA.
(b) Nitromide std sains. — (/) Stock soln. — I mg/mL. Weigh
100 mg nitromide (Salsbury Laboratories, Inc.) into 100 mL
flask and dil. to vol. with MeOH. (2) Working soln. — 20 juig/
mL. Transfer 2.0 mL stock soln to 100 mL vol. flask and dil.
to vol. with MeOH.
B. Preparation of Standard Curve
Place 1 .0, 2.0, 3.0, and 5.0 mL working soln contg 20, 40,
60, and 100 fxg, resp., of nitromide in 4 colorimeter tubes.
Evap. to dryness at 50° in air current. Dissolve residue in 8
mL dimethyl sulfoxide at 70°, cool, and add 2 mL DEA re-
agent. Place in dark at 20-25° and read after 1 hr. Plot std
curve, using A as ordinate and concn as abscissa.
C. Preparation of Sample
Weigh 5.0 g feed, contg 0.025% nitromide, into 100 mL
vol. flask and dil. to vol. with MeOH. Shake frequently 20
min and let stand 40 min to permit feed particles to settle.
If feed contains 0.075% nitromide, use 2 g finely ground
feed; if 0.15%, use 1 g in 100 mL or 5 g in 500 mL MeOH.
Prep, premixes by weighing appropriate sample and serially
dilg MeOH ext.
D. Determination
Pipet 4 mL aliquot of ext into g-s test tube. Place tube in
50° H 2 bath and evap. to dryness with air current directed
onto surface of MeOH. Add 8 mL dimethyl sulfoxide and heat
to 70° to hasten soln; cool, and add 2 mL DEA reagent. Place
in dark 1 hr at 20- 25°. Det. A at 560 nm in Beckman DU
spectrophtr, Klett-Summerson photoelec. colorimeter with No.
56 filter, or similar instrument, against dimethyl sulfoxide as
ref.
Det. amt of nitromide in tube from std curve.
% nitromide in feed
= fxg nitromide in tube x 25 X J 00/5,000,000
or |mg nitromide in tube x 5 = |xg nitro mide/g feed or ppm.
Ref.: JAOAC42, 239(1959).
CAS- 121-8 .1-3 (nitromide)
952.29* Nitrophenide in Feeds
Spectrophotometric Method
Final Action
Surplus 1980
(Applicable in presence of arsanilic acid)
See 42.120-42.122, 13th ed.
967.37 Phenothiazine in Feeds
Spectrophotometric Method
First Action 1967
Final Action 1970
A. Reagent
Phenothiazine std soln. — Dissolve 10 mg recrystd (from 10%
soln in toluene) phenothiazine (1CI Americas, Inc., Eighty Four,
PA 15330) in 50 mL alcohol and dil. to 100 mL with alcohol.
For working stds, dil. with equal vol. of alcohol. (1 mL dild
soln = 50 jxg phenothiazine.) Use freshly prepd soln; ale. solns
gradually develop rose tint within few hr.
B. Determination
Place 1 g ground sample in 100 mL vol. flask, add 50 mL
alcohol, and heat on steam bath 15 min. Cool, dil. to vol. with
alcohol, mix, and let settle (ca 15 min) until supernate is clear.
Place 2 mL aliquot in 25 mL vol. flask and add 10 mL
alcohol. To flask add, in order given, 1 mL ./% ale. p-ami-
nobenzoic acid soln, 1 mL aq. 2% NaN0 2 soln, and 1 mL
HCI (1 + 3). Dil. to vol. with alcohol. Read A of green soln
at 600 nm in spectrophtr against reagent blank. Det. amt of
phenothiazine from std curve.
% Phenothiazine — fxg/200
Prep, std curve, using 1,2, and 3 mL dil. std soln, as above.
Refs.: JAOAC 41, 338(1958); 42, 254(1959).
CAS-92-84-2 (phenothiazine)
967.38 Piperazine in Feeds
Spectrophotometric Method
Final Action
A. Principle
Piperazine or piperazine salt is quant, extd from feed into
slightly acidic aq. soln. Dild filtrate is reacted with equal vol.
benzoquinone soln at 80°. Colored complex formed is detd
spectrophtric at 490 nm.
Applicable to detn of 0.05-0.5% piperazine, usually present
as one of its salts, in animal feeds. Amines give similar color
reaction. Alkalies produce increased color; pH adjustment in
method overcomes interference of this kind.
B. Apparatus
(a) Water hath. — Approx. 25 cm (10") diam. with 15-20
cm (6-8") depth H 2 0. Thermostatically controlled at 80 ± 0.1°.
(Viscosity bath is satisfactory.)
(b) Test tubes.— Pyrex, 15 x 125 mm, with rubber stop-
pers, and rack capable of supporting tubes when immersed in
H 2 bath.
C. Reagents
(a) Qui none soln. — Dissolve 0.5 g p -benzoquinone in 2.5
mL HO Ac and little alcohol in dry 100 mL vol. flask and dil.
to vol. with alcohol. Keep soln in ice bath or refrigerator. Prep.
fresh daily. (Caution: p -Benzoquinone is lachrymator; avoid
AOAC Official Methods of Analysis (1990)
Pyrantel Tartrate 107
breathing vapor and contact with skin and clothing.) If blanks
are high or variable, purify /?-benzoqui none by steam distn in
hood.
(b) Piperazine std solns. — (/) Stock soln. — Dissolve ex-
actly 185 mg pure piperazine. 2HCJ (Salsbury Laboratories, Inc.)
(equiv. to 100 mg piperazine) in H 2 and dil. to 250 mL. (2)
Working soln. — 20 |xg/mL. Dil. 25.0 mL stock soln to vol.
in 500 mL vol. flask.
D. Preparation of Standard Curve
Using working soln, add, by microburet or pipets, 20, 40,
60, 80, and 100 |mg piperazine equiv. and intermediate values,
if required, into test tubes. Dil. to 5 mL in each test tube with
H 2 0. Include H 2 blank with each detn.
Add 5 mL quinone reagent to each std and blank. Stopper
tubes and mix by inverting. Remove stoppers and immerse in
H 2 bath at 80 ± 0.1° exactly 10 min. (Bath temp, can be
varied; use same temp, for samples and stds.) Immediately
immerse tubes in ice bath 3 min. Let stand at room temp. >20
min, but <40 min. Read A at 490 nm in 1.0 cm cells, using
reagent blank to zero instrument. Plot A of each std against
|xg piperazine.
E. Determination
Weigh 10.00 g well mixed feed (grind pellets in mortar) into
500 mL (.1.6 oz) wide- mouth, screw-cap bottle. Add exactly
200 mL H 2 from graduate and adjust to pH 4-5 (0.15 mL
H 2 S0 4 (1 +2) is usually enough for 1 g feed). Cap or stopper
bottle and place in wrist-action shaker 30 min. Add ca 5 g
Celite (Note: Some grades may retain piperazine.) as slurry to
buchner contg 9.0 cm Whatman No. 3 paper and pull down
under full vac. Wash pad with small portion feed ext and dis-
card washing. Rapidly filter remaining feed ext and reserve
filtrate for color development. (Do not delay; turbidity may
form.)
Pipet 25.0 mL filtrate into 250 mL vol.. flask and dil. to vol.
with H 2 0. Pipet 5 mL aliquot into test tube and immediately
proceed with color development as in 967.38D. Include H 2
blank with each detn.
Prep, sample color blank for each feed as follows: Pipet 5
mL aliquot dild ext into test tube and 5 mL H 2 into another
test tube as ref. To each, add 5 mL soln contg 2.5 mL HO Ac
dild to 100 mL with alcohol. Mix by inverting and omit heat-
ing. Read A at same wavelength and subtract from sample
reading. Include 1 or 2 stds with each detn to detect shift in
std curve; adjust accordingly. Obtain |mg piperazine from std
curve.
% Piperazine = (juug piperazine X 10~~ 4 )/g sample in aliquot
Ref.: JAOAC 50, 268(1967).
CAS- 110-85-0 (piperazine)
978.30 Pyrantel Tartrate in Feeds
Spectrophotometric Method
First Action 1978
Final Action 1981
(Applicable to range 0.0106-0.881 1%. Pyrantel tartrate solns
are light sensitive. Exts must be protected from direct sunlight
or artificial light.)
A. Apparatus
(a) High-speed blender.— Waring Blendor, or equiv,
(b) Centrifuge. — International Model EXD (International
Equipment Co.), or equiv. , equipped to hold 50 mL g-s centrf.
tubes.
(c) Filter aid. — Celite 545, acid- washed (Manville Filtra-
tion and Minerals), or Millipore prefilter pad (No. AP2507500,
Millipore Corp.), or equivs.
(d) Filtrator. — Fisher No. 9-788, low form (Fisher Scien-
tific Co.), or equiv.
(e) Mixer. — Vortex Genie mixer (Scientific Products), or
equiv.
(f) Spectrophotometer. — For use at 31 I nm, with 1 cm cells.
B. Reagents
(a) Hydrochloric acid.— AN. Dil. 8.33 mL HC1 to 1 L
with H 2 0.
(b) Leaching soln. — Dissolve 100 g NaCl in 1 L H 2 0, add
1 L MeOH, and mix vigorously. Prep, fresh daily.
(c) Perchloric acid.~~Q.24M. Dil. 20 mL H'CI0 4 to 1 L
with H 2 0.
(d) Sodium hydroxide. — 0.17V. Dissolve 4 g NaOH in H 2
and dil. to 1 L with H 2 0.
(e) Pyrantel tartrate std solns. — (1) Stock soln. — 0.80 mg/
mL. Weigh 80.0 mg Pyrantel Tartrate Ref. Std (available from
Pfizer, Inc., Quality Control, Agricultural Div., 1107 S Rt
291, Lee's Summit, MO 64081) into 100 mL vol. flask, dis-
solve in leaching soln, (b), and dil. to vol. with same solv.
Use ultrasonic bath to speed soln. If std does not dissolve readily,
remake. Prep, fresh daily. (2) Working soln.— 0A6 mg/mL.
Pipet 20 mL stock soln into 100 mL vol. flask, dil. to vol.
with leaching soln, and mix well. Prep, fresh daily.
C. Preparation of Samples
Weigh duplicate portions feed ground in high-speed blender,
(a), into 125 mL erlenmeyers: 15.000 ± 0.010 g for 0.0106%
pyrantel tartrate; 1.800 ± 0.010 g, 0.0881%; and 1.000 ±
0.010 g, 0.881%. Add 60 mL leaching soln to 1 portion; add
50 mL leaching soln and, by pipet, 10 mL working std soln,
(e)(2), to other portion of 0.0106% or 0.0881% pairs or 10
mL stock std soln, (e)(7), to other of 0.881% pair. Cap with
polypropylene stopper and shake mech. 1 hr.
Quant, transfer mixt. to buchner precoated with Celite or
contg prefilter pad, (c), rinsing flask with small portions leach-
ing soln, (b), and collecting filtrate under vac. with Filtrator,
(d), contg 250 mL erlenmeyer, until vol. filtrate is 150 mL.
Transfer filtrate to 200 mL vol. flask, and dil. to vol. with
leaching soln. For 0.881% level, pipet 50 mL dild filtrate into
250 mL vol. flask, and dil. to vol. with leaching soln.
D. Determination
Pipet 25 mL prepd sample soln into 250 mL separator contg
5 g finely ground KI, and shake to dissolve. Pipet in 100 mL
CHC1 3 and shake 45 sec. Transfer lower CHC1 3 layer to an-
other 250 mL separator contg 25 mL 0.24M HC10 4 , and shake
10 sec. Transfer ca 35 mL lower CHCI 3 layer to 50 mL g-s
centrf. tube contg 10.0 mL 0.1N NaOH. Stopper tube, and
mix on vortex mixer, (e), 15 sec while shaking back and forth.
Centrf. 10 min at 990 x g (1800 rpm). Pipet 25 mL lower
CHCI3 layer from tube into another 50 mL g-s centrf. tube
contg 10.0 mL 0. \N HC1. Stopper tube, mix, and centrf. as
before. Rapidly record A of upper aq. layer at 311 nm against
CHCl r satd O.liV HC1 as ref. solv.
% Pyrantel tartrate = (A/W) x \W /(A' - A)] x F x 100
where A and A' refer to absorbance of sample and spiked sam-
ple, resp.; W = g feed, supplement, or cone; W' = g pyrantel
tartrate ref. std used to prep, std soln; F — [(1/100 mL) x
(20 mL/100 mL) x 10 mL] for 0.0106% and 0.0881% levels;
and F - [(1/100) x 10] for 0.881% level.
Ref.: JAOAC 61, 296, 473(1978).
CAS-3340 1-94-4 (pyrantel tartrate)
108
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
966.27a Racephenicol in Feeds
Final Action 1970
Surplus 1975
A. Method I— First Action 1966
(Applicable to levels ^0.002%)
See 42.128-42.131, 12th ed.
B. Method ll—First Action 1968
(Applicable to levels ^0.0005%)
See 42.133-42.136, 12th ed.
960.66* Reserpine in Feeds
Photofluorometric Method
First Action 1960
Final Action 1964
Surplus 1989
(Applicable at 0.2-2.0 ppm level)
5<?e 42.148-42.151, 14th ed.
969.56* Ronnel in Feeds
Gas Chromatographic Method
First Action 1969
Final Action 1970
Surplus 1989
See 42.152-42.154, 14th ed.
970.87* Ronnel in Feeds
Spectrophotometric Method
First Action 1970
Final Action 1972
Surplus 1989
(For mineral feed mixts contg 1-40% ronnel)
See 42.155-42.159, 14th ed.
971.47 Roxarsone in Feeds and Premixes
Spectrophotometric Method
First Action 1971
Final Action 1974
(Not applicable to pelleted feeds contg hemicellulose ext)
A. Principle
Roxarsone is extd from feed with 2% K 2 HP0 4 . Proteins are
pptd at isoelec. point and removed by centrfg. Ext is treated
with activated C at pH 12 to remove interferences and rox-
arsone is detd spectrophtric at 410 nm.
0. Apparatus
(a) Centrifuge. —International Model V, or equiv.
(b) Mechanical shaker. — Burrell wrist-action (Burrell Corp.),
or equiv.
C. Reagents
(a) Potassium phosphate, dibasic, soln. — 2%. Dissolve 2
gK 2 HP0 4 in 100 mL H 2 0.
(b) Dilute hydrochloric acid. — Dil. 45 mL HC1 to 200 mL
with H 2 0.
(c) Sodium hydroxide soln. — Dissolve 24 g NaOH in 100
mL H 2 0.
(d) Charcoal adsorbent. — Activated (Darco G-60, or equiv.).
(e) Roxarsone std soln. — 300 |mg/mL. Accurately weigh 300
mg Roxarsone Ref. Std (Salsbury Laboratories) into 1 L vol.
flask. Dissolve and dil. to vol. with 2% K 2 HP0 4 soln.
D. Preparation of Sample
Grind sample in high-speed blender to pass No. 20 sieve (ca
3 min) and mix thoroly. Weigh 15.0 g ground sample into 125
mL erlenmeyer, add 50.0 mL 2% K 2 HP0 4 soln, place on mech.
shaker, and shake vigorously 5 min at room temp. Immedi-
ately transfer to 100 mL centrf. tube and centrf. 10 min at
3000 rpm. Perform extn and centrfg with min. delay.
Decant 30 mL aliquot supernate into 50 mL graduated centrf.
tube, pipet in 1 mL dil, HO, stopper, and mix thoroly. Let
sample stand until protein flocculates (ca 15 min) and centrf.
10 min at 3000 rpm.
E. Purification
Pipet 25 mL clear supernate into 125 mL erlenmeyer. Pipet
in I mL NaOH soln, and mix thoroly. Add 2.0 g activated C
and swirl sample several times during 30 min standing. Filter
thru Whatman No. 42 fluted paper into 50 mL erlenmeyer.
Repeat C treatment with second 2.0 g portion.
F. Determination
Place 3 mL filtrate in cell and det. A at 410 nm against H 2
blank. Add 2 drops coned HO from dropper (or 5 mL serolog-
ical pipet), mix, and reread ,4. Difference in A of acidic and
basic samples represents roxarsone present. Det. amt from std
curve.
G. Preparation of Standard Curve
Pipet 0, 1, 3, 5, 7, and 10 mL aliquots std roxarsone soln
into 100 mL vol. flasks and dil. to vol. with 2% K 2 HP0 4 soln.
Pipet 30 mL aliquot from each flask into sep. erlenmeyers,
pipet 1 mL dil. HO into each flask, and mix. Pipet 25 mL
aliquot this soln into sep. 50 mL erlenmeyers, pipet in 1 mL
NaOH soln, and mix thoroly. Transfer 3 mL soln from erlen-
meyers into sep. cells and det. A at 410 nm against H 2 blank.
Add 2 drops coned HO from dropper (or 5 mL serological
pipet), mix, and reread A. Plot difference in A of std solns
against concn of solns expressed as 0, 0.001, 0.003, 0.005,
0.007, and 0.010% roxarsone in feed when 15 g sample is
used.
H. Determination in Premix
Weigh appropriate size sample of premix, place in 200 mL
vol. flask, and add 50 mL 2% K 2 HP0 4 soln and 4.0 mL NaOH
soln. Let stand 20 min, shaking occasionally; then dil. to vol.
with 2% K 2 HP0 4 soln and let feed particles settle 30 min.
Proceed as in 971.47D, par. 2, using appropriate aliquots and
dilns to give final conens in range of std curve.
(Example: 5% Premix. — Place 5 g thoroly mixed premix in
200 mL vol. flask and ext as above. Transfer 10 mL aliquot
ext to 100 mL vol. flask, dil. to vol. with H 2 0, and mix tho-
roly. Transfer 10 mL aliquot this soln to another 100 mL vol.
flask and dil. to vol. with H^O. Diln factor for this premix is
1200.)
After diln, transfer 30 mL aliquot to 125 mL erlenmeyer.
Pipet in J mL dil. HO and mix. Transfer 25 mL this soln to
125 mL erlenmeyer, pipet in I mL NaOH soln, mix thoroly,
add 2.0 g activated C, shake several times during 30 min, and
filter. Repeat C treatment. Read A of filtrate and det. concn
of roxarsone as in 971.47F, multiplying by diln factor.
AOAC Official Methods of Analysis (1990)
Roxarsone
109
Ref.: JAOAC 54, 80(1971).
CAS-121-19-7 (roxarsone)
986.39 Roxarsone in Feeds
Atomic Absorption Spectrophotometry Method
First Action 1986
Final Action 1989
(Method dets total As and is not specific for roxarsone. Ap-
plicable range is 0-50 ppm 4-hydroxy-3-nitrobenzene arsonic
acid.)
A. Principle
Sample is extd with aq. ammonium carbonate soln and ana-
lyzed by furnace AAS for total As content, which is converted
by factor to roxarsone concn in finished feed.
0. Reagents
(a) Water. — Super Quality from Millipore Super Q system.
(b) Nitric acid. — Mallinckrodt, ACS grade.
(c) Argon. — Linde purified.
(d) Nickel nitrate.— ~Ni(N0 2 ) 2 .6H 2 (Mallinckrodt AR).
(e) Nickel nitrate soln. — Approximately 2000 ppm Ni. Dis-
solve 10.0 g Ni(N0 3 ) 2 .6H 2 in H 2 and dil. to 1 L with H 2 0.
(f) Ammonium carbonate. — Powder, purified (EM Science
No. AX 1260).
(g) Methanol.— Anhyd., ACS (EM Science No. MX0485).
(h) Diluting soln. — Add 5 mL coned HN0 3 and 150 mL
anhyd. MeOH to 1 L vol. flask, dil. to vol. with H 2 0, and
mix.
(i) Tantalum pentoxide. —99. 99% Ta 2 5 (Aldrich Chemi-
cal Co.).
(j) Tantalum pentoxide soln. — Suspend 2.0 g in 10 mL H 2 0.
(k) Roxarsone std soln. — 1250 ppm roxarsone (356 ppm
As). Accurately weigh 625.0 mg roxarsone ref. std (Salsbury
Laboratories, Inc., 2000 Rockford Rd, Charles City, IA 50616)
into 500 mL vol. flask. Dissolve and dil. to vol. with 2% am-
monium carbonate soln. Caution: Wear protective clothing and
avoid breathing dust.
(1) Dilute roxarsone std soln. — 12.5 ppm roxarsone (3.56
ppm As). Dil. 10.0 mL roxarsone std soln to 1 L with H 2 0.
(m) Control feed extract. — Using typical nonmedicated
poultry or swine ration, prep, feed ext as described under Sam-
ple Preparation. Test suitability of control feed ext by dilg 1
mL aliquot with dilg soln used in sample prepn. Set up AAS
system and furnace conditions as described in procedure. It is
not necessary to perform calibration for this test; absorbance
reading is satisfactory. Zero spectrophtr on 20 jxL injection of
dilg soln and measure A on 20 jxL control feed ext. Absor-
bance reading <0.010 indicates suitability.
(n) Working std soln. — Transfer 1.0 mL dil. roxarsone std
soln to 10 mL vol. flask. Dil. to vol. with control feed ext.
Transfer 1 .0 mL aliquot of this soln to 25 mL erlenmeyer and
add 9.0 mL dilg soln. Twenty |ulL working std soln = 50 ppm
roxarsone in feed sample for wts and vols used in procedure.
C. Apparatus
(a) Atomic absorption spectrophotometer. — Perkin-Elmer
Model 5000, or equiv., with heated graphite atomizer furnace,
autosampler, and printer sequencer.
(b) Mechanical shaker. — Wrist-action.
(c) Pipets.— Eppendorf: 10, 20, 50, and 1000 pJL.
(d) Dispensing pipet. — Repipet (Labindustries, 620 Hearst
Ave, Berkeley, CA 94710), or equiv., 10 mL capacity set to
deliver 9.0 mL dilg soln.
D. Sample Preparation
Grind sample in Wiley mill to pass 20 mesh sieve and tho-
roly mix. Weigh 5.0 g ground sample into 250 mL vol. flask
or 300 mL erlenmeyer. Add 2.0 g ammonium carbonate pow-
der and 200.0 mL H 2 0, place on mech. shaker, and shake
vigorously 5 min at room temp. Remove flask from shaker
and let suspended feed particles settle 15-30 min. Transfer 1.0
mL aliquot of feed ext to 25 mL erlenmeyer and add 9.0 mL
dilg soln. Mix thoroly. Repeat this step on reagent blank and
on std-fortified control feed ext equiv. to 50 ppm roxarsone in
feed. Samples and std are now ready for furnace AAS anal-
ysis.
£ AAS Conditions
Set up graphite furnace and spectrophtr according to follow-
ing conditions and allow 30 min warm-up time. Operating
conditions: lamp, As EDL operated at 8 watts, properly aligned;
lamp current, ma; wavelength, 193.7 nm; slit, 0.7 nm band-
pass, low position; readtime, 5 s; mode, AA-BG; readout, concn;
signal, peak ht on instrument display and A on recorder if used;
std 1, SI — 50.0 ppm ((xg/g) roxarsone (use 3 digits), do not
use S2 and S3.
Install furnace assembly in AAS system and align as in man-
ufacturer's instructions.
F. Furnace Tube Coating Procedure
Prep. 20% Ta 2 5 aq. suspension. Shake suspension vigor-
ously, introduce 50 jxL aliquot into pyrolytically coated graph-
ite furnace tube, and perform following sequence of opera-
tions; H 2 flow, 1-2 L/min to cool furnace; Ar pressure, 35
psi; on/off switch, on; gas control, on.
Step 1 (drying): temp., 100°; ramp time, 10 s; hold time,
90 s.
Step 2 (charring): temp., 1000°; ramp time, 10 s; hold time,
30 s.
Step 3 (atomizing): temp., 2700°; ramp time, 5 s; hold time,
10 s; stop flow, on.
Repeat coating procedure twice (3 applications). Tube is now
ready for furnace AAS use.
(With initial coating, some material may flake after approx.
35 firings. If this happens, pass small brush or Kimwipe thru
tube to remove loose tantalum and apply single recoating.)
G. Furnace Conditions for Sample Assay
Step 1 (drying): temp., 100°; ramp time, 10 s; hold time,
50 s.
Step 2 (charring): temp., 1000°; ramp time, 10 s; hold time,
30 s.
Step 3 (atomizing): temp. , 2300°; ramp time, s; hold time,
5 s; read, on; stop flow, on.
Step 4 (burnout): temp., 2400°; ramp time, s; hold time,
5 s; read, off; flow, 300 mL/min (stop flow, off).
H. Autosampler Conditions
Install autosampler assembly in furnace and align as per
manufacturer's instructions. Operating conditions: power switch,
on, and let autosampler go thru count down; program se-
quence, press standby key to bring programmer into operating
mode; method number, enter 1 and press method # key; re-
calibrate, for full tray recalibrate at 9A, 1.8B, and 27C; last
sample, program number for last sample vol. key; alternate
vol., enter 10 uX and press alternate vol. key (Ni(N0 3 ) 2 .6H 2
soln, 2000 ppm Ni); instrument program, enter 1 and press
instrument program key; HGA program, enter 1 and press HGA
program key.
Use sample tray 1 with method 1 in autosampler sequence.
This method uses external std technic for instrument calibra-
tion. In this procedure, only position SI is used because cal-
110
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
ibration is based on single point std with calibration std equiv.
to 50.0 ppm roxarsone in finished feeds.
/. Sample Analysis
Load sample tray 1 as follows: Place blank of reagents (dilg
soln (h)) in AZ location of tray; place 50.0 ppm roxarsone feed
std in SI location and at position 1 (check sample), then load
samples in sequence around tray, starting at position 2. Place
2000 ppm Ni soln in reagent container and place in appropriate
location for alternate sample in autosampler. Cover sample tray
with cover provided to minimize evapn; these solns contain
MeOH.
Instrument and samples are now ready for calibration and
sample analysis. Press start/stop key to start program in sam-
pling cycle. AZ and SI calibration should be done in dupli-
cate. Observed for duplicate SI values. These values should
be within reasonable agreement (±5%) before program is al-
lowed to proceed with samples. Instrument is recalibrated by
autosampler in setup instructions which will monitor calibra-
tion for any changes and update calibration as time progresses.
J. Calculations
Instrument is programmed to calc. sample ppm on basis of
single point std equiv. to 50 ppm roxarsone. For wts or vols
other than those specified, manual calcn is required.
Ret.: IAOAC 69, 838(1986).
CAS- 121 -19-7 (roxarsone)
970.88 Sulfadimethoxine in Feeds
Colorimetric Method
First Action 1970
A. Reagents and Apparatus
See 969.53A(b), (c), and (d), and in addn:
(a) Fie in soln. — 0.2%. Disperse 500 mg ficin (Calbio-
chem, fig latex) in H 2 (preheated to 40°) and dil. to 250 mL.
Use 10 mL of this warm soln in detn. {Caution: Ficin is very
potent proteolytic enzyme which attacks living tissues. Avoid
contact with skin and eyes and breathing dust.) (Ficin product
listed is no longer available. Method may be satisfactory with-
out ficin treatment, but users should verify recovery.)
(b) Petroleum ether. — Bp 35-60°, purified on silica gel
column.
(c) Trichloroacetic acid soln. — 3%. Dissolve 30 g
CCI3COOH in H 2 and dil. to 1 L. (Caution: See safety notes
on trichloroacetic acid.)
(d) Sulfadimethoxine std soln. — Accurately weigh 125 mg
sulfadimethoxine USP Ref. Std and transfer quant, to 100 mL
vol. flask. Add ca 70 mL acetone and shake until completely
dissolved. Dil. to vol. with acetone and mix. Pipet 20 mL soln
into 200 mL vol. flask, dil. to vol. with acetone, and mix.
Pipet 10 mL (or 5 mL if working at 0.00625% level) of last
diln into 200 mL vol. flask contg 10 mL 0.2% ficin soln. Add
ca 120 mL acetone and 2 mL 40% NaOH soJn, and mix. Dil.
to vol. with acetone and mix. Pipet 25 mL final diln into 50
mL g-s centrf. tube, evap. almost to dryness under N stream
in 50° H 2 bath, and proceed as in 970.88B, beginning "Pipet
15 mL pet ether into centrf. tube ..." Resulting clear filtrate
is std soln.
(e) Reagent blank. — Into 200 mL vol. flask pipet 10 mL
0.2% ficin soln and 2 mL 40% NaOH soln. Dil. to vol. with
acetone and mix. Pipet 25 mL of this soln into 50 mL g-s
centrf. tube, evap. almost to dryness under N stream in 50°
H 2 bath, and proceed as in 970. 88B, beginning "Pipet 15
mL pet ether into centrf. tube ..." Resulting clear filtrate is
blank soln.
(f) Spectrophotometer . — With 5 cm cells, or photoelec.
colorimeter with 540 nm filter, or equiv.
B. Preparation of Sample
(Caution: See safety notes on blenders, pipets, flammable
solvents, and acetone.)
Pipet 10 mL 0.2% ficin soln into high-speed blender. Ac-
curately weigh 10 g sample into blender, spreading carefully
on surface of liq. Let sample soak 10 min.
Add ca 120 mL acetone. Blend 2 min, adjusting speed with
variable transformer, so that acetone does not wet screw cap.
(Note: To release pressure, stop blending after 3-4 sec and
unscrew cap momentarily.) Blend 2 min and remove screw
cap. Push down into acetone all solid particles adhering to con-
tainer wall, using rubber policeman. Replace screw cap and
continue blending 1 min. Remove screw cap, pipet 2 mL 40%
NaOH soln into container, and continue blending 2 min. Push
down into acetone all solid particles adhering to wall of con-
tainer, using rubber policeman.
Quant, transfer blender contents to 250 mL g-s graduate,
using small portions acetone to total vol. of 200 mL. Stopper,
mix well, and let liq. and solids sep. Wrap tip of 50 mL pipet
with glass wool and transfer ca 40 mL ext to 50 mL g-s centrf.
tube. Centrf. 5 min at 2000 rpm. Pipet 25 mL clear acetone
ext into another centrf. tube and evap. almost to dryness (only
few drops of oily, sirupy liq. left) under N stream in 50° H 2
bath. Pipet 15 mL pet ether into centrf. tube and dissolve or
disperse residue in it,
Pipet 25 mL 0.2jV NaOH into centrf. tube, stopper, and shake
mech. 5 min. Centrf. 10 min at 2000 rpm. By pipet, transfer
lower NaOH layer (ca 24 mL) into another centrf. tube and
centrf. 10 min at 2000 rpm. Pipet 20 mL clear soln into 100
mL vol. flask. Dil. to vol. with 3% CCl 3 COOH soln. Mix and
let stand 10 min. Filter entire soln thru Whatman No. 42 pa-
per, discarding first 10 mL filtrate. If turbid, filter thru second
paper. Clear filtrate is sample soln.
C. Determination
(a) Reading on spectrophotometer with 5 cm cells. — Pipet
following vols (mL) of indicated solns into 6 sep. labeled 25
mL vol. flasks:
Soln
Sample
1
Sample
2
Sample
Blank
Std 1
Std 2
Reagent
Blank
Sample
Std
Blank
15
15
15
15
15
15
Pipet 1 mL 0.1% NaN0 2 soln, 969.53A(b), into each, mix,
and let stand 3 min. Pipet 1 mL 0.5% NH 4 suifamate soln,
969.53A(c), into each, mix, and Jet stand 2 min. Pipet 1 mL
0.1% coupling reagent, 969.53A(d), into all. except sample
blank, and 1 mL H 2 into sample blank. Mix and let stand 10
min in dark. Dil. each flask to vol. with H?0 and mix. Mea-
sure A of each soln at 540 nm in 5 cm cell against reagent
blank in ref. cell.
(b) Reading on photoelectric colorimeter . — Proceed as in
(a) except do not dil. after standing, but read at existing vol.;
50 mL g-s centrf. tubes may be used in place of vol. flasks.
Transfer solns from flasks or tubes to matched colorimeter tubes.
Set instrument with 540 nm filter to 100% T (0 A) with tube
contg reagent blank. Det. A (= 2 - log (%T)) of each of other
tubes contg samples, sample blank, and stds.
(c) Calculation. — Higher levels of sulfadimethoxine:
% Sulfadimethoxine = [(A - A ) X S]/(1000 x A' X W)
AOAC Official Methods of Analysis (1990)
Sulfamethazine
111
Lower levels of sulfadimethoxine:
% Sulfadimethoxine = [(A - A ) x S]/(2000 X A' x W)
where A, A (h and A' refer to sample, reagent blank, and std,
resp.; W = g original sample; and S - mg std weighed.
Ref.: JAOAC 53, 638(1970); 72, 106(1989).
CAS- 122- 11-2 (sulfadimethoxine)
951.07* Sulfaguanidine in Feeds
Spectrophotometric Method
First Action
Surplus 1989
See 42 .171, 14th ed.
969.57 Sulfamethazine in Feeds
Spectrophotometric Method
First Action 1969
(Applicable to feeds contg procaine penicillin. Not applicable
to feeds made from granule-stabilized Tylan-Sulfa premixes.)
A. Reagents
See 969.53A(b), (c), and (d) and in addn;
(a) 50% Methanol sola.— 50% (v/v) aq. soln of MeOH.
(b) Sulfamethazine std so Ins . — ( / ) Stock soln . — Accurate! y
weigh 0.100 g pure sulfamethazine (available from American
Cyanamid Co.) into 100 mL vol. flask. Add 50 mL 50% MeOH
soln and shake until dissolved. Dil. to vol. with 50% MeOH
soln. Soln is stable at least several weeks. (2) Intermediate
soln. — Pipet 5 mL stock soln into 200 mL vol. flask, dil. to
vol. with 50% MeOH, and mix well. Soln is also stable sev-
eral weeks. (3) Working soln. — 2.5 |ULg/mL. Pipet 10 mL in-
termediate soln into 100 mL vol. flask, add 1 mL HC1 and 50
mL 50% MeOH, dil. to vol. with H 2 0, and mix well. Soln is
stable ca 2 weeks.
B. Preparation of Sample
Weigh 5.00 g sample into 250 mL g-s erlenmeyer. Add 100.0
mL 50% MeOH soln, shake well on mech. shaker 1 hr, and
centrf. Pipet aliquot supernate contg ca 250 |ig sulfamethazine
into 100 mL vol. flask, add 50% MeOH, if necessary, to vol.
of ca 60 mL, followed by 1.0 mL HC1, and 10 mL 1% ZnS0 4
soln. Let stand 10 min, dil. to vol. with H 2 0, and mix.
C. Determination
Filter portion of prepd soln thru Whatman No. 42 paper, or
equiv., into 250 mL flask. Filtrate should be clear. Pipet two
10 mL aliquots filtrate and 10 mL working std soln into sep.
50 mL centrf. tubes. To each tube add 1.0 mL 0.1% NaN0 2
soln; mix and let stand 3 min. Add 1.0 mL 0.5% NH 4 sul-
famate soln; mix and let stand 2 min. Add 1.0 mL 0.1% N-
(l-naphthyl)ethylenediamine.2HCI soln to one of sample solns
and to std soln. To second sample soln add 1.0 mL H 2 (sam-
ple blank). Mix all solns well and let stand 10 min.
To sample, sample blank, and std soln add ca 10 mL CHCl 3 ,
stopper, and shake vigorously 30 sec (30 sec is required to
ensure complete removal of procaine dye). Add 0.8 mL ION
NaOH to sample, sample blank, and std soln. Stopper and shake
vigorously >1 min to ensure complete removal of procaine
dye. Centrf. solns at 2000 rpm 5 min or until aq. layer is com-
pletely clear. Remove 10.0 mL aq. phase with pipet and trans-
fer to 50 mL erlenmeyer or 50 mL beaker. Add 1.0 mL HO
and remove fumes formed in flask with aspirator or air stream.
Read A of sample, sample blank, and std (A') at 540 nm in
spectrophtr, against H 2 blank. Correct A of sample by sub-
tracting that of sample blank.
% Sulfamethazine = (A /A') X (2,5 fxg/mL)
x (100 mL/mL ext aliquot taken) x (100 mL/5 g)
x (l g/10 6 p,g) x 100
Refs.: JAOAC 51, 1282(1968); 72, 106(1989).
CAS-57-68-1 (sulfamethazine)
963.34 Sulfanitran in Feeds
Spectrophotometric Method
Final Action
A. Reagents
See 969.53 A(b), (c), and (d) and in addn:
Sulfanitran std solns. — (./) Stock soln. — 100 jxg/mL. Ac-
curately weigh 100 mg pure sulfanitran (available from Sals-
bury Laboratories, Inc.) into 1 L vol. flask, add enough IN
NaOH for complete soln, and dil. to vol. with H 2 0. (2) Work-
ing soln. — 10 |xg/mL. Dil. 10 mL stock soln to 100 mL with
H 2 0.
B. Preparation of Standard Curve
Pipet 0, 4, 6, 8, and 10 mL aliquots working std soln into
sep. 50 mL vol. flasks. Add 0.5 mL HO and adjust vol. with
H 2 to ca 15 mL. Place flasks in boiling H 2 bath I hr to
deacetylate. Cool, and dil. to \o\. Transfer 5 mL aliquot from
each flask to sep. colorimeter tubes. Develop color by adding
0.5 mL 0.1% NaN0 2 soln (not >5 days old, stored in refrig-
erator), 0.5 mL 0.5% NH 4 sulfamate soln, and 0.5 mL cou-
pling reagent. Det. A at 540 nm against reagent blank (0 mL
aliquot).
(To establish most reliable std curve, make detns on 3 sep.
days and use av. values.)
C. Extraction and Deacetylation
(For premixes, use proper dilns to give 5-10 (xg sulfanitran in
final aliquot, taking dilns into consideration in final calcn.)
Weigh 5.0 g sample into 100 mL vol. flask and add 80 mL
MeOH. Place flask in 60° H 2 bath until MeOH is hot. Re-
peatedly remove and immerse flask during 20 min, shaking
frequently. Cool to room temp., and dil. to vol. with MeOH.
Shake thoroly, and let stand 40 min to permit particles to set-
tle.
Pipet 25 mL aliquot MeOH ext into 50 mL vol. flask. Add
10 mL H 2 0, 5 mL 7 .0% ZnS0 4 soln, and ca 3 drops IN NaOH
to improve ilocculation. (Keep near neutrality.) Place flask in
boiling H 2 bath 2 min to aid pptn; then cool to room temp.,
dil. to vol., mix thoroly, and filter thru Whatman No. 42 pa-
per, or equiv. Discard first 5 mL filtrate.
Pipet 10 mL aliquot filtrate into 50 mL vol. flask contg 8.0
mL H 2 and 0.5 mL HO. Place flask in boiling H 2 bath 1
hr to evap. MeOH and deacetylate sulfanitran, shaking fre-
quently during first 15 min. Cool to room temp, and dil. to
vol. with H 2 0. Centrf. if turbidity appears.
D. Determination
Place 5.0 mL aliquot in each of 2 colorimeter tubes. To I
tube (blank) add 1.0 mL H 2 and 0.5 mL coupling reagent.
To other tube add 0.5 mL 0. 1 % NaN0 2 soln; after 3 min, add
0.5 mL 0.5% NH 4 sulfamate soln, wait 2 min, and add 0.5
mL coupling reagent. Close tube with thumb and invert im-
mediately after adding each reagent. Let stand 10 min for color
development and det. A of sample and blank at 540 nm in
112
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
spectrophtr or colorimeter against H 2 0. Det. amt sulfanitran
from std curve after subtracting A of blank.
% sulfanitran in sample = (xg sulfanitran in tube
x 200 x 100/ (5,000,000 x Lig sample)
= u,g sulfanitran x 0.004
Ref.: J AOAC 46, 452(1963).
CAS- 122- 16-7 (sulfanitran)
963.35 Sulfaquinoxaline in Feeds
Spectrophotometry Method
First Action 1973
Final Action 1988
Method I
(Applicable only to nonpelleted feeds contg arsanilic acid. In
absence of arsanilic acid, use 963.35F>)
A. Principle
Sulfaquinoxaline is extd from feed with DMF and sepd from
interfering substances by column chromatgy on alumina. Iso-
lated sulfaquinoxaline is acidified, diazotized, and coupled in
presence of Zr, and colored complex is extd with BuOH and
measured at 550 nm. Arsanilic acid remains in final aq. soln
and can be measured at 540 nm and compared with std treated
similarly.
B. Reagents
See 969.53A(b), (c), and (d) and in addn:
(a) Alkaline salt soln. — Dissolve 2.0 g NaOH and 100.0 g
NaCl in 500 mL H 2 0.
(b) Zirconium soln. —Dissolve 5.0 g zirconyl chloride,
ZrOCl 2 .8H 2 (Fisher Scientific Co.), in 100 mL H 2 0.
(c) Sulfaquinoxaline std solns . — (7 ) Stock soln . — Weigh 40 .
mg Sulfaquinoxaline Ref. Std (available from Merck & Co.,
Inc.) and dissolve in 50.0 mL DMF. Soln is stable at least 1
month if kept tightly stoppered and protected from light. (2)
Intermediate soln. — 80 |xg/mL. Dil. 5 mL stock soln to 50
mL with DMF. (3) Working soln. — 8 |xg/mL. Dil. 5 mL in-
termediate soln to 50 mL with DMF. Prep, from freshly prepd
intermediate soln just before use.
(d) Butanol mixture. — Mix 100 mL n-hexane with 400 mL
h-BuOH.
C. Preparation of Sample
Weigh 4.00 g ground feed sample into 100 mL vol. flask.
Add 50.0 mL DMF, stopper, and agitate by mag. stirrer or
mech. shaker 60 min. Transfer mixt. to 50 mL centrf, tube
and centrf. 5 min at 2500 rpm.
D. Chromatography
(a) Preparation of column. — Constrict end of 50-60 cm
length of 9-11 mm id glass tubing by rotating in hot flame
until opening is 4-5 mm. Insert small plug of Pyrex glass wool
in lower end and compress with glass rod to thickness of 2-
3 mm. Transfer 5.0 g alumina, 961.24B(b), to dry tube and
pack by gentle tapping while applying vac.
(b) Separation. — Pipet 10 mL clear ext onto column and
let pass thru by gravity. Do not let column run dry; keep 5
mm head of liq. Wash inner walls with two 5.0 mL portions
CHC1 3 . Let final washing drain until no further liq. appears at
tip. Discard effluent and washings. Attach column tip to vac.
and draw air thru until alumina is dry, indicated by tube re-
turning to room temp. Elute column by gravity with 25 mL
alk. salt soln, collecting eluate in 25 mL vol. flask. Add 1.0
mL HC1 to eluate, dil. to vol. with H 2 0, and mix well.
Prep, reagent blank by transferring 10 mL DMF onto fresh
column and proceeding as for sample. Prep, std by transferring
10.0 mL sulfaquinoxaline working std soln onto fresh column
and proceeding as for sample.
E. Determination
Transfer 10 mL aliquots of each eluate to sep. centrf. tubes.
Add 2.0 mL Zr soln and mix. Add 1.0 mL 0.1% NoN0 2 soln,
mix, and let stand 2 min. Add 1.0 mL 0,5% NH 4 sulfamate
soln, mix, and let stand 2 min. Add 1 .0 mL coupling reagent,
969.53A(d), mix, and let stand 10 min. Add 2.0 g NaCl and
10.0 mL BuOH mixt., stopper, and shake vigorously until NaCl
dissolves. Centrf., carefully transfer portion of clear, colored
top solv. layer to 1 cm cell, and read A at 550 nm against
BuOH mixt. Correct for reagent blank.
% Sulfaquinoxaline = 0.04 x (A/A')/W
where A and A' refer to sample and std (blank corrected), resp. ,
and W = g sample.
Method II— Final Action 1960
(Applicable in absence of arsanilic acid)
F. Determination
Weigh 5 g ground sample into 250 mL vol. flask, add 150
mL H 2 and 5 mL 0.5N NaOH, and place in boiling H 2 bath
15 min. Remove, cool, dil. to vol. with H 2 0, mix, and let
settle. Transfer 50 mL supernatant to 100 mL vol. flask, add
3 mL HC1, and dil. to vol. Mix, and filter thru 18.5 cm What-
man No. 2 paper (or equiv.), discarding first 15 mL filtrate if
turbid.
To 10 mL filtrate in each of two 50 mL beakers add 2 mL
freshly prepd 0.1% NaN0 2 soln and let stand 3 min. Add 2
mL 0.5% NH 4 sulfamate soln and let stand 2 min. Add 1 mL
coupling reagent, 969.53A(d), to first beaker and 1 mL H 2
to second beaker. Mix thoroly after adding each reagent. After
10 min, read A in spectrophtr at 545 nm. Subtract A of feed
blank from sample A and det. amt of sulfaquinoxaline from
std curve. Divide by 1000 to obtain % sulfaquinoxaline.
Prep, std curve as follows: Dissolve 0.250 g pure sulfaquin-
oxaline in 5 mL 0.5N NaOH and 50 mL H 2 in 500 mL vol.
flask, and dil. to vol. with H 2 0. Pipet 5 mL aliquot of this
soln into 100 mL vol. flask and dil. to vol. with H 2 0. Pipet
2, 4, 6, 8, and 10 mL portions of this dild soln (equiv. to 50,
100, 150, 200, and 250 |xg sulfaquinoxaline, resp.) into sep.
100 mL vol. flasks, add 3 mL HC1 to each flask, and dil. to
vol. with H 2 0. Treat 10 mL aliquots of these final dilns as in
second par. Det. A at 545 nm against H 2 blank, and plot A
against u,g sulfaquinoxaline.
Refs.: JAOAC 33, 156(1950); 38, 229(1955); 39, 307(1956);
56, 758(1973); 59, 399(1976); 62, 423(1979).
CAS-59-40-5 (sulfaquinoxaline)
974.46 Sulfonamides in Feeds
Spectrophotometric Method
First Action 1974
(Applicable to premixes and cones)
A. Determination of Absorptivities
Prep. sep. std solns of sulfathiazole (SZ), sulfamerazine (SM),
and sulfamethazine (SH) by accurately weighing ca 50 mg each
AOAC Official Methods of Analysis (1990)
Sulfonamides
113
compd and transferring to sep. 50 mL vol. flasks; add 5 mL
alcohol and 2 mL NH 4 OH, and swirl to dissolve. For
sulfaquinoxaline (SQ), weigh 45 mg, warm on steam bath to
dissolve, and cool. Dil. each flask to vol. with alcohol, and
mix. Eva p. 2 mL aliquots to dryness in sep. small beakers,
transfer to sep. 200 mL vol. flasks (100 mL for SQ) with sev-
eral small portions QAN NaOH, and dil. to vol. with 0.1/V
NaOH. Also dil. 25 mL aliquot SQ soln to 50 mL with 0.17V
NaOH. Obtain spectrum of each soln in 1 cm cell against 0.17V
NaOH from 400 to 220 nm.
Calc. a 255 (1.00 mg/100 mL) = A X 50/W
where A is reading at max., ca 255 nm, corrected for A at 400
nm, if any, and W is mg compd weighed. (For SQ, use A of
dild soln.)
For SQ, also calc. a 35S (2.00 mg/100 mL) -Ax 50/VV,
where A is reading at max., ca 358 nm, corrected for A at 400
nm, if any, of more coned soln.
B. Preparation of Sample
(a) Solids. — Transfer accurately weighed sample contg ca
50 mg sulfonamide with lowest concn to 50 mL vol. flask,
add 5 mL alcohol and 2 mL NH 4 OH, and warm 10 min on
steam bath. Cool to room temp., dil. to vol. with alcohol, and
mix.
(b) Liquid concentrates. — Pipet aliquot contg ca 200 mg
sulfonamide with lowest concn into 200 mL vol. flask, dil. to
vol. with alcohol, and mix.
For each sulfonamide declared, calc. an R value to 2 dec-
imal places by dividing its labeled amt by that of sulfonamide
with lowest labeled amt, whose R = 1.00. Calc. to 2 decimal
places. Designate each ratio as R S q, R S z, ^sm> an ^ ^sh, and
their sum as R T . (If 4 sulfonamides are present in equal amts,
all/? - 1.00 and/? T = 4.00.)
C. Determination of Sulfaquinoxaline
Pipet 5 mL sample soln into g-s flask and add accurately
measured vol. alcohol so that total mL of final soln = 10 x
R T (Soln f). Mix, pipet 10 mL into small beaker, and evap. to
dryness on steam bath. Transfer residue to 100 mL vol. flask
with several small portions 0.1 Af NaOH, dil. to vol. with 0. \N
NaOH, and mix (Soln If). Obtain spectrum from 400 to 300
nm, and det. A at max., ca 358 nm.
% SQ = [A 35g x 2 x R T x (V/5) x 100]/(a 358 X S)
where V = mL original sample soln (50 or 200). and S — mg
original sample (for solids) or mL x 1000 (for liqs).
If Na salt declared, NaSQ - SQ/0.9318.
D. Determination of Total Sulfonamides
Dil. 20.0 mL Soln U to 100 mL with 0.17V NaOH. Obtain
spectrum from 400 to 230 nm and det. A T at max. , ca 255 nm.
% Total sulfonamides - [A r X V x R T 2 x 100]/
l(R x a )sQ H- (R X a ) sz + (R x a ) SM
+ (R x a ) SH \ X S
If Na salts are present, multiply each a by appropriate factor:
NaSQ, 0.9318; NaSZ, 0.9207; NaSM, 0.9232; and NaSH,
0.9268.
Ref.: JAOAC 57, 345(1974).
CAS- 127-79-7 (sulfamerazine)
CAS-57-68-1 (sulfamethazine)
CAS-59-40-5 (sulfaquinoxaline)
CAS-72-14-0 (sulfathiazole)
974.47 Sulfonamides in Feeds
Thin Layer Chromatographic Method
First Action
A. Preparation of Plates
(a) Plates A.— Weigh 30 g silica gel H or HF 254 (Brink-
mann Instruments, Inc.) into 250 mL g-s flask. (Add 100 mg
fluorescent indicator H 254 if silica gel H is used.) Add 70
mL H 2 and shake well 1 min. Coat five 20X20 cm plates
with 0.25 mm layer and air dry. Do not dry in oven and do
not store in presence of drying agent.
(b) Plates B. — Proceed as in (a) but use 0AN NaOH in-
stead of H 2 to slurry silica gel.
B. Preparation of Blanks
Develop a plate A in CHCl 3 -MeOH (97 + 3) to ht of ca 15
cm. Scrape 2 spots, each ca 2 sq cm, from developed section
of plate, into centrf. tubes and ext with 10 mL 0AN NaOH.
Scrape 2 addnl spots, each ca 4 sq cm, from developed portion
and ext with 50 mL 0.UV NaOH. Centrf. the 4 exts 5 min at
high speed and decant most of soln into sep. small beakers,
being careful not to disturb sediment.
Develop a plate B in CH'CJs-M'eOH (90 + 10) and proceed
as above.
Det. A at max. , ca 255 nm, of all 8 exts. A should be <0.04.
If readings are low and reproducible, average each set of 4
values for 2 and 4 sq cm exts, resp. If readings are high or
not reproducible, recentrf. exts at higher speed, and be very
careful to exclude sediment during decanting. Av. blank A may
be used for all detns which use same batch of silica gel and
same speed of centrfg.
C. Thin Layer Chromatography
Spot 100 (xL sample soln, 974. 46B, on plate A and plate B
by repeated application of adjacent drops on line 4 cm long,
drying each drop with gentle air current before applying an-
other drop at same place. Develop plate A in CHCl 3 -MeOH
(93 + 7) and plate B in (90 4- 10) until sol v. front reaches
top of plate.
View plates under shortwave UV light and delineate each
spot with dissecting needle, including small margins whenever
possible. Plate A should have 2 completely sepd spots, des-
ignated as c (lower: SZ) and d (upper: SM + SH + SQ); plate
B should have 3 completely sepd spots designated as /(lower:
SQ + SZ), h (middle: SM), and p (top: SH). If less than in-
dicated no. of spots are sepd, spot another aliquot over slightly
longer line and develop as before.
Use collection tube consisting of 7-8 cm of 8 mm od glass
tubing with short constriction at one end and medium fritted
glass disk fused near center. Clean tube with strong air current
and attach wide tube to vac. Draw as much of spot as possible
into tube, using narrow tube to loosen adsorbent layer. With-
out disconnecting vac, transfer tube to 10 mL vol. flask for
spot c and 50 mL for spot d. Release vac, and transfer ma-
terial to flask with repeated gentle tapping. Repeat until entire
spot has been transferred; then blow out tube into flask with
gentle air current to remove last of particles.
Fill each flask ca V2 full with 0. \N NaOH, swirl well 1 min,
dil. to vol. with 0AN NaOH, and mix. Centrf. all or >10 mL
each soln at same speed used for blanks and carefully decant
ca 8 mL into small beakers. Obtain spectrum of each soln from
400 to 230 nm and record A c of spot c ext and A$ of spot d
ext at max., ca 255 nm. Correct A c for av. blank of 2 sq cm
exts and A d for av. blank of 4 sq cm exts.
114
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
Calc. recovery factor, F = f(5 X A d ) + A C \/(R T X A T )
% SZ - (A c x V x 100)/(a sz ,255 x F x S)
% NaSZ = % SZ/0.9207
From plate B, transfer spot f to 50 mL vol. flask and spot
h and p to 10 mL vol. flasks. Obtain blank corrected A h A h ,
and v4 p as for exts of plate A.
Calc. recovery factor F f - [(5 X A f ) + A h + A P ]/(J? T x A T )
% SM - (A h X V x 100)/(a SM x F' x S)
% SH - 04 p x V x 100)/(a SH x F' x S)
% NaSM = % SM/0.9232
% NaSH = % SH/0.9268
Ref.: J AOAC 57, 345(1974).
CAS- 127-79-7 (sulfamerazine)
CAS-57-68-1 (sulfamethazine)
CAS-59-40-5 (sulfaquinoxaline)
CAS-72-14-0 (sulfathiazole)
966.28 Thiabendazole in Feeds
Spectrophotometric Method
First Action 1966
Final Action 1967
Method I
(Applicable to all feeds)
A. Principle
Thiabendazole is extd from feed with 0A.N HC1. Interfer-
ences are removed by adjusting ext to pH 5-6 with Na citrate
and extg with CHCl 3 . Thiabendazole is re-extd with 0.1N HCl
and reduced with Zn slurry in 30% glycerol in presence of
p-phenylenediamine. Oxidn with ferric iron yields blue com-
plex which is extd with BuOH and measured at 605 nm.
B. Reagents
(a) Zinc dust. — Reagent grade. Crush fine lumps with spat-
ula immediately before use.
(b) Zinc slurry. — Weigh 50 mg /?-phenylenediamine.2HCl
(Caution: /?-Phenylenediamine may be harmful; see safety notes
on toxic dusts.) and 2 g Zn dust into dry 100 mL g-s graduate.
Add 100 mL 30% (v/v) glycerol soln, stopper, and shake ca
30 sec to suspend Zn dust uniformly. (There must be no ag-
glomeration of Zn.) Prep, just before use and use immediately.
(c) Ferric soln.— Dissolve 15.0 g FeNH 4 (S0 4 )?.12H 2 in
75 mL H 2 0, add 10.0 mL \N H 2 S0 4 , dil. to 100 mL, and
mix.
(d) Thiabendazole std solns. — (J) Stock soln. — 0.5 mg/mL.
Dissolve 50.0 mg Thiabendazole Ref. Std (available from Merck
& Co.) in 0.1JV HCl and dil. to 100 mL. Soln is stable >1
month. (2) Intermediate soln. — 50 jxg/mL. Dil. 10 mL stock
soln to 100 mL with 0. 17V HCl. Soln is stable >1 month. (3)
Working soln. — 5 juig/mL. Dil. 20.0 mL intermediate soln to
200 mL with 0. 1W HCl. (Use same 0AN HCl as in extn of
feed.)
C. Determination
Grind ca 100 g well mixed sample to pass No. 30 sieve and
mix. (3 min in high-speed blender should be enough.)
Weigh 2.000 g ground sample into 250 mL I 24/40 flat-
bottom extn flask. (For feeds contg <0.025% thiabendazole,
weigh 5.000 g.)
Add 100.0 mL 0.1N HCl to sample and add mag. stirring
bar. Connect flask to reflux condenser (Allihn, drip tip) and
reflux gently on mag. hot plate, while stirring, 30 min. Cool,
transfer mixt. to centrf. tube, and centrf. ca 5 min. Dil. mea-
sured aliquot of supernate to concn of 5 fig thiabendazole/mL
(serial dilns may be necessary). Such dilns det. u dilution fac-
tor," DF:
Declaration,
%
Sample
wt, g
Dtlution(s)
DF
0.01
5
none
1
0.025
2
none
1
0.1
2
25-100
4
1.0
2
10-100; 25-100
40
6,0
2
10-100; 10-250
250
Mark series of 50 mL centrf. tubes 1 , 2, 3, 4, etc. Place
20.0 mL 0AN HCl in tube 1 and 20.0 mL (100 u.g) working
std soln in tubes 2 and 3. Place 20.0 mL aliquots of sample
solns in tubes 4, 5, etc. Add 3 g Na citrate, 3 g NaCl, and
20.0 mL CHC1 3 to each tube, stopper tightly with polyethylene
stopper, and shake mech. 5 min. Centrf. ca 5 min and discard
top layers. With pipet, transfer 10 mL CHC1 ? ext to dry, marked,
centrf. tubes, add 25.0 mL 0. IN HCl to each, stopper, and
shake 5 min. Centrf., and transfer, with pipet, 15 mL of top
acid layer to another marked tube. (Because of timing, handle
<10 tubes at one time.)
With rapid delivery pipet, add 5 mL freshly prepd Zn slurry,
(b), to each tube. (5 mL pipet with tip cut off to give delivery
in ca 5 sec is suitable. Hold pipet directly over center of soln.)
Do not shake tube but immediately stopper tightly and let stand
4 min. Start timing after delivery of slurry to first tube.
After 4 min, add 5.0 mL ferric soln, (c), to each tube with
rapid pipet, stopper, and mix by inverting tube. Let stand 5
min; then shake vigorously and centrf. ca 3 min. With pipet,
transfer 15 mL clear, colored soln to marked, dry, centrf. tubes.
Let stand 45 min from addn of ferric soln. Then add 5.00 mL
ai-BuOH and 3 g anhyd. Na 2 S0 4 to each tube. Stopper, and
immediately shake each tube ca 5 sec to avoid caking of Na 2 S0 4 ;
then shake all tubes ca 3 min or until Na 2 S0 4 is completely
dissolved, and centrf.
Transfer clear BuOH soln (top layer) to dry 1 cm cell and
read A at 605 nm against n-BuOH as ref.
% Thiabendazole in feed
= (A - A )(C)(DF)/360(A f - A )W
where A refers to sample, A to reagent blank (tube 1), A' to
std, C = |ig thiabendazole std in final 15.0 mL colored soln
(18 |xg), DF = diln factor, and W = g original sample.
Refs.: JAOAC 47, 235(1964); 49, 312(1966).
CAS- 148-79-8 (thiabendazole)
966.29 Thiabendazole in Feeds, Supplements,
and Premixes
Spectrophotometric Method
First Action 1966
Final Action 1967
Method II
(Applicable to cattle supplements and premixes contg >1%
thiabendazole. Principle is same as 966. 28A, except that sin-
gle extn at room temp, with 0.1/V HCl is used. Not applicable
to feeds, premixes, or cattle supplements contg high levels of
protein.)
AOAC Official Methods of Analysis (1990)
Antibiotics
115
A. Reagents
See 966.28B except:
(a) Thiabendazole working soln. — 2 |xg/mL. Dil. 10.0 mL
thiabendazole intermediate soln, 966.28B(d)(2), to 250 mL with
O.IjVHCI.
B. Determination
Prep, sample as in 966*28C, except use ca 50 g represen-
tative sample.
Weigh 2.000 g ground sample into 1 L vol. flask and add
750 mL 0.17V HC1. Add mag. stirring bar, stopper, and mix
vigorously on mag. stirrer 1 hr at room temp. (Mech. shaker
providing vigorous agitation may be used.) Remove and rinse
bar, and dil. to vol. with 0.1JV HCL Mix, centrf., and dil.
aliquots of clear ext with 0. IN HC1 to concn of 2 jxg thiaben-
dazole /mL. Diln factors, DF, are as follows (see 966. 28C):
Declaration,
Dilution
DF
1.0
2.5
6.0
10-100
4.0-100
4.0-250
10
25
62.5
Develop color in exts as soon as possible after extn. (Acid
exts of some feeds deteriorate upon standing.)
Mark series of 50 mL centrf. tubes (<10) as in 966. 28C.
Add 15.0 mL 0. \N HC1 to tube 1 , and 15.0 mL working soln,
(a), to tubes 2 and 3. Add 15.0 mL sample exts to other tubes.
Then with rapid delivery pipet, add 5.0 mL freshly prepd Zn
slurry as in 966. 28C. Proceed as in 966. 28C with addn of
ferric soln, observing same technics and time precautions. Read
final clear BuOH ext, as above, in 1 cm cell at 605 nm.
% Thiabendazole in feed
- (A - A )(C)(DF)/90(A' - A )W
where symbols are as defined in 966. 28C.
Rets.: JAOAC 47, 235(1964); 49, 312(1966).
CAS- 148-79-8 (thiabendazole)
(f ) Zoalene std soln. — 40 |xg/m'L. Weigh 40.0 mg Zoalene
Ref. Std (available from Dow Chemical Co.) into 1 L vol.
flask, dil. to vol. with 85% CH 3 CN, and mix.
C. Determination
Weigh 10.0 g sample into 250 mL erlenmeyer and add 65
mL 85% CH 3 CN. Warm on steam bath to 50 ± 5°, swirling
occasionally. Let cool to room temp, (ca 30 min). Add 20 g
alumina and swirl occasionally ca 3 min. (Addn of alumina is
unnecessary for cones contg >1% zoalene.) Filter with suction
on medium or fine porosity 40 mm diam. fritted glass funnel,
transferring as much solids as possible. Transfer remaining solids
with min. vol. 85% CH 3 CN, and suck dry. Suspend cake in
funnel with min. vol. 85% CH 3 CN and slight stirring but with-
out suction. Then filter with suction and repeat suspension and
filtering, keeping total vol. <100 mL. Transfer combined fil-
trates to 100 mL vol. flask (or vol. flask may be used to collect
filtrates directly), dil. to vol. with 85% CH 3 CN, and mix.
Based on zoalene concn, make addnl dilns with 95% ace-
tone and use aliquots indicated in Table 961.26.
Pipet indicated aliquots into three 50 mL beakers, X, Y, and
Z, for conens <0.25%; omit X for samples >0.25%. Pipet 1
mL std soln into beaker Z and evap. all solns to dryness with
air current. (Heat may be used but temp, must not exceed 60°.)
Pipet 10 mL 95% DMF into X and 2 mL each into Y and Z.
Swirl intermittently during 5 min to dissolve zoalene. Pipet 8
mL ethylenediamine into Y and Z and mix. If turbidity persists
after 2 min, filter thru small Reeve Angel No. 804, or equiv.,
paper. Read A of solns at 560 nm in stoppered 1 cm cells
against 95% DMF 5 min after addn of ethylenediamine. Keep
cell compartment of spectrophtr at <30° to avoid rapid fading
of color. If A is >1 , reanalyze, using greater diln or smaller
aliquot.
% Zoalene = (A Y - A x ) x M/100 (A z - A Y )
Caution: CH 3 CN and ethylenediamine are toxic.
hood and avoid contact with skin.
Handle in
Refs.: JAOAC 44, 18(1961); 45, 294(1962); 51, 501(1968).
CAS- 148-01-6 (zoalene)
961.26 Zoalene in Feeds
Spectrophotometric Method
First Action 1961
Final Action 1962
(Not applicable in presence of furazolidone, nitrofurazone, and
nihydrazone)
A. Principle
Zoalene is extd from feeds, premixes, and cones contg 0.004-
25% with 85% CH 3 CN. For mixes contg <1%, alumina is
added. After filtration and diln, zoalene is detd colorimetrical-
ly after reaction with ethylenediamine.
B. Reagents
(a) Acetone. — 95%. Add 5 mL H 2 Q to 95 mL acetone.
(b) Acetonitrile—%5%. Add 850 mL practical grade CH 3 CN
to 150 mL H 2 (deionized or distd).
(c) Activated alumina. — Alcoa grade F 20, 80-200 mesh.
(Available from Fisher Scientific Co. "Alumina, Adsorption,
Fisher. ")
(d) Dimethylformamide (DMF).— 95%. Add 5 mL H 2 to
95 mL tech. DMF. Prep, fresh daily, since old solns may cause
cloudiness. (Caution: see safety notes on dimethylformamide.)
(e) Ethylenediamine. —98- 100%. (No. EX0510, EM Sci-
ence). Reagent must be practically colorless.
Table 961.26 Dilution of Sample for Determination
% Zoalene
in Sample
Addnl Diln
Aliquot
Size, mL
Multiplication
Factor M
0.004- 0.012
None
4
1
0.012- 0.025
None
2
2
0.025- 0.050
10 to 100
10
4
0.050- 0.10
10 to 100
5
8
0.10 - 0.25
10 to 100
2
20
0.25 - 0.5
1 to 100
10
40
0.5 - 1.0
1 to 100
5
80
1.0 - 2.5
1 to 100
2
200
2.5 - 5.0
1 to 1000
10
400
5.0 -10.0
1 to 1000
5
800
10.0 -25.0
1 to 1000
2
2000
MICROBIOLOGICAL METHODS FOR ANTIBIOTICS
957.23
Antibiotics in Feeds
Microbiological Methods
A. Culture Media
(Deionized H 2 may be used for prepn of media.)
(a) Agar medium A. — (Antibiotic Medium 1 .) Dissolve 6.0
g pancreatic digest of gelatin, 4.0 g pancreatic digest of casein,
116
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
3.0 g yeast ext, 1.5 g beef ext, 1.0 g anhyd. glucose, and 15
g agar in H 2 0, and dil. to 1 L. Adjust with IN NaOH or HC1
(1 + 9) so that after sterilization pH is 6.5-6.6. (Difco Pen-
assay Seed Agar (antibiotic medium I) and BBL Seed Agar
have been found satisfactory.)
(b) Agar medium B. — (Antibiotic Medium 4.) Dissolve 6.0
g pancreatic digest of gelatin, 3.0 g yeast ext, 1.5 g beef ext,
1.0 g anhyd. glucose, and 15 g agar in H 2 0, and dil. to 1 L.
Adjust with IN NaOH or HC1 (1 + 9) so that after sterilization
pH is 6.5-6.6. (Difco and BBL Yeast Beef Agar have been
found satisfactory.)
(c) Agar medium C. — (Antibiotic Medium 2.) Dissolve 6.0
g pancreatic digest of gelatin, 3.0 g yeast ext, 1,5 g beef ext,
and 15 g agar in H 2 0, and dil. to 1 L. Adjust with IN NaOH
or HC1 (1 +9) so that after sterilization pH is 6.5-6.6. (Difco
Penassay Base Agar and BBL Base Agar have been found sat-
isfactory.)
(d) Agar medium D. — (Antibiotic Medium 8.) Use agar
medium C adjusted with 17V NaOH or HC1 (1+9) so that
final pH is 5.7-5.9. (Difco Antibiotic Medium 8 and BBL
Base Agar with low pH have been found satisfactory.)
(e) Agar medium E. — (Antibiotic Medium 5.) Use agar me-
dium C adjusted with IN NaOH so that final pH is 7.8-8.0.
(Difco Streptomycin Assay Agar (antibiotic medium 5) and
BBL Streptomycin Assay Agar with Yeast Extract have been
found satisfactory.)
(f) Agar medium, F. — Adjust agar medium A with 3.57V
NaOH (2.8-3.8 mL/L) so that after sterilization pH is 8.9-
9.1.
(g) Agar medium G. — (Antibiotic Medium 32.) Use agar
medium A to which is added 300 mg MnS0 4 .H 2 or 0.4 ml
.1% MnCl 2 soln/L.
(h) Agar medium H. — Dil. 1 L agar medium A to 1.2 L
and adjust to pH 8.1.
(i) Agar medium. I. — Dissolve 9.4 g pancreatic digest of
gelatin, 4.7 g yeast ext, 2.4 g beef ext, 10. g NaCl, 10.0 g
anhyd. glucose, 13.0 g anhyd. KH 2 P0 4 , 1 g Na 2 HP0 4 .7H 2 0,
and 23.5 g agar in H 2 0, and dil. to 1 L. After sterilization pH
is 5.3-5.5. (BBL Nystatin Assay Agar supplemented with 13.0
g anhyd. KH 2 P0 4 and 1 g Na 2 HP0 4 .7H 2 has been found sat-
isfactory.)
(j) Agar medium J. — (Antibiotic Medium 11.) Use agar
medium A adjusted with IN NaOH so that final pH is 7.9-
8.0. (Difco and BBL Neomycin Assay Agar have been found
satisfactory.)
(k) Agar medium K. — To each L agar medium J add 12.5
mL 2M CaCl 2 after autoclaving and just before pouring plates.
(J) Agar medium L.— Dissolve 0.69 g K 2 HP0 4 , 0.45 g
KH 2 P0 4 , 2.5 g yeast ext, 10.0 g anhyd. glucose, and 15.0 g
Difco Noble agar in H 2 and dil. to 1 L. Adjust to pH 6.0
with HC1 before use.
(m) Agar medium M. —Dissolve 2.5 g yeast ext, 10.0 g
glucose, 0.69 g K 2 HP0 4 , 0.45 g KH 2 P0 4 , and 20.0 g agar in
H 2 and dil. to 1 L. Before adding inoculum, adjust liquified
medium to pH 6.0 with IN HC1 (ca 2 mL/L).
(n) Broth medium A. — (Antibiotic Medium 3.) Dissolve 5.0
g pancreatic digest of gelatin, 1.5 g yeast ext, 1 .5 g beef ext,
3.5 g NaCl, 1.0 g anhyd. glucose, 3.68 g anhyd. K 2 HP0 4 ,
and 1.32 g anhyd. KH 2 P0 4 in H 2 0, and dil. to 1 L. Adjust
with IN NaOH or HCJ (1 +9) so that after sterilization pH
is 6.95-7.05. (Difco Penassay Broth (antibiotic medium 3) and
BBL Antibiotic Assay Broth have been found satisfactory.)
(o) Broth medium B. — Dissolve 5.0 g pancreatic digest of
casein, 5.0 g pancreatic digest of animal tissues, and 20 g
anhyd. glucose in H 2 0, and dil. to 1 L. Adjust with IN NaOH
or with HC1 (1 +11) so that after sterilization pH is 5.6-5.7.
(Difco Fluid Sabouraud Medium and BBL Sabouraud Liquid
Broth Modified have been found satisfactory.)
B. Reagents
(a) Phosphate-bicarbonate buffer. — pH 8. Dissolve 16.73
g anhyd. K 2 HP0 4 , 0.523 g anhyd. KH 2 P0 4 , and 20 g NaHC0 3
in H 2 and dil. to 1 L.
(b) Phosphate buffer.— pH 8; 0.1M. Dissolve 16.73 g
anhyd. K 2 HP0 4 and 0.523 g anhyd. KH 2 P0 4 in H 2 and dil.
to 1 L.
(c) Phosphate buffer. — pH 7.0; 0.IM. Dissolve 13.6 g
anhyd. K 2 HP0 4 and 4.0 g anhyd. KH 2 P0 4 in H 2 and dil. to
1 L.
(d) 5% Phosphate buffer.— pH 6.5. Dissolve 22.15 g
anhyd. K 2 HP0 4 and 27.85 g anhyd. KH 2 P0 4 in H 2 and dil.
to 1 L.
(e) 10% Phosphate buffer. — pH 6. Dissolve 80 g anhyd.
KH 2 P0 4 and 20 g anhyd. K 2 HP0 4 in H 2 and dil. to 1 L.
(f) 1% Phosphate buffer. — pH 6. Dissolve 8.0 g anhyd.
KH 2 P0 4 and 2.0 g anhyd. K 2 HP0 4 in H 2 and dil. to J L.
(g) Phosphate buffer. — pH 4.5; 0.1 A/. Dissolve 13.6 g
anhyd. KH 2 P0 4 in H 2 and dil. to 1 L.
(h) Acid-acetone. — Mix 1 vol. AN HO, 13 vols acetone,
and 6 vols H 2 0.
(i) Acid-methanol. — Mix 1 vol. HC1 and 50 vols MeOH.
(j) Ethyl acetate. — 99% undenatured grade.
(k) Buffer-acetone extractant. — Mix equal vols pH 6 buffer,
(f), and acetone.
(1) Tris buffer.— pH 8.0, 0.05M. Dissolve 6.05 g
tris(hydroxymethyl)aminomethane (THAM, primary std,
available from Fisher Scientific Co.) in 900 mL H 2 0, adjust
pH to 8.0 with HC1, and dil. to 1 L.
(m) Calcium chloride soln. — 2M. Dissolve 294.04 g
CaCl 2 .2H 2 in H 2 and dil. to I L.
(n) Sodium chloride-calcium chloride soln. — Dissolve 200
g NaCL in H 2 0, add 10 mL 2M CaCl 2 , and dil. to 1 L.
(o) Sodium hypochlorite soln. — 5.25%. Use freshly opened
bottle com. soln. (Clorox has been found satisfactory.) Store
in dark at 2-10°.
(p) Sterile isotonic saline soln. — Dissolve 9.0 g NaCl in
H 2 and dil. to I L. Autoclave 20 min at 121°.
(q) Lead acetate soln. — Dissolve 303 mg Pb(OAc) 2 .3H 2
in H 2 and dil. to 1 L with H 2 0.
C. Apparatus
(High-speed blender jars, after disassembling, must be cleaned
with great care to eliminate all traces of antibiotics. All app.
that contacts sample and solns must be thoroly cleaned and be
detergent-free.)
(a) Cylinders. — Polished open stainless steel cylinders, 8 ±
0.1 mm od, 6 ± 0. 1 mm id, and 10 ± 0. 1 mm high (obtainable
from S & L Metal Products Corp., 58-29 57th Drive, Mas-
peth, NY 11378).
(b) Petri dishes {plates). — Glass or plastic; 100 mm wide
X 20 mm deep. Porcelain covers glazed on outside or cover
lids with filter pad inserts are satisfactory for absorbing H 2
of syneresis. Glass or plastic covers may be used if they are
raised slightly to allow escape of H 2 0.
(c) Cylinder dispenser. — May be used to place cylinders on
plates. Shaw Dispenser, available from Arthur E. Farmer, 47
Frazier St, PO Box 1785, Trenton, NJ 08618.
(d) Agar cutter — Used to prep, cups in agar, available from
Biochem. Dept, Purdue Univ., W. Lafayette, IN 47907.
D. Stock Cultures and Preparation of Test Organism
Suspensions
For appropriate test organism designated below, prep, slant
culture on >1 tube of agar medium A. Incubate overnight at
indicated temp, held constant to ± 0.5°, and then store in dark
at 2-10°. Do not use if >2 weeks old.
AOAC Official Methods of Analysis (1990)
Antibiotics
117
Prep, suspensions of test organisms as follows:
(a) Micrococcus flavus . — ATCC No. 10240. Incubate stock
culture at 32-35°. Wash growth from stock culture with ca 3
m'L broth medium A and transfer liq. to surface of 300 mL
agar medium A in Roux bottle. Spread suspension evenly over
entire surface, using sterile glass beads, and incubate over-
night at 32-35°. Wash growth from agar surface with ca 25
mL sterile isotonic saline soln. Store bulk suspension at 2-
10°. Use for bacitracin assay.
(b) Sarcina subflava. — ATCC No. 7468. Incubate stock
culture at 32-35°. Prep, suspension as in (a) and use as al-
ternative organism for bacitracin assay.
(c) Bacillus cereus. — ATCC No. 11778. Incubate stock
culture at 30°. Wash growth from stock culture with ca 3 mL
sterile H 2 0, transfer to surface of 300 mL agar medium A,
and incubate 7 days at 30°. Wash growth from agar surface
with ca 25 mL sterile H 2 and heat suspension 30 min at 65°.
Centrf. and decant. Wash residual spores 3 times with sterile
H 2 0, centrfg and decanting each time. Discard wash H 2 0. Heat
residual spores 30 min at 65° and resuspend in sterile H 2 0.
Store this stock suspension at 2-10°. Use for chlortetracycline
and oxytetracycline assays.
(d) Bacillus sub tills. — ATCC No. 6633. Incubate stock
culture at 37°. Wash growth from stock culture with ca 3 mL
sterile isotonic saline soln, transfer to surface of 300 mL agar
medium G in Roux bottle, and incubate 7 days at 37°. Wash
growth from agar surface with ca 50 mL sterile isotonic saline
soln into centrf. bottle. Heat suspension 30 min in 65° H 2
bath to destroy vegetative cells. Centrf., decant, and resuspend
cells in ca 50 mL sterile isotonic saline soln. Repeat heating,
centrfg, and suspending twice, or until supernate is clear. Final
suspension is stock spore suspension. Store at 2-10°. Use for
hygromycin B, monensin, and streptomycin assays.
(e) Sarcina lutea. — ATCC No. 9341. Incubate stock cul-
ture at 26-30°. Prep, organism suspension by one of following
methods:
(/) Roux bottle culture. — Wash growth from 24 hr slant
culture with ca 3 mL broth medium A, and transfer liq. to
surface of 300 mL agar medium A in Roux bottle. Spread
suspension evenly over entire surface, using sterile glass beads,
and incubate 24 hr at 26-32°. Wash growth from agar surface
with ca 15 mL sterile isotonic saline soln. Store bulk suspen-
sion <2 weeks at 2-10°.
(2) Broth culture. — Wash growth from stock culture with
ca 3 mL broth medium A, and transfer liq. to 100 mL broth
medium A. Incubate 48 hr at 26-32° with continuous mech.
agitation. This 48 hr culture is inoculum. Store <2 weeks at
2-10°.
Use for erythromycin, lincomycin, novobiocin feed supple-
ment, oleandomycin, penicillin, and tylosin assays.
(f) Staphylococcus epidermidis.^ ATCC 12228. Incubate
stock culture at 32°. Inoculate 30 mL broth medium A in 300
mL flask with 1 loop from stock culture, and incubate over-
night at 26-32°. Prep, daily. Use for neomycin and for no-
vobiocin final feed assays.
(g) Saccharomyces cerevisiae. — ATCC No. 9763. Incu-
bate stock culture on agar medium I at 37°. Prep, inoculum
by one of following methods:
(/) Broth culture. — Inoculate 100 mL broth medium B with
1 loop from stock culture and incubate overnight at 37°. This
culture is inoculum. Store <2 weeks at 2-10°.
(2) Roux bottle culture. — Wash growth from stock culture
with ca 3 mL sterile isotonic saline soln, and transfer liq. to
surface of 300 mL agar medium I in Roux bottle. Spread sus-
pension evenly over entire surface, using sterile glass beads,
and incubate 24 hr at 37°. Wash growth from agar surface with
ca 15 mL sterile isotonic saline soln. Store <2 weeks at 2-
10°.
Use for nystatin assay.
(h) Escherichia cob. — UC 527 (available from The Upjohn
Co.). Incubate at 36°. Inoculate 30 mL broth medium A in
250 mL flask from stock culture of E. coli and grow 1.8-24
hr at 36°. Prep, daily. Use for spectinomycin assay.
£. Design and Plotting of Standard Response Line
Prep, concns of ref. std as described for each antibiotic. In
general, it is preferable to use shorter 4-fold range between
lowest and highest doses of std line. Use indicated concn as
ref. concn. (Values of std or ref. concn could slightly vary
from those indicated for each antibiotic without affecting va-
lidity of assay.)
Prep, plates with appropriate base agar layer and /or appro-
priate seed agar layer; one layer of media can be substituted
for 2 layers of media if ref. concn gives adequate zone size
as described for each antibiotic. Distribute agar evenly by tilt-
ing plates from side to side with circular motion and let harden.
Use plates same day prepd.
Place 6 cylinders on each plate at ca 60° intervals on 2.8
cm radius. Fill 3 alternate cylinders with ref. concn and other
3 cylinders with one of other concns of std. Use 3 plates for
each concn required for std response line, except ref. concn.
Incubate plates overnight at appropriate temp., and measure
diams of zones of inhibition as accurately as possible. (In most
cases, it is possible to est. zone diams to nearest 0.1 mm.)
Values given in each method for zones of inhibition to be ob-
tained with ref. concns of antibiotics are for guidance only,
but it is important that lowest concns on std response line give
measurable zone and that slope of response line be adequate.
In each set of 3 plates, average the 9 readings of ref. concn
and the 9 readings of concn being tested. Av. of all 36 read-
ings of ref. concn from 12 plates is correction point for re-
sponse line. Correct av. value obtained for each concn to ap-
propriate figure if ref. concn reading on that set of 3 plates
was same as correction point.
For example, if in correcting second concn of std response
line, av. of 36 readings of ref. concn is 20.0 mm, and av. of
9 readings of ref. concn of this set of 3 plates is 19.8 mm,
correction is + 0.2 mm. If av. reading of second concn on
same 3 plates is 17.0 mm, corrected value is 17.2 mm. Plot
corrected values, including correction point, on semilog graph
paper, using logarithmic scale for concn and arithmetic scale
for av. zone diams. Manual plotting of std lines is possible
but could be subject to large variation. Response lines would
be more accurate if calcd. When std doses are equally spaced,
i.e., interval between successive doses is the same, calc. L
and H (calcd zone diams for low and high concns, resp., of
std response line) as follows:
For method specifying 5 doses of std,
L - (3a + 2b + c - e)/5
H = (3e + 2d + c - a)/5
where a, b, c, d, and e — corrected av. zone diams for each
concn of std.
For methods specifying 4 doses of std,
L = (la + 4b 4- c - 2d)/\0
H = (Id + Ac + b - 2a)/ 10
For methods specifying 3 doses of std,
L - (5a + 2b - c)/6
H - (5c + 2b ~~ a)/6
Plot values for L and H and connect with straight line. Ref.
point is zone size intercept on arithmetic scale. This corrected
ref. point is to be used for sample calcns (if corrected ref. point
diam. varies significantly from av. ref. diam., error in prepn
118
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
of std solns is indicated and validity of assay is in question.)
For more accuracy in calcn, det. slope of std response line B
= (H - L)/(log h — log /), where / and h are high and low
std concns, resp., and B is increase in zone for each 10X in-
crease in drug concn.
Computer or calculator can be used to calc. std lines whether
std concn s are equally spaced or not. Least square fitting using
linear or polynomial equations may be performed based on
best fit (polynomial fitting is most appropriate, especially for
long range 8x or 16x range).
F. Determination of Potency
Use 3 plates of each assay soln. On each plate, fill 3 alter-
nate cylinders with ref. concn and fill other 3 alternate cyl-
inders with assay soln. Incubate plates overnight at appropriate
temp, and measure diam. of zones of inhibition. Average the
9 readings of ref. concn and the 9 readings of assay soln. If
assay soln gives larger av. than ref. concn, add difference be-
tween them to ref. point on std response line. If assay soln
gives smaller value than ref. concn, subtract difference be-
tween them from ref. point on std response line. Using cor-
rected values of assay soln, det. amt of antibiotic by reading
concn from std response line.
Alternatively, det. log relative potency, M' = (Y u - Y s )/B,
where Y u and K s are av. of 9 readings of assay soln and ref.
concn, resp., and B is slope of std response line. Antilog M'
= potency of assay soln relative to std; and (antilog M') x
100 — potency of assay soln as % of std ref. concn.
For calcn of sample potency by computer or calculator, en-
ter sample data and calc. antibiotic potency based on least square
linear or polynomial lines.
For calcns, 1 ton = 908 000 g; 1 lb = 454 g.
Refs.: JAOAC 40, 857(1957); 72, 105(1989).
982.43 Bacitracin in Premix Feeds
Cylinder Plate Method
First Action 1982
(Applicable to premixes contg ^10 g bacitracin/lb)
A. Principle
Bacitracin is extd from feeds into acidified org. solv. sys-
tem. Ext is centrfd, and supernate is dild in phosphate buffer
and analyzed by cylinder plate assay with M. flavus as detec-
tion organism.
B. Reagents and Apparatus
(a) Microorganism. — Micrococcus flavus ATCC 10240.
Maintain culture as indicated in 957.23D(a).
(b) Extracting solv.— -Mix, by vol., 27% CH 3 CN, 27%
MeOH, 3% pH 6.0 phosphate buffer (957.236(f)), 41% H 2 0,
and 2% H 3 P0 4 (85%); add 0.5 g EDTA/L. (Extg solv. is satd
with EDTA.).
(c) Phosphate buffer. -—5%, pH 6.5. See 957.238(d).
(d) Diluting solvent.—- Methanol -5% pH 6.5 phosphate
buffer (12 + 88).
(e) Dilute HCL— Carefully add 89 mL HC1 to H 2 and dil.
to 1 L (IN). Further dil. soln 1:100 (0.01N).
(f) Cylinders. —See 957.23C(a).
(g) Cylinder dispenser. — Optional: see 957.23C(c).
C. Standard Solutions
See 957.24*(a) and (b). Also prep. 0.30 and 0.16 unit/mL
solns to be plated as samples to monitor assay.
D. Preparation of Plates
Use one layer (ca 15 mL) of agar antibiotic medium, 1,
957.23A(a). Det. by trial plates optimum concn (usually 0.02-
0.05%) of M. flavus ATCC 10240 to be added to agar to ob-
tain zones of inhibition 15-17 mm for 0.2 unit bacitracin/mL.
Pour 4 plates for each point on std curve (i.e., 16 plates) and
4 plates for each sample soln. Std curve will be plated twice
(i.e., 32 plates) as will check samples 0.30 and 0.16 unit/mL.
Therefore, total of 48 plates will be needed for 2 curves and
check samples, plus 4 addnl plates for each sample.
Let agar harden on level surface. Transfer to refrigerator and
cool ^1 h before dosing. Use plates same day prepd.
£ Extraction
Accurately weigh amt feed contg ca 4600 units of bacitracin
into 300 mL erlenmeyer flask, or equiv.
Add 100 mL extg solv. with 100 mL vol. pipet and ext feeds
5=5 min by shaking flask or mixing on mag. stirrer.
Transfer supernate to plastic centrif. tubes and centrif. 10
min at 2000 rpm. Filter supernate thru glass wool into grad-
uate. Use vol. glassware and dilg solv. to prep, final diln 0.2
± 0.05 unit/mL.
F. Plating
Use 16 seeded plates for first curve. Use 0.20 unit/mL as
plate ref. On each plate, fill 3 alternate stainless steel cylinders
with plate ref. and the 3 remaining cylinders with 1 std. Be
sure all cylinders are filled with const vol. (i.e., 0.25 mL).
Preset Eppendorf pipet is best for this purpose. Use 4 plates
for each sample, including 0.3 and 0.16 unit/mL check sam-
ples.
Use 16 seeded plates for second curve, to be plated after all
samples are plated. Use 8 plates for second plating of 0.3 and
0.16 unit/mL check samples.
incubate dosed plates 16-18 h at 37 ± 2°. Read zones of
inhibition to nearest mm, using Fisher-Lily zone reader.
G. Determination
Det. corrected av. zone diams for std (Z') and sample (Z)
solns according to 957. 23E. Det. response line as least squares
linear regression of following equation:
Z' = mlog P' + b
where P' = potency in unit/mL of std soln associated with
Z'; m, b = are least squares fitted slope and intercept param-
eters. Calc. potency of sample by following equation:
g bacitracin/lb = [antilog(Z - b/m)
x D x 0.0108]/sample wt
where D = total sample diln; 0.0108 - 453.6 (g/lb)/42 000
(units/g bacitracin).
Ref.: JAOAC 65, 1168(1982).
CAS-1405-87-4 (bacitracin)
957,24* Bacitracin
in Feed Supplements
Microbiological Method
First Action 1957
Final Action 1960
Surplus 1981
(Applicable to supplements contg 5=6 g/lb)
See 42.223-42.226, 14th ed.
AOAC Official Methods of Analysis (1990)
Chlortetracycline
119
965.48* Bacitracin in Mixed Feeds
Microbiological Method
First Action 1965
Surplus 1981
(Applicable to feeds contg ^20 g bacitracin /ton)
See 42.227-42.231, 14th ed.
967.39 Chlortetracycline HCI in Feeds
Microbiological Method
First Action 1967
Final Action 1968
(Applicable to feeds contg >:10 ppm)
A. Standard Solutions
(a) Chlortetracycline (CTC) stock soln. — Accurately weigh
ca 40 mg CTC. HCI USP Ref. Std and dissolve in enough 0.0 IN
HCI to give concn of exactly 1000 jxg/mL. Store in dark <5
days at 2-10°.
(b) Std solns and response line for samples containing more
than 50 ppm chlortetracycline. HCI. — Dil. appropriate ali-
quots of stock soln, (a), with enough pH 4.5 buffer, 957.23B(g),
to obtain conens of 0.01, 0.02, 0.04, 0.08, and 0.16 >xg/mL.
Ref. concn is 0.04 |xg/mL.
(c) Std solns and response line for samples containing 10—
50 ppm chlortetracycline .HCI. — Prep, as in (b), but dil. with
inactivated diluent, (d), instead of buffer soln and include conens
of 0, 0.005, and 0.32 \ig CTC.HCl/mL. Draw best line of fit
by inspection.
(d) Inactivated diluent. — To 10 ml acid-acetone feed ext
(prepd from feed under test as in 967.39C(b)) in 600 mL beaker,
add 90 mL pH 4.5 phosphate buffer, 957.23B(g), and adjust
to pH 4.5-4.7 with IN NaOH. Add 1.0 mL fresh 5.25% NaOCl
soln, 957.23B(o), and stir 1-2 min, rinsing sides of beaker.
Heat, stirring thoroly at 10 min intervals, in uncovered beaker
in boiling H 2 bath 30 min. Cool to room temp, under tap
H 2 stream and transfer quant, to 100 mL vol. flask. Rinse
beaker with 6 mL acetone, add rinsings to vol. flask, and dil.
to vol. with pH 4.5 buffer. Transfer quant, to another flask
and dil. with enough pH 4.5 buffer so that final concn of feed
ext is same as that in assay soln.
B. Plates
(a) Base layer. — Add 6.0 mL melted agar medium D to
sterile petri dishes, distribute evenly, and let harden on per-
fectly level surface.
(b) Seed layer.— Before assay, det. by prepn of trial plates
optimum concn (usually 0.03-0. 10%) of organism suspension
of B. cereus, 957.23D(c), to be added to agar medium D to
obtain zones of inhibition with as little as 0.01 |xg CTC. HCI/
mL for assaying samples contg >50 ppm and 0.005 |xg/mL
for samples contg <50 ppm CTC. HCI. Zone of 20 mm ± 10%
should be obtained with 0.04 |mg/mL. For actual assay add
appropriate amt of suspension to agar medium D previously
melted and cooled to 48°. Mix thoroly and add 4.0 mL to each
of plates contg base layer. Alternatively, use 10-12 |xL agar
medium D (single layer).
C. Assay Solution
(Solv. losses may occur from evapn of volatile solvs in open
containers. Carefully measure and record vols of solvs, and
make appropriate mathematical corrections for any losses in
vol.)
(a) Samples containing more than 50 ppm chlortetracy-
cline. HCI. — Obtain and prep, sample as in 965.16 and 950.02.
Place 2, 10, or 20 g sample, resp., contg CTC. HCI >10 g/lb
(>2%), >400 ppm to 2%, or 50-400 ppm in 150 mL beaker
and pipet in 40 mL acid-acetone soln, 957.23B(h). Stir ca 2
min with glass rod, let stand 2 min, and stir. Adjust pH to
1.0-1.2 with HCI, if necessary, and note vol. HCI added.
Transfer to 1 qt (1 L) high-speed blender jar, using addni 20
mL (minus vol. equiv. to HCI added in adjusting pH) acid-
acetone to rinse beaker and pH meter electrodes. Cover jar and
blend 3 min at high speed. Transfer mixt. to 100 mL centrf.
tubes. Wash blender jar with 40 mL acid-acetone and combine
washings with ext in centrf. tubes. Shake well 5 min. Centrf.
ca 15 min at 2000 rpm. Combine and mix clarified exts. Ad-
just 10 mL aliquot to pH 4.5 with IN NaOH. Dil. adjusted
soln with enough pH 4.5 buffer, 957.23B(g), to obtain estd
concn of 0.04 jjig/mL. Designate soln as assay soln.
(b) Samples containing 10-50 ppm chlortetracycline.
HCI. — Obtain and prep, sample as in 956.16 and 950.02. Place
50 g sample in 250 mL beaker and pipet in 100 mL acid-
acetone soln, 957.236(h). Stir, adjust pH, and blend as in (a),
using 50 mL (less vol. equiv. to HCI added in adjusting pH)
acid-acetone to transfer to blender jar. After blending, transfer
quant, to 250 mL centrf. bottle, rinsing jar with 50 mL acid-
acetone soln. Shake thoroly and centrf. ca 15 min at 2000 rpm.
Pipet 5 mL clear supernate into 50 mL beaker, add ca 40 mL
pH 4.5 buffer, 957.23B(g), mix, and adjust pH to 4.5-4.7
with IN NaOH. Transfer quant, to flask, rinse beaker and pH
meter electrodes with pH 4.5 buffer, and add rinsings to flask.
Add enough pH 4.5 buffer to obtain estd concn of 0.04 [xg/
mL. Designate as assay soln.
D. Assay
Using CTC. HCI std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating at 30°.
Refs.: JAOAC 40, 857(1957); 50, 446(1967).
CAS-64-72-2 (chlortetracycline. HCI)
977.37 Chlortetracycline HCI in Feeds
Turbidi metric Method
First Action 1977
(Applicable to feed supplements contg ^20 g/lb)
A. Apparatus and Reagents
(a) Assay broth. — Prep, as in 957.23A(n), but in 1.7x
quantity.
(b) Homogenizer. — Omni-Mixer (Du Pont Instrument Co.,
Sorvall Operations, Peck's Ln, Newtown, CT 06470), or equiv.
(c) For manual assay . — (7 ) Spectrophotometer . — Sequoia-
Turner Model 330 (replacement Model 340, Sequoia-Turner
Corp., 850 Maude Ave, Mountain View, CA 94043), or equiv.
Response time must be rapid, <4 sec. (2) Flowcell. — 10 mm
light path and 0.25 mL vol. (No. 8495-L10, Thomas Scien-
tific, or equiv.) and adapter (No. 8475-F10, Thomas Scien-
tific, or equiv.) to hold cell assembly in spectrophtr. Polyeth-
ylene tubing, 0.055" (1 .4 mm) id, is used as inlet and outlet.
Fit inlet tube with short length of stainless steel tube and con-
nect outlet to vac. thru solenoid valve (AU-0034, Elanco Prod-
ucts Co., or equiv.). Adjust vac. to obtain flow rate of 1.0
mL/sec. Flowcell must be rigidly held in its holder and holder
rigidly fixed in adapter. (3) Water bath and heater. — Part of
AUTOTURB System, (d), or equiv. (4) Constant voltage
transformer. — Sola transformer, wave form corrected, for sta-
120
Drugs in Feeds
AOAC Official Methods of Analysis (1B90)
bilizing current voltage (EU-0020, Elanco Products Co., or
equiv.); connected to spectrophtr. (5) Filling unit. — Filamatic
single nozzle liquid filler (National Instrument Co., Inc., 41 19
Fordleigh Rd, Baltimore, MD 21215), or equiv. Use with Te-
flon tubing, 0.063" (1.6 mm) id, to fill assay tubes. (6) Digital
voltmeter. — 3 l / 2 or 4 l / 2 digit. Newport Model 400AS3 (New-
port Electronics, Inc., 630 E Young St, Santa Ana, CA 92705),
or equiv. Connect to spectrophtr output to measure % T.
(d) For automated assay. — Autoturb System (Elanco Prod-
ucts Co.).
B. Standard Solutions
(a) For manual assay. — Dil. aliquots of stock soln,
967.39A(a), in enough pH 4.5 buffer, 957.23B(g), to give
concns of 0.02, 0.04, 0.06, 0.08, and 0.1.0 fxg CTC.HCl/mL.
(b) For automated system. — Prep, concns of 0.2, 0.4, 0.6,
and 0.8 p,g CTC.HCl/mL as in (a).
C. Preparation of Inoculum
Inoculate 200 mL broth medium A, 957.23A(n), with 1 loop
from 24 hr stock culture of Staphylococcus aureus, ATCC 9144,
and incubate overnight at 37° on rotary shaker. Store <2 weeks
at 2-10°.
D. Preparation of Samples
Weigh 2 g sample into 250 mL glass or plastic centrf. bottle.
Add 50 mL acid-acetone, 957.23B(h). Stopper or cap imme-
diately, agitate intermittently 5 min, and adjust to pH 1.0-1.2
with HC1, if necessary, using pH meter. Note vol. HC1 used.
Add 50 mL (minus vol. HCJ used) of acid-acetone. Insert blades
of homogenizer into centrf. bottle and blend 3 min at high
speed while keeping bottle covered. Rinse blades into bottle
with 100 mL acid-acetone. Tightly cap bottle and centrf. ca
15 min at 2000 rpm. Filter thru Whatman No. 2V paper, or
equiv. Make further dilns with pH 4.5 buffer, 957.23B(g), to
ca 0.09 and 0.06 jxg CTC.HCl/mL for manual assay, and 0.6
for automated assay.
E. Assay
(a) Manual method. — Inoculate assay broth, 957.23A(n),
with 0.5-1 .0 mL inoculum, 977.37C/100 mL. Incubate at 37°
(20-30 min) until A is ca 0.05 at 600 nm in 10 mm flowcell,
using uninoculated broth as blank.
Completely fill test tube carrier with 18 X 150 mm test tubes
contg medium even tho assay may require only portion of these
tubes. These tubes are included only to maintain uniform H 2
flow in bath.
Pipet 1 mL pH 4.5 buffer, 957.23B(g), into each of 4 blank
tubes and into each of 4 zero level std tubes. Pipet into each
of 4 tubes 1 mL of each std and each sample soln. Add 9.0
mL inoculated assay broth to all tubes. Refrigerate blank tubes.
Incubate all other tubes at 37° until % T of zero level tubes is
ca 30 at 600 nin (3-4 hr). Do not remove tubes from bath
during incubation to observe growth. Use extra tubes for this
purpose and after inspection, replace in bath but do not mea-
sure. Stop growth in all tubes by heating 1-2 min at 80°; then
cool rapidly in cold H 2 0. Shake each tube by placing thumb
over tube and inverting once. Do not shake mech. Measure
turbidity at 600 nm in static suspension. Let culture flow ca
4 sec, stop flow ca 2 sec to dislodge air bubbles, and let flow
again ca 3 sec. Stop flow and read % T. Average the 4 read-
ings for each std and sample.
(b) Automated method. — System pipets two 0.10 and two
0.15 mL portions of sample soln and std solns into assay tubes,
dils with inoculated broth, and reads % T at 600 nm. Average
the 2 readings.
F. Calculations
(a) Manual assay. — Convert av. % T to A and plot log A-
against jxg CTC.HCl/mL on semi- log paper. Draw std re-
sponse line. Read jxg CTC.HCl/mL in sample from line.
(b) Automated assay . — Read |xg CTC.HCl/mL from graph
made as in (a) for 0.10 mL vols and for 0.15 mL vols. Av-
erage results.
g/lb - jxg CTC.HC1 (from curve) x D x 454
x 10~ 6 /g sample
where D = diln factor, 454 X 1 0~ 6 - conversion of |xg/g to
g/lb-
Ref.: JAOAC60, 1119(1977).
CAS-64-72-2 (chlortetracycline.HCl)
971.48 Erythromycin in Feeds
Microbiological Method
First Action 1971
(Applicable to feeds contg 9.25 and 92.5 g/ton without
pelleting adjuvants and ^92.5 g/ton with bentonite or Ma-
sonex)
A. Reagent
Dimethoxy methane {methylal). — Tech., CH 2 (OCH 3 ) 2 (Aid-
rich Chemical Co., Inc.; or Eastman Kodak No. 525).
B. Standard Solution
(a) Erythromycin stock soln. — Accurately weigh amt USP
Erythromycin Ref. Std and dissolve in enough methylal -MeOH
(4 + 1) to give concn of 1000 (xg erythromycin base/mL. (1
(xg base is equiv, to 1.08 |xg of the thiocyanate.) Dil. further
with pH 8 buffer, 957.23B(b), to final concn of 100 p,g/mL.
Store in refrigerator ^1 week.
(b) Std response line. — Dil. appropriate aliquots of stock
soln, (a), with enough pH 8 buffer, 957.23B(b), to obtain concns
of 0.05, 0.1, 0.2, 0.4, and 0.8 u.g erythromycin base/mL.
Ref. concn is 0.2 jxg/mL.
C. Plates
Before assay, det. by prepn of trial plates optimum concn
(usually 0.05-0.2%) of organism suspension of Sarcina lutea,
957.23D(e)(i), to be added to agar medium J, 957.23A(j), to
obtain zones of inhibition of adequate size (17.5 mm ± 10%
with ref. concn) and sharpness. For actual assay add appro-
priate amt suspension to agar medium J previously melted and
cooled to 48°. Place 10 mL inoculated medium in each of re-
quired number of plates, let harden, and refrigerate in inverted
position until just before use.
D. Assay Solution
Accurately weigh ca 10 g sample contg equiv. of >92.5 g
erythromycin base /ton or 40 g contg equiv. of 9.25 g eryth-
romycin base /ton and transfer to 250 mL g-s erlenmeyer.
For feeds contg no pelleting adjuvants add 20.0 mL H 2
and 80.0 mL methylal-MeOH (4 + 1) soln. For feeds contg
bentonite or Masonex, add 20.0 mL 5% phosphate buffer,
957.23B(b), and 15.0 mL MeOH. Mix and let feed slurry stand
10 min before adding 65.0 mL methylal. Stopper and mix 1
hr on mag. stirrer or mech. shaker (add glass beads for ade-
quate mixing on shaker).
Let feed settle and dil. to 0.203 p,g erythromycin base/mL
as in (a) or (b) below:
AOAC Official Methods of Analysis (1990)
Hygromycin B
121
(a) Feeds containing equivalent of, or more than 92.5 g
erythromycin base /ton. — Dil. 2.0 ml ext to 100 mL with pH
8 buffer, 957.238(b).
(b) Feeds containing equivalent of 925 g erythromycin base/
ton.— Dil. 5 mL ext to 100 mL with pH 8 buffer.
E. Assay
Using erythromycin std response line, 971.48B(b), assay soln,
971.48D, and plates, 971. 48C, proceed as in 957.23E-F, ex-
cept use 4 plates for each concn required for std response line
(total of 16 plates) and for each assay soln. Incubate at 30°.
Refs.: JAOAC 54, 940, 944(1971); 60, 176(1977).
CAS- 11 4-07-8 (erythromycin)
960.67 Hygromycin B in Feeds
Microbiological Method
Final Action 1965
(Applicable to feeds contg >6000 units /lb)
A. Standard Solutions
(a) Hygromycin B stock soln. — Accurately weigh amt of
Hygromycin B Ref. Std (available from Elanco Products Co.)
contg 50,000 units, transfer to 50 mL vol. flask, and dil. to
vol. with pH 7 phosphate buffer, 957.23B(c). Store in refrig-
erator <2 weeks.
(b) Std response line. — Dil. appropriate aliquots of stock
soln daily with enough pH 7 buffer, 957.23B(c), to obtain
concns of 15, 25, 50, and 75 units/mL. Ref. concn is 25 units/
mL.
B. Plates
(a) Base layer. — Add 10 mL melted agar medium E to ster-
ile petri dishes, distribute evenly, and let harden on perfectly
level surface.
(b) Seed layer. — Before assay, det. by prepn of trial plates
optimum concn (usually 0.2% of 1:10 diin) of spore suspen-
sion of B. subtilis, 957.23D(d), to be added to agar medium
E. Zone of 16 mm ± 10% should be obtained with 25 units/
mL. For actual assay add appropriate amt of spore suspension
to agar medium E which has been melted and cooled to 48°.
Mix thoroly and add 4.0 mL to each plate contg base layer.
Store plates at 2-10° until just before use.
C. Assay Solution
(a) Preparation of ion exchange resin column. — Slurry ca
1 lb (450 g) Amberlite IRC-50 ion exchange resin with 2 L
IN H 2 S0 4 3 hr. Wash until neut. with H 2 and gradually add
solid LiOH with stirring until pH remains at 7-8. Let stand
overnight and wash with H 2 ^5 times. Neutze to pH 7.0 with
IN H3PO4. Store under H 2 in glass container.
Place glass wool plug at bottom of 6 mm id X 140 mm long
tube fitted with valve to control flow and 50 mL reservoir at
top. Fill tube with H 2 and add wet resin to within 20 mm of
top of tube. Drain H 2 to within 5 mm of resin surface. Wash
with 25 mL sterile H 2 immediately before use.
(b) Preparation of assay soln. — Obtain and prep, sample
as in 965.16 and 950.02. Weigh 50 g sample contg 6000-
12,000 units/lb (30 g for 18,000-24,000 units/lb, 20 g for
> 24, 000) into jar of high-speed blender. Add 300 mL (500
for the higher potency feeds) pH 7 phosphate buffer, 957.23B(c),
and blend 5 min, operating blender from variable transformer
set at 70. Centrf. 10 min at 2600 rpm. Adjust 125 mL super-
nate to pH 5.0 with HC1 (ca 0.5 mL). Add 50 mL CHC1 3
previously washed with pH 7.0 buffer, stopper, and shake tho-
roly. Centrf. mixt. 10 min at 2600 rpm. Remove aq. phase,
adjust to pH 7.0 with 40% NaOH soln (ca 0.7 mL), and centrf.
Transfer 100 mL neutzd soln (75 mL if feed contains > 42, 000
units /lb) to ion exchange column and adjust flow rate to 40
drops/min. Wash column with four 20 mL portions sterile H 2 0.
Elute hygromycin B with 50 mL NH 4 OH (1 + 9) into 100 mL
Pyrex beaker. Evap. to 3-5 mL and adjust to pH 7.0 with IN
HC1. Transfer to 10 mL vol. flask, dil. to vol. with pH 7.0
phosphate buffer, and designate as assay soln. (Final concn
should be ca 25 units/mL.)
D. Assay
Using hygromycin std response line, assay soln, and plates,
proceed as in 957.23E-F, except use 6 plates for each concn
required for std response line (total of 18 plates) and for each
assay soln. Equations forL and H cannot be used. Incubate at
37°.
E. Calculation
Units/lb = [1.1 x (units/mL assay soln) x 454 x mL pH
7 buffer (300 or 500) x 10 x (125 + mL HC1 + mL 40%
NaOH)] /[ 125 mL x g sample x mL neutzd soln put on col-
umn].
Ref.: JAOAC 43, 213(1960).
CAS-3 1282-04-9 (hygromycin)
975.60
Lasalocid in Feeds
Microbiological Method
First Action 1975
A. Reagents and Apparatus
See 957.23C(a)-(c), 975.61C(a), and following:
(a) Ethyl acetate. — Purify by passage over silica gel and
distil.
(b) Methanol.— 75% and 19.4% by vol. in H 2 0.
(c) Automatic pipetting machine. — Brewer (available from
Scientific Equipment Products (SEPCO), or equiv.
B. Standard Solutions
(a) Lasalocid sodium stock soln. — 100 /xg/mL. Accurately
weigh suitable amt Lasalocid Na Ref. Std (available from
Hoffmann-La Roche Inc.) and dil. to appropriate vol. with an-
hyd. MeOH.
(b) Std response line. — Dil. aliquots stock soln, (a), using
anhyd. MeOH and H 2 0, to obtain concns of 0.25, 0.5, 1.0,
2.0, and 4.0 [xg/mL in 25% MeOH (v/v). Ref. concn is I
[xg/mL. Solns are stable <1 month at room temp.
C. Stock Culture and Preparation of Inoculum
Prep, slant culture of Bacillus subtilis, ATCC 6633, on >1
tube of agar medium A, 957.23A(a). Incubate 16-24 hr at
37°. Wash growth from stock culture with ca 3 mL sterile distd
H 2 0, transfer liq. to surface of 300 mL agar medium G,
957.23A(g), in Roux bottle, and incubate 7 days at 37°. Wash
growth from agar surface with ca 25 mL sterile distd H 2 into
centrf. bottle. Heat suspension 30 min in 65° H 2 bath. Centrf.,
decant, and resuspend cells in ca 25 mL sterile distd H 2 0.
Repeat centrfg and suspending 3 times, discarding H 2 wash-
ings. Heat residual spores 30 min in 65° H 2 bath. Resuspend
spores in ca 35 mL sterile distd H 2 0. Suspension may be kept
1 yr at ca 5°. Before use, dil. suspension with sterile distd H 2
(usually 1 + 50) to read 20% Ton spectrophtr at 530 nm; store
<1 week at 5°.
122
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
D. Plates
Seed layer. — Use single inoculated agar layer. Before as-
say, det. by prepn of trial plates optimum concn (usually 5 mL
for each 100 mL seed agar) of dild suspension of B. subtilis,
975. 60C, to be added to agar medium M to obtain zones of
inhibition of adequate size (17.5 ± 2.5 mm with 1 .0 |mg/mL)
and sharpness. For actual assay, add appropriate amt of sus-
pension to agar medium M previously melted, adjusted to pH
6.0, and cooled to 60°. Mix thoroly and add 6.0 mL to each
plate. Distribute evenly and let harden on perfectly level sur-
face. Prep plates 2.5-3 hr before use.
E. Assay Solution
(a) Premixes, 15%. — Accurately weigh 1.0 g premix,
transfer to 200 mL vol. flask, add 100 mL MeOH, shake vig-
orously 3 min, and dil. to vol. with MeOH. Dil. 4.0 mL of
this diln to 100 mL with MeOH. Further dil. 3.0 mL of last
diln with 22 mL MeOH and H 2 to 100 mL (1 mL = ca 1
u.g lasalocid Na/mL 25% MeOH).
(b) Final feed, 0.0075%.— Weigh 20 g mash feed or pel-
lets ground to pass No. 20 sieve and transfer to 500 mL vol.
flask. Add 12 mL pH 4.7 buffer (975.61C(a)) and wet feed
thoroly. Immerse flask 5 min in 70° H 2 bath. Cool to room
temp. Add 200 mL EtOAc, stopper, and shake mech. 10 min.
Centrf. ca 100 mL EtOAc ext 10 min at 2000 rpm. Pipet 60
mL clear EtOAc ext into 200 mL vol. flask, add 8 mL 1.57V
HC1, and shake 10 min. Let layers sep., transfer EtOAc layer
to 100 mL g-s centrf. tube, and centrf. 10 min at 2000 rpm.
Pipet 40 mL clear EtOAc ext into another 100 mL g-s centrf.
tube and add 2 mL 40% NaOH soln. Stopper and shake briefly
by hand, add 8 g anhyd. Na 2 S0 4 , and shake again. Centrf. 10
min at 2000 rpm and decant 25 mL clear supernate into 50
mL g-s graduate. Evap. all EtOAc under stream of N with
graduate immersed in 60° H 2 bath. Dissolve residue in 5 mL
hexane, add exactly 25 mL 75% MeOH (v/v), stopper, and
shake vigorously 1 min. Transfer to 125 mL separator and let
stand ca 1 hr. Withdraw lower (MeOH) layer into 25 mL beaker,
pipet 5 mL into 50 mL vol. flask, and dil. to vol. with 19.4%
(v/v) MeOH.
F. Assay
Using lasalocid Na std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating at 35 ± 1°. Calc. L and
H and fit straight line by simplified least square method,
957.23E.
Refs.: JAOAC 57, 978(1974); 58, 941(1975); 59, 398,
1286(1976).
CAS-25999-31-9 (lasalocid)
967.40 Lincomycin in Feeds
Microbiological Method
First Action 1967
Method I
(Applicable to feeds contg ^3.63 g/ton)
A. Standard Solutions
(a) Lincomycin stock soln. — Accurately weigh ca 40 mg USP
Lincomycin. HO Ref. Std and dissolve in enough pH 8 buffer,
957.23B(b), to give concn of exactly 100 jxg lincomycin base/
mL. Store <30 days at 2-10°.
(b) Std response line. — Dil. aliquots stock soln, (a), with
enough pH 8 phosphate buffer, 957.23B(b), to obtain concns
of 0.2, 0.4, 0.8, 1.6, and 3.2 |mg lincomycin base/mL. Ref.
concn is 0.8 |xg/mL.
B. Plates
(a) Base layer. — Add 10 mL melted agar medium E to ster-
ile petri dishes, distribute evenly, and let harden on perfectly
level surface.
(b) Seed layer. — Before assay, det. by prepn of trial plates
optimum concn of organism suspension of S. lutea (usually
0.02-0.05% of suspension prepd as in 957.23D(e)(7) or 0.2-
1% as in 957. 2310(e)(2)) to be added to agar medium J to
obtain zones of inhibition of adequate size (16 mm ± 10%
with 0.8 |xg/mL) and sharpness. For assay, add appropriate
amt of organism suspension to agar medium J previously melted
and cooled to 48°. Mix thoroly and add 4.0 mL to each plate
contg base layer.
C. Assay Solution
Obtain and prep, sample as in 965.16 and 950.02. Accu-
rately weigh ca 10 g ground sample and transfer to 250 mL
g-s, r-b flask, add 20 mL H 2 0, and shake 10 min on wrist-
action shaker. Add 50 mL 0.1 N HCl-MeOH (1 + 4) and shake
10 min. Filter thru Whatman No. 4 paper, using 42 mm buch-
ner and 500 mL flask. Repeat extn twice, using 50 mL HCl-
MeOH each time. (Do not add more H 2 0.) Alternatively, con-
duct extns in 250 mL centrf. bottle and centrf. to clarify.
Transfer combined filtrates to 500 mL r-b flask and evap.
to 15-20 mL, using rotary evaporator. (Do not heat >60°.)
Transfer aq. ext to 125 mL separator. Rinse flask successively
with 10 mL Skelly solve B, 951.01 A(o), 7-8 mL phosphate
buffer, 957.236(b), and 10 mL Skellysolve B. Add all rins-
ings to separator, shake, and let sep. Drain aq. phase, ext
Skellysolve B twice with 7-8 mL buffer, and adjust combined
exts to pH 8.0 with dil. NaOH soln. Adjust vol. with pH 8
buffer to 0.6-1.0 u>g lincomycin base/mL.
D. Assay
Using lincomycin std response line and assay soln, proceed
as in 957.23E-F, incubating at 32°.
Refs.: JAOAC 50, 442(1967); 61, 1107(1978).
CAS-154-21-2 (lincomycin)
978.31 Lincomycin in Feeds
Microbiological Method
First Action 1978
Method II
(Applicable to feeds, feed supplements, and vitamin-mineral
premixes contg 20-2600 g/ton)
A. Standard Solutions
Std response line (for monolayer plates). — Dil. aliquots stock
soln, 967.40A(a), with enough pH 8 phosphate buffer,
957.23B(b), to obtain concns of 0.15, 0.3, 0.6, 1.2, and 2.4
|xg lincomycin base/mL. Ref. concn is 0.6 |mg/mL.
B. Plates
Monolayer. — Proceed as in 967.40B(b), except zones of in-
hibition should be 18.5 mm ± 10% with 0.6 |xg/mL; add 7,5
mL final mixt. to each plate, distribute evenly, and let harden
on perfectly level surface.
C. Assay Solution
Obtain and prep, sample as in 965.16 and 950.02.
(a) For 20-80 g/ton.— Accurately weigh ca 10-20 g ground
sample (see Table 978.31), and transfer quant, to 250 mL centrf.
bottle. Add 75.0 mL 0.1N HCl-M'eOH (1 + 4), and shake 20
min on mech. shaker. Centrf. 5 min at ca 2500 rpm to clarify.
AOAC Official Methods of Analysis (1990)
Monensin 123
Decant supernate into 250 mL separator, add 75 mL hexane,
and shake moderately 1 min. Let layers sep. >15 min. Drain
lower aq. layer into 100 mL beaker, and pipet 2.5-5 mL ali-
quot into 50 mL mixing cylinder. Adjust vol. to ca 40 mL
with pH 8 phosphate buffer, 957.236(b), and add 1 drop 4/V
NaOH. Stopper and shake vigorously. If necessary, adjust to
pH 8 and dil. to 50 mL with p.H 8 buffer. Final concn should
be 0.5-0.8 jutg lincomycin base/mL.
(b) For 80-2600 g/ton. — Accurately weigh 4-6 g ground
sample (see Table 978.31), and transfer quant, to 250 mL centrf.
bottle. Add 50.0 mL 0AN HCl-MeOH (1 + 4), shake 20 min
on mech. shaker, and add 50 mL pH 8 buffer. Shake mech.
5 min and centrf. 5 min at ca 2500 rpm to clarify. Dil. 1.0
mL aliquot with pH 8 buffer to 0.4-0.8 jULg lincomycin base/
mL.
D. Assay
Using lincomycin std response line and assay soln, proceed
as in 957.23E-F, incubating at 32°, except use 2 plates instead
of 3 and 6 readings instead of 9. Av. of all 24 readings of ref.
concn from 8 plates is correction point for response line.
Refs.: JAOAC 50, 442(1967); 61, 1 107(1978).
CAS-154-21-2 (lincomycin)
972.56 Monensin in Feeds
Microbiological Method
First Action 1972
(Applicable to feeds contg >90 g/ton)
A. Standard Solutions
(a) Monensin stdsolns. — (1) Stock soln. — 1 mg/mL. Ac-
curately weigh enough Monensin Na Salt Ref. Std (Elanco
Products Co.) into 100 mL vol. flask to give concn of 1 mg
free acid/mL. Dil. to vol. with anhyd. MeOH. Store at 5°;
discard after 2 weeks. (2) Working soln. — 100 juug/ mL. Dil.
.1 mL stock soln to 10 mL with aq. MeOH (1 + 1). Prep.
fresh daily.
(b) Std response line. — Dil. aliquots of working soln, (a)(2),
with enough aq. MeOH (I + 1) to obtain concns of 0.25, 0.5,
1.0, and 2.0 jixg monensin/mL. Ref. concn is 0.5 u,g/mL.
B. Plates
Seed layer. — Use single inoculated agar layer. Before as-
say, det. by prepn of trial plates optimum concn (usually 0.5
mL for each 100 mL seed agar) of suspension of B. subtilis,
957.23D(d), to be added to agar medium L to obtain zones of
inhibition of adequate size (17.5 ± 2.5 mm with 0.5 (xg/mL)
and sharpness. Before use, dil. suspension with sterile H 2 to
read 20% T on spectrophtr at 530 nm; prep, dild suspension
daily. For actual assay, add appropriate amt of suspension to
agar medium L previously melted and cooled to 48-50°. Mix
thoroly and add 6.0 mL to each plate. Cover and refrigerate
>1 hr before use.
C. Assay Solution
Place glass wool plug at bottom of 19 mm id X 500 mm
chromatgc tube and add ca 75 mm alumina, 961.23C(a), with
gentle tapping. Accurately weigh sample (20 g finished feed,
5 g premix) and add to column. Elute column with MeOH-
H 2 (9 + 1), using 200 mL vol. flask as receiver. Do not
restrict flow. Collect 200 mL eluate, mix, and dil. with aq.
MeOH (1 + 1) to 0.5 |xg monensin/mL. Designate soln ob-
tained as assay soln.
D. Assay
Use 10 seeded agar plates for std curve. Place 4 stainless
steel cylinders, 957.23C(a), on each plate at 90° intervals. Fill
1 cylinder on each plate with different concn of std soln, (b),
and incubate all plates 16-18 hr at 35-37°. Measure diams
of zones of inhibition. Calc. av. zone diam. at each std concn
and fit straight line by simplified least squares method, 957. 23E.
E. Determination
Use 5 plates for each assay soln and align cylinders at 90°
intervals. On each plate fill alternate cylinders with ref. concn
and fill other cylinders with assay soln. Incubate plates 16-
18 hr at 35-37° and measure diams of zones of inhibition to
nearest 0.1 mm. Average 10 readings of ref. concn and 10
readings of assay soln. Proceed as in 957. 23F.
Ref.: JAOAC 55, 718(1972).
CAS- 17090-79-8 (monensin)
976.37
Monensin in Feeds
Turbldimetric Method
First Action 1976
A. Apparatus and Reagents
(a) Assay broth. — In 500 mL erlenmeyer, dissolve 9.0 g
low K ion medium (N-Z Case, Humko-Sheffield Chemical,
PO Box 398, Memphis TN 38101), 3.0 g yeast ext, and 8.0
g glucose in 100 mL H 2 while heating to bp. Cool imme-
diately, and add to bottle contg 1500 mL sterile H 2 0, 4.5 mL
10% soln of polysorbate 80, and 1 2.5 mL citrate buffer soln,
(c). pH should be 5.2 ± 0.1; adjust if necessary.
(b) Automated turbldimetric system. — Autoturb® (Elanco
Products Co.), or equiv. System pipets samples into assay tubes,
dils with inoculated broth, incubates, and measures turbidity.
(c) Citrate buffer soln. — pH 4.0. Dissolve 105 g citric
acid.fLO, 142 g Na citrate. 2H 2 0, and 1.9 g KC1 in H 2 and
dil. to 1 L with H 2 0.
(d) Monensin std solns. — (J) Stock soln. — 1 mg free acid/
mL. Accurately weigh enough Monensin Na Salt Ref. Std
(Elanco Products Co., Department MC757) into 100 mL vol.
Table 978.31 Examples of Sample Size and Dilution of Extract
Assay
Feed Level of
Lincomycin,
Sample
Total
Final Concn,
Soln.
Extn Vol.,
Aliquot
Final Vol.,
978.31 C
g/ton
^g/g
Size, g
mL
Vol., mL
mL
j^g/mL
(a)
20
22
20.0
75
5.0
50
0.59
(a)
40
44
10.0
75
5.0
50
0.59
(a)
80
88
10.0
75
2.5
50
0.59
(b)
80
88
6.0
100
1.0
10
0.53
(b)
140
154
6.0
100
1.0
15
0.62
(b)
200
220
6.0
100
1.0
25
0.53
(b)
400
440
6.0
100
1.0
50
0.53
(b)
1000
1100
4.0
100
1.0
100
0.44
(b)
2600
2860
4.0
100
1.0
200
0.57
124
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
flask to give concn of 1 mg free acid/mL. Add MeOH to dis-
solve salt, dil. to vol. with MeOH, and stopper tightly. (Soln
is stable at room or refrigerator temp. 1 month, except for gain
in potency from loss of MeOH. Warm refrigerated soln to room
temp, before pipetting.) (2) Intermediate soln. — 10 ixg/mL.
Dil. 1.0 mL stock soln to 100 mL with MeOH. (3) Autoturb
working solns. — Using pipets and vol. flasks, prep, solns contg
0.25, 0.50, 0.75, and 1 .0 'p,g/mL MeOH. (4) Manual working
solns. — Dil. stock soln to 10.0 \Lg/mL with MeOH. Prep, std
solns of 0.0, 0.05, 0.10, 0.15, and 0.20 fxg/mL by adding
appropriate vols of 10.0 \xg/mL soln to vol. flasks, adding
enough MeOH so that final MeOH concn is 20% in each flask,
and adjusting to vol. with H 2 0.
B. Preparation of Inoculum
Use Streptococcus faecalis (ATCC 8043) as assay organism
in either frozen suspension or freshly grown inoculum. Prep,
latter by inoculating flask of broth medium A, 957.23 A(n)
(Difco No. 3 Broth), in afternoon and leaving flask overnight
at room temp. Use 10-20 mL inoculum/L assay medium. Let
assay medium inoculated with frozen suspension stand at room
temp. 45 min before using. (This treatment prevents drift within
test.)
C. Preparation of Samples
Grind >100 g feed sample, and mix. Accurately weigh 10.0
g representative well ground sample, and transfer to 4 oz (125
mL) jar fitted with Al foil-lined cap. Add 100.0 mL MeOH
and tightly cap jar. Shake well and let stand overnight or >8
hr. Approx. 30 min before dilg, shake once more and let solids
settle.
(a) Autoturb system measurement. — Dil. 1 .0 mL ext to 20.0
mL with MeOH to prep, soln to be dild by system.
(b) Manual measurement. — Dil. ext 100-fold (1 + 99) and
66-fold (3 + 197) to obtain approx. assay concns of 0.10 and
0.15 |ULg/mL by adding aliquots of sample to sep. vol. flasks,
adding MeOH to 20% final concn, and dilg to vol. with H 2 0.
These solns are stable >1 week at room temp.
D. Assay
(a) Autoturb system measurement. — Place assay tube car-
rier in diluter unit. Place total of 20 sample tubes in sample
turntable (including 5 tubes contg the 4 std levels and 1 tube
with MeOH) in middle of test series. Fill remaining places in
turntable with samples dild to estd concn of 0.6 jig/mL, When
diluter unit has processed tubes, place carrier in 37.5° H 2
bath 3-4 hr or until turbidity of tube measures 40% T or
slightly less. Stop growth by heating carrier with tubes in 80°
H 2 bath 1-2 min. Cool and read at 650 nm.
(b) Manual measurement. — (1) Std curve. — Prep, series
of culture tubes, in triplicate, by adding 0.5 mL of each work-
ing std soln to sep. tubes. Add 9.5 mL inoculated assay broth
to each tube. (2) Samples. — Prep. 2 dilns for each sample to
contain ca 0.1 and 0.15 |xg monensin activity /mL soln. Add
0.5 mL of each diln to 3 sep. tubes, and add 9.5 mL inoculated
assay broth to each tube. Sample levels are c a 0.05 and 0.075
jjig/tube.
Incubate std and sample tubes 4-5 hr at 37.5° or until tur-
bidity of control std tubes (0.0 monensin) measures ca 30% T
at 650 nm. Terminate growth by heating tubes >2 min at 80°.
Cool tubes, and measure turbidity of each tube at 650 nm.
E. Preparation of Standard Curve
(a) Autoturb system measurement. — Prep. 2 dose-response
curves, 1 for 0.1 mL loop and other for 0.15 mL loop. Prep.
graph of log % T against concn. Use av. turbidity of each pair
of tubes in prepg std curves. Assay each sample in duplicate
at 2 concns, 0.10 mL and 0.15 mL sample. Average readings
from 0.10 mL loop for sample and obtain monensin equiv. by
interpolation from 0.10 mL std curve. Repeat for 0.15 mL
loop and use 0.15 mL std curve.
(b) Manual measurement. — Prep, only single std curve.
Prep, graph of log % T against concn. Average 3 readings
obtained for each diln of sample. Obtain monensin equiv. for
each sample diln by interpolation from std curve.
F. Calculations
(a) Autoturb system measurement. — Average 2 readings of
0.10 mL vols for feed sample and obtain monensin equiv. by
interpolation from 0.10 mL std curve (/*,). Repeat for 0.15
mL sample vols to obtain P 2 .
|mg Monensin activity /mL = (Pi + Pi)/2 - P A
fig Monensin/g feed = P A X 200
g Monensin activity (free acid/ton feed)
= P A x 200 x 0.908
(b) Manual measurement. — Multiply av. monensin equiv.
for each of the 2 sample dilns (M, = 0. 10 mL diln, M 2 — 0. 15
mL diln) by its respective diln factor (e.g., 100 and 66). Av-
erage the 2 values to obtain potency of original material (P A ).
(xg Monensin activity /mL = (M ] + M 2 )/2 = P A
u,g Monensin/g feed = P A x 10
g Monensin activity (free acid/ton feed)
= P A x 10 x 0.908
Refs.: JAOAC 55, 114(1972); 60, 179(1977).
CAS- 17090-79-8 (monensin)
970.89
Neomycin in Feeds
Microbiological Method
First Action 1970
(Applicable to feeds contg >28 g neomycin base /ton; soybean
content >40% reduces accuracy of method.)
A. Standard Solutions
(a) Stock soln.— Dry USP Neomycin Sulfate Ref. Std 3 hr
in vac. oven at <5 mm (0.66 kPa). Accurately weigh enough
dried std (10-50 mg) and dissolve in Tris buffer, 957.23B(t),
to give concn of 100 jxg neomycin base/mL. (Neomycin sul-
fate equiv. to neomycin base is given on container.) Store <4
weeks at 2-10°.
(b) Std response line. — Dil. aliquots of stock soln (a) with
enough inactivated feed ext, 970.89C(b), to obtain 0.50, 0.75,
1,13, 1.69, and 2.53 \xg neomycin base/mL. Prep, std re-
sponse line for each feed sample.
a Plates
(a) Base layer. — Add 10 mL melted agar medium K to petri
dishes, distribute evenly, and let harden on perfectly level sur-
face .
(b) Seed layer. — Add appropriate amt (usually 0.5- 2%)
broth culture of S. epidermidis, 957.23D(f), to agar medium
K previously melted and cooled to 48°. Mix thoroly and add
4.0 mL to each plate contg base layer. Distribute agar evenly
by tilting plates from side to side with circular motion and let
harden. Use plates same day prepd. Zone of inhibition of 18
mm ± 15% should be obtained with 1.13 jxg/mL.
C. Assay Solution
(a) Preparation of sample. — Obtain and prep, sample as in
965.16 and 950.02. Weigh 20 g sample into 500 mL r-b flask.
AOAC Official Methods of Analysis (1990)
Novobiocin
125
Table 970.89
Dilution of Extract
Neomycin
Base,
g/ton
Sample
Extract,
Diln I
Neomycin
Base, Final
Concn,
^g/mi_
Inactivated
Extract,
Diln II
140
70
35
1 to 20
1 to 10
1 to 10
1.54
1.54
0.77
10 to 200
20 to 200
20 to 200
Add 100.0 ml NaCl-CaCl 2 soln, 957.23B(n), and shake 15
min on wrist-action shaker. Transfer to 250 mL centrf . bottle
(do not rinse), centrf. 15 rnin at 1800-2000 rpm, and decant
supemate into beaker. Transfer 20 mL aliquot to 100 mL beaker
and set aside to prep, std response line diluent. Using pH me-
ter, adjust remaining portion with HO to pH 2.0. Wait >5
min; then readjust to pH 8.0 with ION NaOH. (High concns
of acid and base are used to avoid significant changes in vol.)
Centrf. 30-35 mL 15 min at 1800-2000 rpm. Dil. ext soln
with Tris buffer according to neomycin content in feed as in
Table 970.89 (diln I).
(b) Preparation of std response line diluent. — Inactivate the
20 mL aliquot from (a) by adjusting to pH 4.5-4.7 with 2N
HC1. Add 1.5 mL fresh 5.25% NaOCl soln, 957.23B(o), and
heat 45 min in boiling H 2 bath, stirring thoroly at least every
10 min during heating period. Cool to room temp., adjust to
pH 8.0 with 3.5N NaOH, and dil. to 20 mL with H 2 0. Dil.
inactivated ext with Tris buffer, 957.236(1), according to neo-
mycin content in feed as in diln II column of Table 970.89.
Use dild soln to prep, std response line solns.
D. Assay
Using neomycin std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating at 32-35°. Result may
be calcd as neomycin sulfate by multiplying neomycin base by
1.428 (1.0 mg neomycin sulfate is equiv. to 0.7 mg neomycin
base).
Ref.: JAOAC53, 60(1970).
CAS- 1404-04-2 (neomycin)
962.25 Novobiocin in Feeds
Microbiological Method
First Action 1962
Final Action 1965
(Applicable to feeds contg ^350 ppm)
A. Standard Solutions
(a) Novobiocin stock soln. — Dry USP Novobiocin Ref. Std
3 hr at 60° in vac. oven at <5 mm (0.66 kPa). Accurately
weigh ca 30 mg dried std, dissolve in 10 mL absolute alcohol,
and dil. with enough pH 8 phosphate buffer, 957.236(b), to
give concn of 1 mg/mL. Store <3 weeks at 2-10°,
(b) Std response line for feed supplements. — Dil. aliquots
of stock soln, (a), with enough pH 6 buffer, 957.23B(f), to
obtain concns of 1.9, 2.4, 3.0, 3.8, and 4.7 u^g/mL. Ref.
concn is 3.0 |ULg/mL.
(c) Std response line for finished feeds. — Dil. aliquots of
stock soln, (a), with enough pH 6 buffer, 957.236(f), to ob-
tain concns of 0.128, 0.16, 0.20, 0.25, and 0.312 [xg/mL.
Ref. concn is 0.20 |xg/mL.
0. Plates
(a) For feed supplements. — (7) Base layer. — Add 21 mL
melted agar medium C to sterile petri dishes, distribute evenly,
and let harden on perfectly level surface.
(2) Seed layer. — Before assay, det. by prepn of trial plates
optimum concn of organism suspension of S. lutea (usually
0.2-0.5% of suspension prepd as in 957. 230(e)(7) or 2-5%
as in 957.23D(e)(2)) to be added to agar medium C to obtain
zones of inhibition of adequate size (14 mm ± 10% with 3.0
|xg/mL) and sharpness. For actual assay, add appropriate amt
of organism suspension to agar medium C, previously melted
and cooled to 48°. Mix thoroly and add 5.0 mL to each plate
contg base layer.
(b) For final feed. — (/) Base layer. — Prep, as in (a) (7),
using 15 mL melted agar medium C.
(2) Seed layer. — Add appropriate amt (usually 0.5- 2%)
broth culture of S. epidermidis, 957.23D(f), to agar medium
C previously melted and cooled to 48°. Mix thoroly and add
5 mL to each plate contg base layer. Zone of 14 mm ± 10%
should be obtained with 0.20 (xg/mL.
C. Assay Solution
(a) For feed supplements containing 50 mg/g. — Obtain and
prep, sample as in 965.16 and 950.02. Accurately weigh suit-
able size sample and add enough absolute alcohol to give estd
concn of 2 mg/mL. Let stand 30 min, shaking occasionally.
Add equal vol. pH 8 phosphate buffer, 957.236(b), and mix.
Dil. to estd concn of 3 u.g/mL with pH 6 buffer, 957.236(f),
and use as assay soln.
(b) For final feed containing not less than 350 pg/g. — Ob-
tain and prep, sample as in 965.16 and 950.02. Weigh 1 g
sample into 50 mL g-s graduate; ext twice with 20 mL EtOAc,
shaking vigorously 2 min. Decant supernate into second 50
mL g-s graduate. Dil. to 50 mL with EtOAc. Transfer 2.0-
4.0 mL aliquot to 100 mL vol. flask, add 5.0 mL pH 8 phos-
phate buffer, 957.236(b), and mix thoroly. Dil. to vol. with
pH 6 buffer, 957.236(f), and shake vigorously to dissolve all
EtOAc. Final concn of novobiocin should be 0.15-0.30 jxg/
mL.
D. Assay
Using proper novobiocin std response line, assay soln, and
plates, proceed as in 957.23E-F, incubating at 32-35°.
Ref.: JAOAC 45, 310(1962).
CAS-303-81-1 (novobiocin)
974.48 Nystatin in Feeds
Microbiological Method
First Action 1974
(For feeds contg >50 g/ton)
A. Standard Solutions
(a) Nystatin stock soln. — Dry ca 25 mg USP Nystatin Ref.
Std 2 hr at 40° in vac. oven at <5 mm (0.66 kPa). Accurately
det. dry wt and add enough MeOH to give concn of exactly
500 units/mL. Dissolve by shaking on mech. shaker 0.5 hr
(soln may be slightly hazy). Prep, fresh daily. (1 g nystatin =
2,800,000 units.)
(b) Std response line. — Dil. aliquots stock soln, (a), with
enough inactivated feed ext, 974.48C(b), to obtain concns of
10, 20, and 40 units/mL. Prep, std response line for each feed
sample. Ref. concn is 20 units/mL.
B. Plates
Seed agar. — Use single inoculated agar layer. Before assay,
det. by prepn of trial plates optimum concn of organism sus-
pension of Sacch. cerevisiae (usually 2.5% of suspension prepd
as in 957.23D(g)(7)), to be added to agar medium I, 957.23A(i),
to obtain zones of inhibition of adequate size (18 mm ± 10%
126
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
for 20 units/mL) and sharpness. For actual assay, add appro-
priate amt of organism suspension to agar medium I, previ-
ously melted and cooled to 48°. Mix thoroly and add 10 mL
to each sterile petri dish.
C. Assay Solution
(a) Preparation of sample. — Obtain and prep, sample as in
965.16 and 950.02. Weigh amt sample contg ca 50 g nystatin/
ton (ca 7700 units total), into 500 mL erlenmeyer. Add 150
mL anhyd. MeOH, mix thoroly by hand, and then shake 1 hr
on rotary shaker. Centrf. briefly. Dil. 4 parts ext with 6 parts
10% phosphate buffer, 957.23B(e). Set aside 10 mL as sample
test soln and use remainder to prep, response line diluent.
(b) Preparation of std response line diluent. — Measure vol.
of remaining diid ext soln from (a). Place soln in cotton-plugged
erlenmeyer having twice capacity of vol. to be contained, and
autoclave 15 min at 121° (slow exhaust). Cool to room temp,
and restore to original vol. by adding anhyd. MeOH. Mix tho-
roly and use dild soln to prep, std response line solns.
D. Assay
Use 10 plates for each sample. On each plate, fill alternate
cylinders, each with I of std response line solns, and fill other
3 cylinders with sample test soln. Incubate plates overnight at
30-37°; then measure diams of zones of inhibition to nearest
0.1 mm. Calc. av. zone diam. at each std concn; plot values
on semilog graph paper and draw line as in 957. 23E. Calc.
av. zone diam. of sample test soln. Using this value, det. po-
tency of antibiotic from std response line.
Ref.: JAOAC 57, 536(1974).
CAS- 1400-6 1-9 (nystatin)
974.49 Oleandomycin in Feeds
Microbiological Method
First Action 1974
Final Action 1975
(For feeds contg >2 g/ton)
A. Standard Solutions
(a) Oleandomycin stock soln. — 250 |xg/mL. Accurately
weigh Oleandomycin Chloroform Adduct Ref. Std (available
from Pfizer, Inc., 235 E. 42nd St, New York, NY 10017) or
USP Ref. Std. Dissolve in ca 5 mL MeOH and dil. with enough
pH 8.0 phosphate-bicarbonate buffer, 957.236(a), to give concn
of 250 |xg/mL. Prep, fresh daily.
(b) Intermediate std soln. — 5 |xg/mL. Dil. 5.0 mL stock
soln to 250 mL in vol. flask with pH 8.0 phosphate-bicarbon-
ate buffer, 957.23B(a).
(c) Std response line. — Dil. intermediate std soln with pH
8.0 buffer, 957.23B(a), to obtain concns of 0.025, 0.05, 0.10,
0.20, and 0.40 |xg/mL. Ref. concn is 0.10 fxg/mL.
B. Assay Solution
Obtain and prep, sample as in 965.16 and 950.02. Accu-
rately weigh 20 g sample contg >2 g oleandomycin/ ton into
500 mL boiling flask, add 200 mL pH 8.0 phosphate-bicar-
bonate buffer, 957.238(a), and shake mech. 45 min. Let set-
tle, decant into 50 mL centrf. tube, and centrf. 5 min at 2000
rpm. Dil. supernate with addnl pH 8.0 buffer to approx. ref.
concn of 0.1 u^g/mL. Use sample ext same day prepd.
C. Plates
Use single inoculated agar layer. Before assay, det. by prepn
of trial plates optimum concn of organism suspension of 5.
lutea (usually 0.05-0.1% of suspension prepd as in
957.23D(e)(7)) to be added to agar medium J to obtain zones
of inhibition of adequate size (16 mm ± 10% with 0.1 (xg/
mL) and sharpness. For actual assay, add appropriate amt of
organism suspension to agar medium J previously melted and
cooled to 48°. Mix thoroly and add 10.0 mL to each petri dish.
D. Assay
Using oleandomycin std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating plates at 37°,
Refs.: JAOAC 56, 1149(1973); 57, 823(1974).
CAS-3922-90-5 (oleandomycin)
968.50 Oxytetracycline In Feeds
First Action 1968
Final Action 1970
(Applicable to feeds contg >10 g/ton)
A. Standard Solutions
(a) Oxytetracycline stock soln. — Accurately weigh ca 40 mg
Oxytetracycline USP Ref. Std and dissolve in enough 0.17V
HC1 to give exact concn of 100 jxg oxytetracycline /mL. (1 |xg
base is equiv. to 1.08 |xg of the hydrochloride.) Store in dark
<5 days at 2-10°.
(b) Std response line. — Dil. appropriate aliquots stock soln,
(a), with enough pH 4.5 buffer, 957.23B(g), to obtain concns
of 0.05, 0.10, 0.20, 0.40, and 0.80 \xg oxytetracycline/mL.
Ref. concn is 0.20 jxg/mL.
Microbiological Method I
(Applicable to >220 mg/lb)
B. Assay Solution
(Solv. losses may occur from evapn of volatile soJvs in open
containers. Carefully measure and record vols, of solvs, and
make appropriate mathematical corrections for any losses
in vol.)
Obtain and prep, sample as in 965.16 and 950.02. Using
mortar and pestle or high-speed blender, grind 2 g sample with
50 mL acid-MeOH, 957.23B(i), and transfer mixt. to 100 mL
centrf. tube. Wash mortar and pestle or blender jar with 50
mL acid-MeOH and combine washings with ext in centrf. tube.
Shake well 5 min. Centrf. ca 15 min at 2000 rpm. Remove
10 mL clear soln and adjust to pH 4.5 with 1/V NaOH. Dil.
adjusted soln with enough pH 4.5 buffer, 957.236(g), to ob-
tain estd concn of 0.20 fjig/mL. Designate as assay soln.
C. Assay
Using oxytetracycline std response line and assay soln, and
chlortetracycline plates, 967.39B(b), proceed as in 957.23E-
F, incubating at 30°.
Microbiological Method II
(Applicable to <220 rag/lb)
D. Plates
Use single inoculated agar layer. Before assay, det. by prepn
of trial plates optimum concn (usually 0.03-0.10%) of stock
suspension of B. cereus, 957.23D(c), to be added to agar me-
dium D, 957.23A(d), to obtain zones of inhibition with as lit-
tle as 0.05 |xg oxytetracycline/mL. Zone of 18 mm ± 10%
AOAC Official Methods of Analysis (1990)
Procaine Penicillin
127
should be obtained with 0.20 (xg/mL. For actual assay add
appropriate amt of inoculum to agar medium D previously
melted and cooled to 48°. Mix thoroly, and add 9.0 mL to
each plate.
E. Assay Solution
(Solv. losses may occur from evapn of volatile solvs in open
containers. Carefully measure and record vols of solvs, and
make appropriate mathematical corrections for any losses
in vol.)
Accurately weigh 20 g ground finished feed into 250 mL
extn flask, add 100 mL acid-MeOH, 957.23B(i), stopper, and
shake mech. 5 min. Centrf. ca 5 min at 2000 rpm. Remove
20 mL supernate and adjust to pH 4.5 with 1W NaOH. Dil.
adjusted soln with enough pH 4.5 buffer, 957.23B(g), to ob-
tain estd concn of 0.20 jxg/mL and filter thru Whatman No.
2V paper, or equiv. Designate as assay soln.
F. Assay
Using oxytetracycline std response line, assay soln, 968.50E,
and plates, proceed as in 957.23E-F, incubating at 28-30°.
Calc. result as oxytetracycline. HC1 by multiplying oxytetra-
cycline base by 1.08.
Ref.: JAOAC 51, 548(1968).
CAS-79-57-2 (oxytetracycline)
967.41 Procaine Penicillin in Feeds
Microbiological Method
First Action 1967
Final Action 1968
(Applicable to feeds contg ^1.5 g/ton)
A. Standard Solutions
(a) Penicillin stock soln. — Accurately weigh, in atm. of
<50% relative humidity, ca 30 mg USP Potassium or Sodium
Penicillin G Ref. Std. Dissolve in enough pH 6 buffer,
957.23B(f ), to give known concn of 100-1000 units/mL. Store
in dark <2 days at 2-10°.
(b) Std response line. — Dil. appropriate aliquots of stock
soln, (a), with enough pH 6 buffer, 957.23B(f), to obtain concns
of 0.0125, 0.025, 0.05, 0.10, and 0.20 unit/mL. Ref. concn
is 0.05 unit/mL.
B. Plates
(a) Base layer. — Add 10 mL melted agar medium A to sterile
petri dishes, distribute evenly, and let harden on perfectly level
surface.
(b) Seed layer. — Before assay, det. by prepn of trial plates
optimum concn of organism suspension of S. lutea (usually
0.2-0.5% of suspension prepd as in 957.23D(e)(7) or 2-5%
as in 957.230(e)(2)) to be added to agar medium B to obtain
zones of inhibition of adequate size (19 mm ± 10% with 0.05
unit/mL) and sharpness.
For actual assay, add appropriate amt of organism suspen-
sion to agar medium B previously melted and cooled to 48°.
Mix thoroly and add 4.0 mL to each plate contg base layer.
C. Assay Solution
(Solv. losses may occur from evapn of volatile solvs in open
containers. Carefully measure and record vols of solvs, and
make appropriate mathematical corrections for any losses in
vol.)
Obtain and prep, sample as in 965.16 and 950.02. Vary amts
of sample and extractant according to penicillin content of feed
as follows:
Penicillin Content
Sample
Size, g
Vol.
Extractant,
mL
>100 g/lb
1
100
1-100 g/lb
3
100
0.1-1 g/lb
10
100
<0.1 g/lb (200 g/ton)
50
200
Ext appropriate amt of sample with pH 6 buffer-acetone ex-
tractant, 957.23B(k), in suitable container, using either wrist-
action or reciprocating mech. shaker 30 min or high-speed
blender 2 min; let settle and decant supernate. Centrf. if more
than slightly turbid. Dil. aliquot of supernate with enough pH
6 buffer, 957.238(f), to obtain estd concn of 0.05 unit/mL.
D. Assay
Using penicillin std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating at 26-32°. Calc. result
in terms of units or wt (1 mg Na penicillin G = 1667 units;
1 mg K salt - 1595 units; 1 mg procaine salt = 1009 units).
E. Identity
To aliquot of supernate assay soln, 967.41 C, add enough
penicillinase soln to inactivate penicillin by incubating mixt.
1 hr at 37°. Further dil. with enough pH 6 buffer, 957.238(f),
to give same diln factor as in 967. 41C. Assay as in 967. 41D.
Absence of zone of inhibition indicates that activity in sample
is due to penicillin. (See 962.14E.)
Ref.: JAOAC 50, 450(1967).
CAS-54-35-3 (penicillin G procaine)
973.81 Spectinomycin in Feeds
Microbiological Method
First Action 1973
(Applicable in presence of lincomycin to feeds contg 5:18
g/ton)
A. Standard Solutions
(a) Spectinomycin stock soln. — 1 mg spectinomycin base/
mL. Accurately weigh amt of Spectinomycin. HC1.5H 2 Ref.
Std (available from Agricultural Division, The Upjohn Co.)
and dissolve in enough H 2 to give concn of exactly 1.0 mg
spectinomycin base/mL. Store in dark <30 days at 2-10°.
(b) Std response line. — Dil. stock soln with Tris buffer,
957.238(1), to obtain concn of 100 fxg/mL. Dil. this soln with
Tris buffer to obtain 2.8, 4.4, 7.0, 11.0, and 17.4 \ig spec-
tinomycin base/mL. Prep, daily. Ref. concn is 7.0 jxg/mL.
B. Plates
Before assay, det. by prepn of trial plates optimum concn
of culture of E. coli, 957.23D(h) (usually ca 0.04%) to be
added to agar medium F to obtain zones of inhibition of ad-
equate size and sharpness. Zones of 13 ± 1 and 23 ± 1 mm
should be obtained for 2.8 and 17.4 juug/mL, resp.
For actual assay, add appropriate amt of culture to agar me-
dium F, previously melted and cooled to 48°. Mix thoroly and
add 7.0 mL to each plate.
C. Assay Solution
Weigh 20 g feed into 250 mL centrf. tube and add 100 mL
acid-MeOH (20 mL 1/V HC1 dild to 1 L with MeOH). Shake
128
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
15 min on wrist-action shaker, or equiv., centrf. 5 min at 2000
rpm, and decant into 500 mL r-b flask. Repeat extn twice,
combining exts in r-b flask. Evap. ext under vac. until all MeOH
and most of H 2 have been removed. Do not exceed 60° and
do not evap. to complete dryness. Add 30 mL Pb(OAc) 2 soln,
957.23B(q), to flask, shake vigorously 2 min, and transfer quant,
to 100 mL beaker. Rinse flask with 10-15 mL Tris buffer and
add rinse to beaker. Adjust to pH 8.0 with 3.5N NaOH. Trans-
fer quant, to 100 mL graduate, rinsing beaker with buffer, and
dil. to 100 mL with buffer. Mix thoroly, and let sample stand
30 min. Centrf. 30-50 mL 10 min at 2000 rpm. Decant su-
pernate into test vials for storage (<2 days) until assay. Des-
ignate soln obtained as assay soln.
D. Assay
Using spectinomycin std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating at 26°.
g Spectinomycin /ton
= (fig spectinomycin base/mL assay soln) X 4.54
If zone size is plotted against g/ton instead of jmg base/mL
working stds as follows, calcns are not necessary.
\xg Base/mL
g/ton
2.8
12.7
4.4
20.0
7.0
31.8
11.0
50.0
17.4
79.0
Ref.: JAOAC 56, 834(1973).
CAS-1695-77-8 (spectinomycin)
971.49 Streptomycin in Feeds
Microbiological Method
First Action 1971
Final Action 1973
Method I
(Applicable to feeds contg >30 g/ton)
A. Standard Solutions
(a) Streptomycin stock soln. — Dry ca 40 mg USP Strep-
tomycin Sulfate Ref. Std 3 hr at 60° in vac. oven at <5 mm
(0.66 kPa). Det. accurate dry wt and dissolve in enough H 2
to give concn of exactly 100 u,g streptomycin base/mL. Store
<30 days at 2-10°.
(b) Std response line. — Dil. aliquots of stock soln, (a), with
enough pK 8 buffer, 957.23B(b), to obtain concns of 0.64,
0.80, 1.0, 1.25, and 1.56 p,g streptomycin base/mL. Ref. concn
is 1.0 (xg/mL.
B. Plates
(a) Base layer. — Add 12 mL melted agar medium E to ster-
ile petri dishes. Distribute agar evenly and let harden on per-
fectly level surface.
(b) Seed layer. — Before assay, det. by prepn of trial plates
optimum concn (usually 0.05-0.2%) of spore suspension of
B. subtilis, 957.23D(d), to be added to agar medium E. For
actual assay, sharp zones of inhibition (14 mm ± 10%) should
be obtained with 0.64 jxg streptomycin base/mL. Add appro-
priate amt of spore suspension to agar medium E which has
been melted and cooled to 48°. Mix thoroly and add 4.0 mL
to each plate contg base layer.
C. Assay Solution
Obtain and prep, sample as in 965.16 and 950.02. Using
10.0 g sample and 200 mL 0.5N HC1, shake mech. 30 min or
blend 2 min in high-speed blender. Centrf. ca 15 min at 2000
rpm. Transfer aliquot of supernate to beaker, add ca 25 mL
pH 8 buffer, 957.238(b), and adjust to pH 8 ± 0.1 with 5N
and IN NaOH. Transfer quant, to suitable vol. flask, dil. to
vol. with pH 8 buffer, 957.23B(b), and mix. Dil. aliquot with
enough pH 8 phosphate buffer to obtain estd concn of 1.0 fxg
streptomycin base/mL. Filter dild soln without suction thru
Whatman No. 2V paper, or equiv. Designate as assay soln.
D. Assay
Using streptomycin std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating at 37°.
Refs.: JAOAC 54, 116(1971); 55, 714(1972).
CAS-57-92-1 (streptomycin)
972.57 Streptomycin in Feeds
Microbiological Method
First Action 1972
Final Action 1973
Method II
(Applicable to feeds contg 5-30 g/ton)
A. Standard Solutions
Std response line. — Dil. aliquots of stock soln, 971.49A(a),
with enough pH 8 buffer, 957*23B(b), to obtain concns of
0.19, 0.24, 0.30, 0.38, and 0.47 fxg streptomycin base/mL.
Ref. concn is 0.30 (xg/mL.
& Plates
(a) Base layer. — Prep, as in 971.49B(a), except use 10 mL
agar medium E.
(b) Seed layer. — Prep, as in 971.49B(b), except that sharp
zones of inhibition (11 mm ± 10%) should be obtained with
0.19 (Jtg/mL std.
C. Assay Solution
Proceed as in 971. 49C, using 40 g sample and dilg to estd
final concn of 0.3 fxg streptomycin base/mL. Let soln stand
1 hr before filtering.
Note: Feeds may produce considerable gas during acid extn.
When using mech. shaker, let each sample stand in acid ex-
tractant ca 1 hr with occasional swirling before placing on
shaker. When using high-speed blender, use jar with lid cover
rather than sealed jar, and hold lid down by hand at start of
blending.
D. Assay
Using streptomycin std response line, assay soln, and plates,
proceed as in 957.23E-F, incubating at 22-25°.
Refs.: JAOAC 54, 116(1971); 55, 714(1972).
CAS-57-92-1 (streptomycin)
962.26
Tylosin in Feeds
Microbiological Method
First Action 1962
Final Action 1965
g/ton)
(Applicable to feeds containing
A. Standard Solutions
(a) Tylosin stock soln. — Dry Tylosin base Ref. Std (avail-
able from Elanco Products Co.) 4 hr at 70° and store in des-
AOAC Official Methods of Analysis (1990)
Bacitracin
129
iccator over fresh P 2 5 . Accurately weigh suitable amt (10-
15 mg) of dried std and dissolve in 5 mL MeOH. Adjust vol.
with pH 7 phosphate buffer, 957.236(c), to give concn of 1000
|xg/mL. Store in refrigerator <2 weeks.
(b) Std response line. — 'Oil. appropriate aliquots of stock
soln with mixt. of MeOH and pH 8 phosphate buffer,
957.23B(b), (4 + 6), to obtain concns of 0.125, 0.25, 0.50,
1.0, and 2.0 (xg/mL. Ref. concn is 0.50 |xg/mL.'
B. Plates
(a) Base layer. — Add 10 mL melted agar medium H to petri
dishes, distribute evenly, and let harden on perfectly level sur-
face.
(b) Seed layer. — Before assay, det. by prepn of trial plates
optimum concn of organism suspension of S. lutea (usually
0.05-0.2% of suspension prepd as in 957.23D(e)(7) or 0.5-
2% as in 957.23D(e)(2)) to be added to agar medium H to
obtain zones of inhibition of adequate size (15 mm ± 10%
with 0.50 (xg/mL) and sharpness. For actual assay, add ap-
propriate amt of organism suspension to agar medium H melted
and cooled to 48°. Mix thoroly and add 5.0 mL to each plate
contg base layer. Refrigerate plates until just before applica-
tion of assay solns.
C. Assay Solution
Obtain and prep, sample as in 965-16 and 950.02. Accu-
rately weigh 10 g feed premix or 20 g final feed into 250 mL
homogenizer cup or blender jar. Add 90 mL hot (70-80°) pH
8 phosphate buffer, 957.23B(b), and place on steam bath 10
min. Blend 5 min, add 60 mL MeOH, and blend addnl 5 min.
Centrf. or filter thru Whatman No. 1 paper and dil., if nec-
essary, with mixt. of MeOH and pH 8 phosphate buffer,
957.238(b), (4 + 6), to concn of 0.5 \xg tylosin/mL.
D. Assay
Prep. 10 plates for std response line and 5 for each sample.
Place 5 cylinders on each std response line plate at 72° inter-
vals on 2.8 cm radius. Place 4 cylinders on each sample plate
at 90° intervals. Fill cylinders on each of 10 std plates with
each concn of std response line. On each sample plate fill 2
diagonally opposite cylinders with ref. concn and remaining 2
cylinders with assay soln. Incubate plates overnight at 30°.
Measure zones of inhibition to nearest 0.1 mm. Record av.
zone diam. for each concn of std on std plates and proceed as
in 957.23E.
Average the 10 readings of ref. concn on sample plates and
the 10 readings of assay soln. Proceed as in 957. 23F.
Ref.: JAOAC45, 317(1962).
CAS-1401-69-0 (tylosin)
CHEMICAL METHODS FOR ANTIBIOTICS
982.44 Bacitracin in Premix Feeds
Liquid Chromatographic Method
First Action 1982
A. Principle
Bacitracin is extd from feed into acidified org. solv. system.
Ext is centrfgd, and supernate is analyzed by ion-suppressed
reverse phase LC with photometric detection at 254 nm.
B. Reagents and Apparatus
(a) Liquid chromatography — Hewlett-Packard Model 1084-
A, equipped with UV photometric detector. Operating con-
dition: flow rate 2.0 mL/min; detector wavelength 254 nm;
20 jxL loop injection valve (Valco Instruments Co., Inc., PO
Box 55603, Houston, TX 77255); ambient temperature.
(b) Chromatographic column. — 15 cm X 4.6 mm id, con-
taining 5 |xm Supelcosil LC-8 reverse phase packing. Use col-
umn for bacitracin analysis only.
(c) Phosphate-EDTA buffer.— pB. 4.5. Dissolve 13.6 g
KH 2 P0 4 and 2.5 g EDTA in 1 L H 2 0.
(d) Phosphate buffer. —pH 6.0. Dissolve 1.5 g K 2 HP0 4 and
8.5 g KH 2 P0 4 in 1 L H 2 0.
(e) Solvent systems. — Measure vol. indicated below with
graduate (except where noted otherwise) into 100 mL vol. flask
and dil. to vol. with H 2 0:
Solvent
A Solv.
Vol.%
B. Solv.
Extg Solv.
CH 3 CN
MeOH
Phosphate-EDTA buffer
Phosphate buffer 3
Coned phosphoric acid 3
20
40
12
20
28
28
3
1.2
a Use vol. pipet
(f) Mobile phase.— Mix 59% (v/v) B solv. with 41% (v/
v) A solv. Mix, and adjust pH to 6.8 with NaOH. Slight ad-
justment to % vol. of B solv. may be required to obtain desired
sepn.
C. Preparation of Standard
(a) Drying of std. — Caution: Bacitracin is very hydro-
scopic. Dry std day before use and store in desiccator over-
night. Accurately weigh 130-140 mg bacitracin ref. std (IMC,
Pitman-Moore Inc., 1331 First St, PO Box 207, Terre Haute,
IN 47808; 56.3 units/mg) into tared (= A) 50 mL vol. flask.
Dry std 3 h at 60° under vac. at <5 mm pressure. Remove
from oven and place in desiccator to cool. Reweigh (- B).
Amt bacitracin std = B — A.
(b) Preparation of std soln. — Note: Store stds under refrig-
eration if not analyzed within 3 h of prepn. Preferably, prep,
std, store in refrigerator >30 min before analysis, and remove
from refrigerator just before analysis. Std soln I: Dissolve bac-
itracin std in 50 mL vol. flask with ca 20 mL extg solv. and
dil. to vol. Prep, following dilns from this soln. Std soln 2:
Pipet 20 mL std soln 1 into 25 mL vol. flask; dil. to vol. with
extg solv. Std soln 3: Pipet 15 mL std soln 1 into 25 mL vol.
flask; dil. to vol. with extg solv.
D. Extraction
Accurately weigh amt of feed contg ca 6000 units bacitracin
activity into 125 mL erlenmeyer. Add 50 mL extg solv. with
vol. pipet and ext with wrist-action shaking >5 min. Centri-
fuge 10 mL portion of ext 2-3 min at 2000-3000 rpm. Use
clear supernate for assay. Note: Store extd sample soln under
refrigeration if not analyzed within 3 h. Preferably, prep, sam-
ple solns, store in refrigerator >30 min before analysis, and
remove from refrigerator just before analysis.
E. Determination
Inject clear supernate from centrfgd feed and std solns into
chromatograph, starting with std soln, then 2 sample solns,
and then another std soln, until all samples and stds have been
injected. Measure and total peak hts of the 3 active component
peaks (Fig. 982.44) for sample (PH) and std (PH') solns.
Calc. response line for stds, using least squares linear fitting
of following equation:
PH* = w(P') + b
where PH' = peak hts of std solns 1 , 2, and 3; P' — potency
of std soln in units/50 mL for std solns 1, 2, and 3; m, b =..
least squares detd slope and intercept.
130
Drugs in Feeds
AOAC Official Methods of Analysis (1 990)
= TIME
FIG. 982.44— Liquid chromatogram of bacitracin active com-
ponents (indicated by arrows)
Det. bacitracin content of feed from:
0.01080 (PH - b)
g bacitracin /lb =
m x sample wt
where 0.01080 = 453.6 (g/lb)/42 000 (units/g bacitracin).
Ref.: JAOAC65, 1178(1982).
CAS- 1405-87-4 (bacitracin)
968.49 Chlortetracycline in Feeds
Microscopic Test
First Action 1968
Final Action 1969
A. Apparatus
Microscopes.— See 964.07A(a) and (b).
B. Reagent
Modified Sakaguchi reagent. — Dissolve 5 g H3BO3 in 150
mL H 2 and add 350 mL H 2 S0 4 . Store in g-s bottle in re-
frigerator. Use cold.
C. Determination
Grind sample to pass sieve with circular openings 1 mm (V25
in.) diam. and mix thoroly. If sample cannot be ground, re-
duce to as fine condition as possible. Do not grind molasses
feeds. Pipet ca 10 mL Sakaguchi reagent into 9 cm petri dish.
Place No. 60 sieve over petri dish. With top of spatula, sprin-
kle ca 0.5 g sample on sieve, and gently tap it to obtain good
distribution of particles over liq. surface. Place under stereo-
scopic microscope and examine with transmitted light at ca
15 X . If substage illumination is not available, place petri dish
on white surface and illuminate with blue light.
As particles of antibiotic slowly dissolve, diffusing chlor-
tetracycline turns intense purple. Color fades in 5-10 min.
Refs.: JAOAC 51, 750(1968); 59, 357(1976).
CAS-57-62-5 (chlortetracycline)
966.30 Griseofulvin in Feeds
Spectrophotometric Method
First Action 1966
Final Action 1975
(Applicable to concns >10 mg/oz.)
{Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
A. Reagents
(a) Activated alumina. — Alcoa grade F-20.
(b) Solvent mixture.— Pet ether-CHCl 3 (65 + 35).
(c) Griseofulvin std soln. — 10 |mg/mL. Accurately weigh
ca 25 mg USP Griseofulvin Ref. Std into 250 mL vol. flask;
dissolve and dil. to vol. with solv. mixt. Dil. 10 mL of this
soln to 100 mL in vol. flask.
B. Apparatus
(a) Chromatographic tube. — 20 X 400 mm, with fritted
disk and stopcock.
(b) High-speed blender. — Waring-type, or equiv., 1 L
capacity.
(c) Spectrophotometer. — Capable of accurate readings at
290 and 320 nm.
C. Preparation of Sample
Grind 250 g feed pellets or mash in high-speed blender 5
min. Accurately weigh ca 14 g finely powd feed into fat-free
thimble and ext in Soxhlet app. 2 hr with 100 mL CHC1 3 .
Evap. ext to 10 mL on steam bath, dil. with 100 mL pet ether,
and chromatograph.
D. Preparation of Chromatographic Column
Place 50 mL solv. mixt., (b), in tube and add 45 mL
activated alumina portionwise, with tapping to ensure uniform
packing. Place small glass wool pad on top of alumina and
drain solv. to just below top of pad.
E. Determination
Add pet ether-CHCl 3 sample ext to column. As last of ext
passes thru glass wool pad, rinse sample flask with solv. mixt.,
add to column, and begin elution with solv. mixt. Adjust liq.
head to give flow rate of 15-20 mL/min. Start collecting 25
mL fractions when green eluate first appears (discard yellow
and almost colorless eluates which precede). When A of frac-
tions at 290 nm exceeds A at 320 nm, stop fractionating, and
collect next 700 mL eluate. Dil. eluate to vol. in 1 L vol. flask
with solv. mixt. Det. A of this soln and of griseofulvin std
soln at 290 and 320 nm against solv. mixt. blank.
mg Griseofulvin/oz = (A? 90 — A 32 o)(W)
(10) (28.35)/(A 290 - ^20) (25) (g sample)
where A refers to sample eluate, A 1 to std soln, and W
ref. std griseofulvin used to prep, std soln.
mg
AOAC Official Methods of Analysis (1990)
Lasalocid
131
Ref.: JAOAC 49, 494(1966).
CAS- 126-07-8 (griseofulvin)
975.61 Lasalocid in Feeds
Spectrofluorometric Method
First Action 1975
Final Action 1977
A. Principle
Compd is extd from pH 4.7 soln with EtOAc, fluorescent
impurities are removed by acid and alkali treatments, and compd
is detd fluorometrically, correcting for nonspecific fluores-
cence by complexing with H.3BO3. Monensin and ethoxyquin
do not interfere.
B. Apparatus
Spectrofluorometer . — With 10 mm fused quartz cells.
Excitation and emission wavelengths, ca 310 and 419 nm, resp.
Accurately det. peak excitation and emission wavelengths us-
ing std soln I, following manufacturer's directions. Do not
change wavelength settings between readings.
For routine setting and checking of instrument, use std
soln I.
Adjust settings to compensate for decreased intensity with
age from dulled reflecting surfaces and lamp.
C. Reagents
(a) Acetate buffer soln. — pH 4.7. Dissolve 5.0 g NaOAc
in ca 50 mL H 2 0, adjust to pH 4.7 with HO Ac, and dil. to
100 mL with H 2 0.
(b) Ethyl acetate. — Must have ca fluorescence. If nec-
essary, purify as follows: Elute 1 gal. EtOAc thru 9-10 cm
(od) column packed with ca 100 cm silica gel (activated des-
iccant, 100-200 mesh, Grade H, W. R. Grace & Co., Davison
Chemical Div., 10 E. Baltimore St, PO Box 2117, Baltimore,
MD) topped with 5 cm layer of NaHCO> Redistil eluate from
all-glass app. with 60 cm jacketed distg column, discarding
first and last 10%. {Caution: See safety notes on distillation
and ethyl acetate.) To redistd EtOAc add 40% aq. NaOH and
mix briefly. Follow with anhyd. Na 2 S0 4 and shake again. (Ra-
tio of EtOAc:40% NaOH:Na 2 S0 4 is 1000:50:200 or multiple
thereof.)
(c) Methanolic boric acid soln. — Dissolve 20.0 g H 3 B0 3
in MeOH and dil. to 500 mL with MeOH. Prep, fresh daily.
(d) Lasalocid std solns. — (1) Std soln I. — Dissolve 30.0
mg Lasalocid Ref. Std (available from Hoffmann-La Roche
Inc.) in EtOAc and dil. to 100 mL with EtOAc. Pipet 4 mL
into 100 mL vol. flask, dil. to vol. with EtOAc, and mix. Pipet
25 mL final diln into 50 mL g-s centrf. tube contg 2.4 mL pH
4.7 buffer. Shake mech. 25-30 min and centrf. 10 min at 2000
rpm. Pipet 2 mL clear EtOAc ext into 100 mL vol. flask, dil.
to vol. with EtOAc, and mix. (2) Std soln II. — Pipet addnl 2
mL clear EtOAc ext into another 100 mL vol. flask contg 10
mL methanolic H 3 B0 3 soln, dil. to vol. with EtOAc, and mix.
D. Preparation of Sampie
(a) Feeds. — Grind 200 g sample (mash, pellets, or crum-
bles) to pass No. 30 sieve, and mix thoroly. Accurately weigh
ca 4 g sample into 50 mL g-s centrf. tube contg 2.4 mL pH
4.7 buffer. Turn and shake tube by hand to wet uniformly.
Immerse tube 4-5 min in 70° H 2 bath. Cool to room temp,
and add 25 mL EtOAc by pipet. Stopper tube and shake briefly
but vigorously by hand to disperse sample. If necessary, break
up lumps with narrow-tip spatula or glass rod. Stopper tube
and shake mech. 25-30 min. Centrf. 10 min at 2000 rpm.
Pipet 15 mL clear EtOAc ext into 50 mL centrf. tube. Add 2
mL L5/V HC1 and shake 10 min. Centrf. 10 min at 2000 rpm.
Pipet 10 mL clear EtOAc ext into another 50 mL g-s centrf.
tube and add 0.5 mL 40% NaOH soln. Shake briefly by hand,
add 2 g anhyd, Na 2 S0 4 , and shake again. Centrf. 10 min at
2000 rpm. If EtOAc soln is not clear or fine particles are pres-
ent at surface, swirl tube gently by hand and recentrf. Pipet 2
mL EtOAc layer into 100 mL vol. flask without disturbing aq.
alk. soln, dil. to vol. with EtOAc, and mix. Designate as Sam-
ple soln I. Pipet another 2 mL aliquot EtOAc layer into second
100 mL vol. flask contg 10 mL methanolic H 3 B0 3 soln, dil.
to vol. with EtOAc, and mix. Designate as Sample soln II.
Pipet 25 mL EtOAc into 50 mL g-s centrf. tube contg 2.4
mL pH 4.7 buffer and proceed as above, beginning "Stopper
tube and shake briefly ..." Designate final solns as Reagent
blank soln I and Reagent blank soln II.
(b) Premixes. — Accurately weigh 2.00 g 15% premix and
transfer into 500 mL vol. flask. Add exactly 250 mL EtOAc
and shake 25 min on mech. shaker. Centrf. aliquot, and dil.
with EtOAc as in (a) to obtain Sample soln I and Sample soln
II (complex) contg ca 0.24 u.g lasalocid/mL EtOAc. Proceed
with fluorescence measurements as in 975. 61E. (Note: Omit
treatment with pH 4.7 buffer for both premix and std.)
E. Determination
Set excitation and emission wavelengths of app. at max.
Adjust instrument with std. soln I, (d)(/), in cell to microam-
meter reading of 0.400. Check this ref. point before and after
each reading, using same cell for all ref. readings. Because of
decomposition in UV, discard and replace std soln I after every
second reading. Measure fluorescence at 419 nm in order:
Sample soln I (U\), Std soln I (Si), Reagent blank soln I (R\),
Sample soln II (U 2 ), Std soln II (5 2 ), and Reagent blank soln
II (R 2 ). If reading of std soln I drifts, adjust gain to initial
setting. If drift is beyond 0.393-0.407, recheck readings of all
solns.
Altho fluorescence response of std is linear from 0.12 to
0.48 |ag/mL, concn of lasalocid in sample soln should be ±
25% of that of std soln.
F. Calculations
(a) % Lasalocid Na in feed = [(17, - Rj)
- (U 2 - R 2 )} x D x S/[(S, - R } ) - (S 2 - R 2 )}
x W x 96 x 100
where V , S u S 2 , andtf are defined in 975. 61E; D - diln factor
(25 x 100/2 = 1250); S = concn of lasalocid in Std soln I ( =
0.24 |xg/mL); W = g sample; and 96 = % recovery. When S
- 0.24 |xg/mL, W = 4.00 g, and tf , and R 2 = 0,
% Lasalocid Na in feed - (U t - U 2 ) X 0.00781/(5, -5 2 )
(b) % Lasalocid Na in premix - \(U , - R f ) ~ (U 2 - R 2 )\
x D x Si\(S } - Ri) - (S 2 - R 2 )] x W x 10,000
where V, S U S 2 , and R are defined in 975.61E; D = diln factor
(250 x 50 x 50 x 100/5 x 5 x 2 = 1,250,000); S = concn
of lasalocid in Std soln I (= 0.24 |xg/mL); W - g sample.
When S = 0.24 |ULg/mL, W = 2.00 g and /?, and R 2 = 0,
% Lasalocid Na in 15% premix
- (U } - U 2 ) x 15/(5, - S 2 )
Ref.: JAOAC 58, 507(1975).
CAS-25999-31-9 (lasalocid)
132
Drugs in Feeds
AOAC Official Methods of Analysis (1990)
Common and Chemical Names of Drugs in this Chapter
Common Name
Chemical Name
Aklomide
Amprolium
Arprinocid
Arsanilic acid
Bithionol
Buquinolate
Carbadox
Chlortetracycline
hydrochloride
Decoquinate
Dibutyltin diiaurate
Diethylstilbestrol
Dimetridazole
Ethopabate
Furazolidone
Glycarbylamide
Griseofulvin
Hygromycin B
Ipronidazole
Lasalocid
Lincomycin
Melengestrol (acetate)
Nequinate
Nicarbazin
Nicotine
Nifursoi
Nihydrazone
Nitarsone
Nithiazide
Nitrodan
Nitrofurazone
Nitromide
Nitrophenide
Oxy tetracycline
Penicillin G procaine
Phenothiazine
Pyrantel tartrate
Racephenicol
Reserpine
Ronnel
Roxarsone
Spectinomycin
Streptomycin
Sulfadimethoxine
Sulfaguanidine
Sulfamerazine
Sulfamethazine
Sulfanitran
Sulfaquinoxaline
Sulfathiazole
Thiabendazole
Zoalene
2-Chloro-4-nitrobenzamide
1-[(4-Amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride
9-[2-Chloro-6-fluorophenyl)methy|]-9H-purin-6-amine
(4-Aminophenyl)arsonic acid
2,2'-Thiobis(4,6-dichlorophenol)
4-Hydroxy-6,7-bis(2-methylpropoxy)-3-quinolinecarboxylic acid ethyl ester
2-(2-Quinoxalinylmethy)ene)hydrazinecarboxylic acid methyl ester N\/V 4 -dioxide
(4a,4aa,5aa,60,1 2aa)-7-Chloro-4-(dimethylamino)-1 ,4,43,5,53,6,1 1 ,1 2a-octahydro-3,6,10,12,1 2a-pentahydroxy-6-methyl-1 ,1 1 -dioxo-
2-naphthacenecarboxamide monohydrochloride
6-(Decyloxy)-7-ethoxy-4-hydroxy-3-quinolinecarboxyiic acid ethyl ester
Dibutylbis[(1-oxododecyl)oxy]stannane
4,4'-(1 ,2-Diethyl-1 ,2-ethenediyl)bisphenol
1 ,2-Dimethyl-5-nitro-1 H-imidazole
4-(Acetamido)-2-ethoxybenzoic acid methyl ester
3-[[(5-Nitro-2-furanyl)methylene]amino]-2-oxazolidinone
1 H-lmidazole-4,5-dicarboxamide
7-Chloro-2',4,6-trimethoxy-6'-methyl-spiro[benzofuran-2(3H),1'-[2]cyclohexene]-3,4'-dione
C^6-Amino-6-deoxy-L-glycero-D-galacto-heptopyranosylidene-(1~^2-3)-0-p-D-talopyranosyl-(1-^5)-2-deoxy-/V 3 -methyl-
D-streptamine
1-Methyl-2-(1-methylethyl)-5-nitro-1H-imdazole
6-[7(/?)-[5(S)-Ethyl-5-(5{fl)-ethyltetrahy
4-(S)-hydroxy-3-(R),5(S)-dimethyl-6-oxononyl]-2-hydroxy-3-methylbenzoic acid
Methyl-6,8-dideoxy-6[[1-methyl-4-propyl-2-pyrrolidinyl)carbonyl]amino)-1-thio-D-ery^ro-a-D-gia/acto-octopyranoside
1 7-Hydroxy-6-methyl-1 6-methylenepregna- 4,6-diene-3,20-dione
6-Butyl-1,4-dihydro-4-oxo-7-(phenylmethoxy)-3-quinolinecarboxylic acid
N,/V'-Bis(4-nitrophenyl)urea compd with 4,6-dimethyt-2(1H)-pyrimidinone (1:1)
3-(1-Methyl-2-pyrrolidinyl)pyridine
[(5-Nitro-2-furanyl)methylene]hydrazide-2-hydroxy-3,5-dinitrobenzoic acid
5-Nitrofurfurylidenehydrazide acetic acid
(4-Nitrophenyl)-arsonic acid
W-Ethyl-A/'-(5-nitro-2-thiazolyl)urea
3-Methyl-5-[(4-nitrophenyi)azo]-2-thioxo-4-thiazolidinone
2-[(5-Nitro-2-furanyl)methylene]hydrazinecarboxamide
3,5-Dinitrobenzamide
Bis(m-nitrophenyi)disulfide
(4a,4aa,5a,5aa,63,12aa)-4-(Dimethylamino)-1,4,4a,5,5a ! 6,11,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxo-2-
naphthacenecarboxamide dihydrate
(2tt,5a,6p)-3,3-Dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyc!o[3.2.0]heptane-2-carboxylic acid
Thiodiphenylamine
E-1,4,5,6-Tetrahydro-1-methyl-2-[2-(2-thienyl)vin]pyrimidine tartrate (1:1)
2,2-Dichloro-A/-[2-hydroxy-1-(hydroxymethyl)-2~[4-(methylsulfonyl)phenyl]ethyl]-acetamide
(3p,16p,17a,18|3,20a)-1 1-17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester
0,0-Dimethyl 0-(2,4,5-trichlorophenyl) phosphorothioic acid ester
(4-Hydroxy-3-nitrophenyl)-arsonic acid
Decahydro^a^.g-trihydroxy^-methyl-e^-bistmethylaminoJ^H-pyrano^.S-oKl^lbenzodioxin^-one
0-2-Deoxy-2-(methylamino)-a-L"glucopyranosyl-(1^2)-05-deoxy-3-C-formyl-a-L-lyxofuranosyl-(1-^4)-A/ J A/'-bis(aminoiminomethyl)-
D-streptamine
4-Amino-A/-(2,6-dimethoxy-4-pyrimidinyl)-benzenesulfonamide
4-Amino-/V-(diaminomethy!ene)-benzenesulfonamide
4-Amino-/V-(4-methyl-2-pyrimidinyl)-benzenesulfonamide
4-Amino-A/-(4,6-dimethyl-2-pyrimidinyl)-benzenesulfonamide
N-[4-[[(4-Nitrophenyl)amino]sulfonyl]phenyl]-acetamide
4-Amino-A/-2-quinoxalinyl-benzenesulfonamide
4-Amino-/V-2-thiazolyl-benzenesulfonamide
2-(4-Thiazolyl)-1H-benzimidazole
2-Methyl-3,5-dinitrobenzamide
Sources: U SAN and the (JSP Dictionary of Drug Names (1989). U.S. Pharmacopeia! Convention, Rockville, MD; The Merck index (1989) 10th ed.,
Co., Inc., Rahway, NJ.
Merck &
6 B Disinfectants
Aram BeSosan, Associate Chapter Editor
Environmental Protection Agency
PHENOL COEFFICIENT METHODS
955.11 Testing Disinfectants
against Salmonella typhi
Phenol Coefficient Method
Final Action 1964
(Applicable to testing disinfectants miscible with H 2 that do
not exert bacteriostatic effects that cannot be neutzd by one of
subculture media specified, or overcome by suitable subtrans-
fer procedures. The 95% confidence limits are ±12%.)
A. Culture Media
(a) Nutrient broth. — Boil 5 g beef ext (Difco), 5 g NaCl,
and 10 g peptone (Anatone, peptic hydrolysate of pork tissues,
manufactured by American Laboratories, Inc., 4410 S 102nd
St, Omaha, NE 68127) in 1 L H 2 20 min, and dil. to vol.
with H 2 0; adjust to pH 6.8. (If colorimetric method is used,
adjust broth to give dark green with bromothymol blue.) Filter
thru paper, place 10 mL portions in 20 x 150 mm test tubes,
and autoclave 20 min at 121°. Use this broth for daily transfers
of test cultures.
(b) Synthetic broth. — Soln A: Dissolve 0.05 g L-cystine, 0.37
g DL-methionine, 0.4 g L-arginine.HCl, 0.3 g DL-histi-
dine.HCI, 0.85 g t-lysine.HCl, 0.21 g L-tyrosine, 0.5 g dl-
threonine, 1 .0 g dl- valine, 0.8 g L-leucine, 0.44 g DL-isoleu-
cine, 0.06 g glycine, 0.61 g DL-serine, 0.43 g DL-alanine, 1 .3
g L-glutamic acid. HO, 0.45 g L-aspartic acid, 0.26 g DL-phen-
ylalanine, 0.05 g dl- tryptophan, and 0.05 g l- proline in 500
mL H 2 contg 18 mL IN NaOH.
Soln B: Dissolve 3.0 g NaCl, 0.2 g KC1, 0. 1 g MgS0 4 .7H 2 0,
1.5 g KH 2 P0 4 , 4.0 g Na 2 HP0 4 , 0.01 g thiamine.HCl, and 0.01
g niacinamide in 500 mL H 2 0.
Mix Solns A and B, dispense in 10 mL portions in 20 X
150 mm tubes, and autoclave 20 min at 121°. Before using
for daily transfers of test cultures, aseptically add 0. 1 mL ster-
ile 1.0% glucose soln per tube. Grow cultures with tube slanted
8° from horizontal.
(c) Nutrient agar. — Dissolve 1.5% Bacto agar (Difco) in
nutrient broth and adjust to pH 1 .2-1 A (blue-green with
bromothymol blue) or in synthetic broth, tube, autoclave, and
slant,
(d) Subculture media. — Use (/), (2), or (3), whichever gives
lowest result. (Com. dehydrated brands made to conform with
preceding specifications may be used.) With oxidizing prod-
ucts and products formulated with toxic compds contg certain
heavy metals like Hg, (2) will usually give lowest result. With
products contg cationic surface active materials, (3) will usu-
ally give lowest result. See also 955. 14C, par. 5
(J) Nutrient broth described in (a).
(2) Fluid thioglycolate medium USP XX: Mix 0.5 g L-cys-
tine, 0.75 g agar, 2.5 g NaCl, 5.5 g glucose. H 2 0, 5.0 g H 2 0-
sol. yeast ext, and 15.0 g pancreatic digest of casein with 1
L H 2 0. Heat on H 2 bath to dissolve, add 0.5 g Na thiogly-
colate or 0.3 g thioglycolic acid, and adjust with 17V NaOH to
pH 7.1 ± 0.2. If filtration is necessary, reheat without boiling
and filter hot thru moistened filter paper. Add 1.0 mL freshly
prepd 0.1% Na resazurin soln, transfer 10 mL portions to 20
X 150 mm tubes, and autoclave 20 min at 121°. Cool at once
to 25° and store at 20-30°, protected from light.
(3) "Letheen broth" : Dissolve 0.7 g lecithin (Asolectin, As-
sociated Concentrates, 32-34 61st St, Woodside, NY 11377)
and 5.0 g polysorbate 80 (Tween 80, or equiv.) in 400 mL
hot H 2 and boil until clear. Add 600 mL soln of 5.0 g beef
ext (Difco), 10.0 g peptone (Anatone, (a)), and 5 g NaCl in
H 2 0, and boil 10 min. Adjust with IN NaOH and /or IN HO
to pH 7.0 ± 0.2 and filter thru coarse paper; transfer 10 mL
portions to 20 x 150 mm tubes, and autoclave 20 min at 121°.
(4) Cystine trypticase agar (BBL Microbiology Systems):
Suspend 29.5 g in 1 L H 2 0. Heat gently with frequent agi-
tation and boil ca 1 min or until soln is complete. Transfer 10
mL portions to 20 x 150 mm tubes, and autoclave 15 min at
12 lb pressure. Cool in upright position and store <25 days at
20-30°. Use for monthly transfer of stab stock cultures of Ps.
aeruginosa PRD 10 (ATCC 15442).
(5) Other subculture media: Use (d)(2) with 0.7 g lecithin
(Asolectin, Associated Concentrates, Inc.) and 5.0 g polysor-
bate 80 (Tween 80, or equiv.) added; or suspend 29.8 g prepd
fluid thioglycolate medium (Difco), 0.7 g lecithin, and 5.0 g
polysorbate 80 in 1 L H 2 0, and boil until soln is clear. Cool,
dispense in 10 mL portions in 20 x 150 mm tubes, and au-
toclave 20 min at 121°. Store at 20-30°. Protect from light.
B. Apparatus and Reagents
(a) Glassware. — 1, 5, and 10 mL vol. pipets; 1, 5, and 10
mL Mohr pipets graduated to 0. 1 mL or less; 100 mL g-s cyl-
inders graduated in 1 mL divisions; Pyrex lipped test tubes,
25 x 150 mm (medication tubes) reusable or disposable bo-
rosilicate; bacteriological culture tubes, 20 x 150 mm (test
culture and subculture tubes). Plug medication tubes with cot-
ton wrapped in 1 layer of cheesecloth (tubes capped with Mor-
ton closures are an acceptable alternative). Sterilize all glass-
ware 2 hr in hot air oven at 180°. Loosely plug pipets with
cotton at mouth and place in closed metal containers before
sterilizing.
(b) Water bath. — Const temp, relatively deep H 2 bath ca-
pable of maintaining 20 ± 0.2°, with cover having > 10 well-
spaced holes which admit medication tubes but not their lips.
(c) Racks. — Any convenient style. Blocks of wood (size
depending on space in incubator) with deep holes are satis-
factory. Have holes well spaced to ensure quick manipulation
of tubes. It is convenient to have them large enough to admit
medication tubes while dilns are being made.
(d) Transfer loop. — Make 4 mm id single loop at end of
50^75 mm (2-3") Pt or Pt alloy wire No. 23 B&S gage or 4
mm loop fused on 75 mm (3") shaft (available from Matthey-
Bishop, Inc., 1401 King Rd, West Chester, PA 19380). Fit
other end in suitable holder (glass or A J rod). Bend loop at
30° angle with stem, Fig. 955.11.
(e) Test organism. — Hopkins strain 26 of Salmonella typhi
(Schroeter) Warren and Scott, ATCC No. 6539 (formerly called
Bac. typhosus and E be r the I la typhosa). Maintain stock culture
on nutrient agar slants by monthly transfers. Incubate new stock
transfer 2 days at 37°; then store at 2-5°. From stock culture
inoculate tube of nutrient broth and make at least 4 consecutive
133
134
Disinfectants
AOAC Official Methods of Analysis (1990)
FIG. 955.11 — Transfer loop and manner of using in phenol
coefficient technic
daily transfers (^30) in nutrient broth, incubating at 37°, be-
fore using culture for testing. (If only 1 daily transfer has been
missed, it is not necessary to repeat the 4 consecutive trans-
fers.) Use 22-26 hr culture of organism grown in nutrient broth
at 37° in test. Shake, and let settle 15 min before using.
With Ps. aeruginosa PRD 10, proceed as in 964.02.
(f) Phenol stock soln. — 5% (w/v). Weigh 50 g USP phenol,
which congeals at >40°, in beaker. Dissolve in H 2 0, rinse soln
into 1 L vol. flask, and dil. to vol. Stdze with 0.17V KBr-
KBr0 3 soln, (g), as follows: Transfer 25 mL stock soln to 500
mL vol. flask and dil. to vol. with H 2 0. Transfer 15 mL ali-
quot of dild soln to 500 mL 1 flask and add 30 mL std KBr-
KBr0 3 soln. Add 5 mL HO and immediately insert stopper.
Shake frequently during 30 min and let stand 15 min. Remove
stopper just enough to quickly add 5 mL 20% KI soln, taking
care that no Br vapors escape, and immediately stopper flask.
Shake thoroly, remove stopper, and rinse it and neck of flask
with little H 2 so that washings flow into flask. Titr. with
0.1/V Na 2 S 2 3 , using starch indicator: Mix ca 2 g finely powd.
potato starch with cold H 2 to thin paste; add ca 200 mL boil-
ing H 2 0, stirring constantly, and immediately discontinue
heating. Add ca 1 mL Hg, shake, and let stand over the Hg.
1 mL 0. 1/V KBr-KBr0 3 = 0.001569 g phenol.
% phenol in stock soln = (30 - mL 0.LV Na 2 S 2 3
soln from titrn) X 0.001569 x 1333 X 100/1000
where 30 = mL 0AN KBr-KBr0 3 soln added, 0.001569 - g
phenol equiv. to 1 mL 0AN KBr-KBr0 3 soln, 1333 = diln
factor, and 1000 = original vol. phenol stock soln.
If necessary, adjust stock soln to 5.00 ± 0.05% phenol by
adding H 2 or phenol. Keep in well stoppered amber bottles
in cool place, protected from light.
(g) Potassium hromide-hr ornate soln. — 0.17V. Prep, as in
947. 13A. Stdze as follows: Transfer 30 mL to I flask, and add
25 mL H 2 0, 5 mL 20% KI soln, and 5 mL HO. Shake thoroly
and titr. with O.lvV Na 2 S 2 3 , using starch indicator.
C. Operating Technic
Make 1% stock diln of substance to be tested (or any other
convenient diln, depending on anticipated concn) in g-s cyl-
inder. Make final dilns, from 1% stock diln, directly into med-
ication tubes and remove all excess >5 mL. (Range of dilns
should cover killing limits of disinfectant in 5-15 min and
should at same time be close enough for accuracy.) From 5%
stock phenol soln (1-20) dil. further to make 1-90 and 1-1.00
dilns, and place in medication tubes. Place these tubes, contg
5 mL each of final dilns of disinfectant and of phenol, and
tube contg test culture in H 2 bath at 20° and leave 5 min.
Add 0.5 mL test culture to each of dilns at time intervals cor-
responding to intervals at which transfers are to be made. (Thus,
by time 10 tubes have been seeded at 30 sec intervals, 4.5 min
has elapsed, and 30 sec interval intervenes before transference
to subculture begins.) Add culture from graduated pipet large
enough to seed all tubes in any one set. In using Ps. aerugi-
nosa PRD 10 (ATCC 15442), proceed as in 964.02.
In inoculating medication tubes, hold them in slanting po-
sition after removal from bath, insert pipet to just above sur-
face of disinfectant, and run in culture without letting tip touch
disinfectant. After adding culture, agitate tubes gently but tho-
roly to ensure even distribution of bacteria, and replace in bath;
5 min after seeding first medication tube, transfer 1 loopful of
mixt. of culture and dild disinfectant from medication tube to
corresponding subculture tube. To facilitate transfer of uni-
form drops of medication mixt., hold tube at 60° angle, and
withdraw loop so that plane of loop is parallel with surface of
liq. (Fig. 955.11). After 30 sec, transfer loopful from second
medication tube to second subculture tube and continue pro-
cess for each successive diln; 5 min after making first transfer,
begin second set of transfers for 10 min period, and finally
repeat for 15 min. period.
Gently agitate medication tubes before taking each interval
loop subsample for transfer to subculture medium. Before each
transfer, heat loop to redness in flame and flame mouth of
every tube. Sterilize loop immediately after each transfer (be-
fore replugging tubes) to allow time for cooling. Use care in
transferring and seeding to prevent pipet or needle from touch-
ing sides or mouth of medication tube, and see that no cotton
threads adhere to inner sides or mouths of tubes. Incubate sub-
culture 48 hr at 37° and read results. Thoroly agitate individual
subculture tubes before incubation. Macroscopic examination
is usually sufficient. Occasionally 3-day incubation period, agar
streak, microscopic examination, or agglutination with anti-
typhoid serum may be necessary to det. feeble growth or sus-
pected contamination.
D. Calculation
Express results in terms of phenol coefficient number, or
highest diln killing test organism in 10 min but not in 5 min,
whichever most accurately reflects germicidal value of disin-
fectant. Phenol coefficient is number obtained by dividing nu-
merical value of greatest diln (denominator of fraction ex-
pressing diln) of disinfectant capable of killing S. typhi in 10
min but not in 5 min by greatest diln of phenol showing same
results.
Example:
Disinfectant (X):
Diln
5 Min 10 Min 15 Min
1-300
1-325
-t-0
1-350
+ 00
1-375
+ +0
1-400
+ + +
Phenol:
1- 90
+ 00
1-100
+ + +
350
Phenol coefficient would be - 3.89
90
Test is satisfactory only when phenol control gives one of fol-
lowing readings:
AOAC Official Methods of Analysis (1990)
Use Dilution
135
Phenol
5 Min
10 Min
15 Min
1- 90
+ or
+ or
1-100
+
+
+ or
If none of dilns of disinfectant shows growth in 5 min and
killing in 10 min, est. hypothetical diln only when any 3 con-
secutive dilns show following results: first, no growth in 5
min; second, growth in 5 and 10 min but not in 15 min; and
third, growth in 5, 10, and 15 min.
Example:
Disinfectant (X):
Diln
5 Min
10 Min
15 Min
1-300
1-350
+
+
1-400
+
Phenol -
+
+
1- 90
1-100
+
+
Phenol coefficient would be -
325
95
^3.42
To avoid giving impression of fictitious accuracy, calc. phenol
coefficient to nearest 0.1. Thus, in examples cited above, phenol
coefficients would be reported as 3.9 and 3.4, instead of 3.89
and 3.42.
Note: Although it is commonly accepted criterion that dis-
infectants be at diln equiv. in germicidal efficiency to phenol
against S. typhi by calcg 20 x S. typhi coefficient to det. num-
ber of parts H 2 in which 1 part disinfectant may be mixed,
this should be regarded as presumptive and is subject to con-
firmation by use-dilution method.
Refs.: J. Roy. Sanit. Inst. 24, 424(1903). Am. J. Public Health
3, 575(1913). U.S. Dept. Agric. Circ. 198 (1931).
JAOAC 32, 408(1949); 38, 465(1955). Soap Chem.
Spec. 34, No. 10, 79(1958); 47, 176(1964); 53,
860(1970); 56, 308(1973).
955.12 Testing Disinfectants against
Staphylococcus aureus
Phenol Coefficient Method
Final Action
Proceed as in 955.11, except change phenol dilns and test
organisms. Use 22-26 hr culture of Staph, aureus FDA 209,
ATCC No. 6538, having at 20° at least resistance indicated by
following:
Phenol
5 Min
10 Min
15 Min
1-60
+ or
+ or
1-70
+
+
+
Refs.: J. Roy. Sanit, Inst. 24, 424(1903). Am. J. Public Health
3, 575(1913). U.S. Dept. Agric. Circ. 198 (1931).
JAOAC 32, 408(1949); 38, 465(1955). Soap Chem.
Spec. 34, No. 10, 79(1958).
955.13 Testing Disinfectants against
Pseudomonas aeruginosa
Phenol Coefficient Method
First Action
Proceed as in 955.11. Use 22-26 hr culture of Ps. aeru-
ginosa PRD 10 (ATCC 15442), having resistance to phenol at
20° at least as follows:
Phenol
1-80
1-90
5 Min
+ or
+
10 Min
+ or
+
15 Min
+
USE-DILUTION METHODS 1
(Applicable to testing disinfectants miscible with H 2 to
confirm phenol coefficient results and to det. max. dilns ef-
fective for practical disinfection. These microbiological meth-
ods are technique-sensitive methods in which extreme adher-
ence to the method with identified critical control points, good
microbiological techniques, and quality controls is required for
proficiency and validity of results. These methods have been
validated using distd H 2 only without soil challenge.)
955.14 Testing Disinfectants against
Salmonella choleraesuis
Use-Dilution Method
First Action 1953
Final Action 1959
Repealed First Action 1988
A. Reagents
(a) Culture media, — See 955.11 A.
(b) Test organism, Salmonella choleraesuis. — (ATCC
10708). Obtain annually directly from ATCC. Maintain stock
culture on nutrient agar slants by monthly transfers. Incubate
new stock transfer 2 days at 37°; then store at 2-5°. From stock
culture inoculate tube of nutrient broth and incubate at 37°.
Make 3 consecutive 24 hr transfers; then inoculate tubes of
nutrient broth (2 for each 10 carriers to be tested), using one
loop of inoculum with each tube; incubate 48-54 hr at 37°.
(c) Phenol.— See 955.11B(f).
(d) Sterile distilled water. — Prep, stock supply of H 2 in
1 L flasks, plug with cotton, sterilize 20 min at 121°, and use
to prep, dilns of medicants.
(e) Asparagine soln. — Make stock supply of 0.1% aspar-
agine ("Bacto") soln in H 2 in erlenmeyer of convenient size,
plug with cotton, and sterilize 20 min at 121°. Use to cover
metal carriers for sterilization and storage.
(f) Sodium hydroxide soln. — Approx. \N (4%). (For clean-
ing metal carriers before use.)
6. Apparatus
(a) Glassware. — As in 955.11B(a). Also: straight side Pyrex
test tubes, 20 X 150 mm; 15 X 1 10 mm petri dishes; 100 mL,
300 mL, and 1 L erlenmeyers. Sterilize petri dishes in closed
metal containers. Use 25 x 150 mm straight side tubes for
disinfectant soln. (Smaller tubes can give high percentage of
false positives when sides are touched.)
(b) Water bath and racks.— See 955.11B(b) and (c).
(c) Transfer loops and needles. — (7) See 955.11B(d). (2)
Make 3 mm right angle bend at end of 50-75 mm nichrome
wire No. 18 B&S gage. Have other end in suitable holder (glass
or Al rod).
(d) Carriers. — Polished stainless steel cylinders (penicillin
cups), 8 ± 1 mm od, 6 ± 1 mm id, length 10 ± 1 mm, of
type 304 stainless steel, SS 18-8. (Obtainable from S. & L.
Metal Products Corp., 58-29 57 Drive, Maspeth, NY 11378.)
'The use-dilution methods for testing disinfectants, 955.14, 955A5, and 964.02,
are technique-sensitive and may produce questionable results unless conducted
by experienced, trained analysts under strictly controlled conditions. Users of
the methods are advised to consult the following reports of recent studies for
current scientific data and interpretation: JAOAC 69, 1003(1986); 70, 635(1987);
70, 903(1987); 71, 9(1988); 71, 288(1988); 71, 868(1988); 71, 1187(1988).
Infect. Control 8, 501(1987).
136
Disinfectants
AOAC Official Methods of Analysis (1990)
Discard cylinders that are visibly damaged (dull, chipped,
dented, or gouged). Biologically screen remaining cylinders
by performing use-dilution test with Staphylococcus aureus
ATCC 6538 and 500 ppm alkyldimethylammonium chloride
with alkyl chain distribution C14, 50%; C12, 40%; C16, 10%
(e.g., BTC-835 Onyx Chemical Co., Jersey City, NJ 07302).
Discard those cylinders giving pos. results in screening pro-
cedure. In subsequent testing of samples, cylinders in tubes
showing growth must be rescreened and may not be reused
unless screen tests result in no growth.
(e) Petri dishes. — Have available ca 6 sterile petri dishes
matted with 2 layers of S&S No. 597 or Whatman No. 2, 9
cm filter paper.
(f) Pipets. — Use only disposable pipets. (Reusable pipets
may have residues or chips.)
C. Operating Technic
Soak ring carriers overnight in IN NaOH, rinse with tap
H 2 until rinse H 2 is neut. to phthln, then rinse twice with
distd H 2 0; place cleaned ring carriers in multiples of 10 in
cotton-plugged erlenmeyers or 25 x 150 mm cotton plugged
Pyrex test tubes, cover with asparagine soln, 955.14A(e), ster-
ilize 20 min at 121°, cool, and hold at room temp. Vortex-
mix nutrient broth test culture 3-4 s and let stand 10 min at
room temp, before continuing. Transfer 20 sterile ring car-
riers, using flamed nichrome wire hook, into 20 mL 38-54 hr
nutrient broth test culture in sterile 25 X 150 mm medication
tube. One or 2 addnl carriers may be added at same inoculum
rate to serve as reserves. Carriers that fall over in petri dishes
cannot be used in test. After 15 min contact period remove
cylinders, using flamed nichrome wire hook, shake carrier vig-
orously against side of tube to remove excess culture, and place
on end in vertical position in sterile petri dish matted with filter
paper, 955.14B(e), making sure that carriers do not touch to
prevent improper drying. Cover and place in incubator at 37°
and let dry 40 min. Hold broth culture for detn of its resistance
to phenol by phenol coefficient method, 955. 11C.
From 5% stock phenol soln (1-20) make 1-90 and 1-100
dilns directly into medication tubes. Place tube for each diln
in H 2 bath and let come to 20° (10 min). Make stock soln
of germicide to be tested in sterile g-s cylinder. From this soln
make 10 mL dilns to be tested, depending upon phenol coef-
ficient found and /or claimed against S. typhi at 20°, directly
into each of ten 25 X 150 mm medication tubes; place the 10
tubes in H 2 bath at 20° and let come to temp. Prep, diln of
germicide to be tested by diln in sterile H 2 0, 955.14A(d). Diln
of sample should be made using >1.0 mL of sample. Use v/
v dilns for liq. products and w/v dilns for solids. Round to 2
decimal places toward a stronger product. To ensure stable
product, soln should be prepd <3 hr prior to use. Place tubes
in 20° H 2 bath >10 min. Det. diln to be tested by multiplying
phenol coefficient number found and/or claimed by 20 to det.
number of parts H 2 in which 1 part germicide is to be in-
corporated. This detn is not required when disinfectant under
test yields phenol coefficient that cannot be converted validly
to presumptive use-diln, or when analyst dets that use-diln range
can be found without resort to phenol coefficient test.
Add 0.5 mL of test culture suspension to 1-90 diln of phenol
control; after 30 sec interval, add 0.5 mL to 1-100 diln of
control, using sterile cotton-plugged pipets. After adding cul-
ture, agitate tubes gently but thoroly to distribute bacteria evenly,
and replace in bath; 5 min after seeding first medication tube,
transfer 1 loopful of mixt. of culture and dild phenol from
medication tube to corresponding subculture tube. After 30 sec,
transfer loopful from second medication tube; 5 min after mak-
ing first set of transfers begin second set of transfers for 10
min period; and finally repeat for 15 min period. Use technic
of loop sampling, flaming loop and mouths of tubes, and ag-
itating medication and subculture tubes as in phenol coefficient
method, 955. 11C. Incubate subcultures 48 hr at 37° and read
results. Resistance in 48-54 hr culture of S. choleraesuis should
fall within range specified for 24 hr culture of S. typhi in phenol
coefficient method.
Without touching sides of tube with contaminated carrier or
hook, either when placing carrier in tube or when withdrawing
hook, add 1 contaminated dried cylinder carrier at 1 min in-
tervals to each of the .10 tubes of use-diln of germicide to be
tested. (Note: Proper execution of transfer step is one of the
most critical, technique-sensitive areas of method. False pos-
itives will result if sides of tube are touched.) Thus, by time
10 tubes have been seeded, 9 min will have elapsed, plus 1
min interval before transfer of first carrier in series to individ-
ual tube of subculture broth. This interval is const for each
tube with prescribed exposure period of 10 min. The 1 min
interval between transfers allows adequate time for flaming and
cooling nichrome wire hook and making transfer in manner so
as to drain all excess medication from carrier by shaking car-
rier against side of tube. Shorter intervals may be used in add-
ing and removing carriers if 2 alternately flamed and cooled
hooks are used. Individual manipulation of carriers is required;
use of semiautomated ring carrier is prohibited. (Note: Above
step is one of the most critical, technique- sensitive areas of
method. False positives can result from transfer of live organ-
isms to sides of tube due to aerosol formation.) Flame lips of
medication and subculture tubes in conventional manner. Im-
mediately after placing carrier in medication tube, swirl tube
3 times before placing it back in bath. Thoroly shake subcul-
ture tubes, incubate 48 hr at 37°, and report results as + (growth)
or — (no growth) values. Growth in tubes should be checked
by gram stain to ensure that no contamination is present. Check
>:20% of pos. tubes. Confirm all pos. results by duplicate test-
ing to assure against false pos. tests.
Where there is reason to suspect that lack of growth at con-
clusion of incubation period may be due to bacteriostatic ac-
tion of medicant adsorbed on carrier that has not been neutzd
by subculture medium used, transfer each ring to new tube of
sterile medium and reincubate for addnl 48 hr at 37°. Where
soln under test is such that material adsorbed on ring carriers
and transferred into subculture medium makes it unsuitable for
growth of test organism, as may be case with coned acids and
alkalies, products carrying antibiotics, and wax emulsions,
transfer each ring to new tube of sterile medium 30 min after
initial transfer and incubate both primary and secondary sub-
culture tubes 48 hr at 37°. Results showing no growth on all
10 carriers will confirm phenol coefficient number found. Re-
sults showing growth on any of the 10 carriers indicate phenol
coefficient number to be unsafe guide to diln for use. In latter
case, repeat test, using lower dilns (higher conens) of germi-
cide under study. Max. diln of germicide which kills test or-
ganism on 10 carriers in 10 min interval represents presumed
max. safe use-diln for practical disinfection.
Refs.: J. Bacteriol. 49, 526(1945). Am. J. Vet. Res. 9,
104(1948). JAOAC 36, 466(1953); 70, 318(1987); 71,
117(1988); 72, 116(1989).
955.15 Testing Disinfectants against
Staphylococcus aureus
Use-Dilution Method
First Action 1953
Final Action 1959
Repealed First Action 1988
Proceed as in 955. 14C except change phenol dilns and test
organism to those specified in 955.12. Use 48-54 hr culture
AOAC Official Methods of Analysis (1990)
Chlorine
137
of Staph, aureus FDA 209, ATCC No. 6538, having at least
resistance specified for 24 hr culture at 20° in phenol coeffi-
cient method, 955.12. Obtain organism annually, directly from
ATCC. Prior to beginning use-dilution test, vortex-mix nu-
trient broth culture as in 955.14. Results showing growth on
any of 10 carriers indicate that diln is too high for use in dis-
infecting where pyogenic bacteria must be killed. In such cases
repeat test, using lower dilns (higher concns). Max. diln of
germicide which kills both this test organism and S. choler-
aesuis on 10 carriers in 10 min interval represents max. pre-
sumed safe use-diln for disinfecting in hospitals, clinics, and
other places where pyogenic bacteria have special signifi-
cance.
Note: While killing in 10 of 10 replicates specified provides
reasonably reliable index in most cases, killing in 59 of 60
replicates is necessary for confidence level of 95%.
Refs.: J. Bacteriol. 49, 526(1945). Am. J. Vet. Res. 9,
104(1948). JAOAC 36, 466(1953); 70, 318(1987); 71,
117(1988); 72, 116(1989).
964.02 Testing Disinfectants against
Pseudomonas aeruginosa
Use-Dilution Method
First Action 1964
Proceed as in 955. 14C. Use 48-54 hr nutrient broth culture
Ps. aeruginosa PRD 10 (ATCC 15442). Carry stock culture
on BBL CTA (cystine trypticase agar) in stab culture incubated
48 hr at 37° and stored at 5° with transfer every 30 days. Trans-
fer nutrient broth test cultures daily for 30-day intervals with
incubation at 37°. Make fresh transfer from stock culture every
30 days. Do not shake 48-54 hr test culture but decant liq.
culture aseptically, leaving pellicle behind, to obtain 20 mL
culture for inoculating 20 carriers in medication tube.
Proceed with vortex-mixing as in 955. 14C prior to use of
culture. Alternatively, pellicle may be carefully suctioned off,
and culture can be poured into clean, sterile tube before vor-
tex-mixing. Any disruption of pellicle resulting in dropping,
breaking up, or stringing of pellicle in culture before or during
its removal renders that culture unusable in use-dilution test.
This is extremely critical because any pellicle fragments re-
maining will result in uneven clumping and layering of or-
ganism on cylinder, allowing unfair exposure to disinfectant
and causing false pos. results.
Refs.: J. Bacteriol. 49, 526(1945). Am. J. Vet. Res. 9,
104(1948); JAOAC 47, 29, 176(1964); 70, 318(1987);
72, 116(1989).
OTHER TESTS
955.16 Chiorine (Available) in Disinfectants
Germicidal Equivalent Concentration
Final Action
(Applicable to H 2 0-miscible disinfectants for detg available CI
germicidal equiv. concns with products offered for use as san-
itizing rinses for previously cleaned nonporous surfaces, es-
pecially where speed of action and capacity are essential con-
siderations)
A. Reagents
Use reagents specified in 955. 11 A and 955.11B(e) and (f),
and in addn:
(a) Sterile distilled H 2 0.—See 955.14A(d).
(b) Sterile phosphate buffer soln. — pH 8.0. Add 97.5 mL
soln contg 11.61 g anhyd. K 2 HP0 4 in 1 L H 2 to 2.5 mL soln
contg 9.08 g anhyd. KH 2 P0 4 in 1 L H 2 and autoclave 20
min at 121° in cotton-plugged erlenmeyer.
(c) NaOCl std stock soln. — Approx. 5%. Store NaOCl stock
soln in tightly closed bottle in refrigerator, and det. exact
available CI concn at frequent intervals by As 2 3 titrn: Trans-
fer 20 mL sample to 1 L vol. flask and dil. to vol. Pipet 50
mL aliquot of mixt. into 200 mL erlenmeyer. Add excess As 2 3
soln and then decided excess NaHC0 3 . Titr. excess As 2 3 with
std 1 soln, using starch soln (mix ca 2 g finely powd. potato
starch with cold H 2 to thin paste; add ca 200 mL boiling
H 2 0, stirring const., and immediately discontinue heating; add
ca 1 mL Hg, shake, and let soln stand over Hg), or use the 1
as its own indicator. Subtract vol. 1 soln, corrected to 0.L/V,
from vol. As 2 3 soln used, and from this value and sp gr of
soln, calc.% NaOCl.
1 mL O.liV As 2 3 = 0.003722 g NaOCl
(d) Test organisms.— Use S. typhi ATCC No. 6539 or Staph,
aureus ATCC No. 6538 or both.
B. Apparatus
See 955. 11B.
C. Operating Technic
Det. resistance of test culture to phenol as in 955.11, and
use cultures with resistance specified. Prep., in sterile g-s cyl-
inders, NaOCl solns contg 200, 100, and 50 ppm available CI
in sterile buffer soln, 955.16A(b). Transfer 10 mL of each soln
to 25 x 150 mm medication tubes, place tubes in 20° H 2
bath, and let come to temp.
Starting with tube contg 200 ppm available CI, add 0.05 mL
test culture prepd as in 955.11B(e), shake, and return to H 2
bath. After 1 min, make transfer to tube of appropriate sub-
culture medium, 955.11A(d), using flamed 4 mm loop. At 1.5
min, add another 0.05 mL culture to the 200 ppm CI soln,
shake, and return to bath. After addnl 1 min interval (2.5 min
in test), make second subculture in same manner, and in 30
sec, or at 3 min time in test, add another 0.05 mL culture,
shaking and returning to H 2 bath. After another 1 min in-
terval (4 min in test), make another transfer to tube of sub-
culture medium.
Repeat operation to give total of 10 added increments. This
requires total time of 14.5 min for each soln and addn of 0.5
mL total culture with subculture at std 1 min intervals after
addn of culture aliquots. At conclusion of test shake all sub-
culture tubes and incubate 48 hr at 37°.
Repeat operation with solns contg 100 and 50 ppm available
CI. Prep, soln of germicide to be tested at concn recommended
or selected for study in sterile H 2 in g-s graduate. Transfer
10 mL to 25 x 150 mm medication tubes, place in H 2 bath,
and let come to temp. Repeat operation with this soln.
To be considered equiv. in disinfecting activity to 200 ppm
available CI, unknown germicide must show absence of growth
in as many consecutive tubes of subculture tube series as 200
ppm available CI std. Det. activity equiv. to 100 and 50 ppm
available CI in same manner. See example, Table 955.16.
In this example, 25 ppm soln of germicide X could be con-
sidered equiv. to 200 ppm soln of available CI, and 20 ppm
soln equiv. to 100 ppm of available CI, but 10 ppm soln of
germicide X would not be considered equiv. in germicidal ac-
tivity to 50 ppm of available CI.
Draw conclusions relative to germicidal equiv. concns only
when resistance of test culture to NaOCl control is such that
^1 neg. increment is obtained at 50 ppm concn and 1 pos.
increment is obtained at 200 ppm level.
Refs.: Soap Sanit. Chem. 27, No. 2, 133(1951). JAOAC 38,
274(1955); 40, 755(1957).
138
Disinfectants
AOAC Official Methods of Analysis (1990)
Table 955.16 Example for Determination of Chlorine Germicidal Equivalent Concentration
No growth
Subculture Series
Concn, ppm
Germicide
Avail. CI
1
2
3
4
5
6
7
8
9
10
200
-
__
-
-
_
+
+
+
+
+
NaOCI control
100
~
-
-
+
+
+
+
+
+
+
50
-
~-
+
+
+
+
+
+
+
+
25
-
_
-
_
_
+
+
+
+
+
Unknown (X)
20
-
-
_
-
+
+
+
+
+
+
10
—
+
+
+
+
+
+
+
+
+
growth
955.17 Fungicidal Activity of Disinfectants
Using Trichophyton mentagrophytes
Final Action
(Applicable for use with H 2 0-miscible type fungicides used to
disinfect inanimate objects)
A. Test Organism
Use as test fungus typical strain of Trichophyton mentagro-
phytes isolated from dermatophytosis of foot. Strain must spo-
rulate freely on artificial media, presence of abundant conidia
being manifested by powdery appearance on surface of 10-day
culture, particularly at top of agar slant, and confirmed by mi-
croscopic examination. Conidia-bearing mycelium should peel
easily from surface of glucose agar. Conidia of required re-
sistance survive 10 min exposure at 20° to phenol diln of 1 :70,
but not to one of 1:60. Strain No. 640, ATCC No. 9533, is
suitable.
B. Culture Medium
Carry fungus on agar slants of following composition: Glu-
cose 2%, Neopeptone (Difco) 1%, agar 2%, adjusted to pH
6.1-6.3. Use same culture medium to prep, cultures for ob-
taining conidial suspension, and use fluid medium of same nu-
trient composition (without agar) to test survival and viability
of conidia after exposure to fungicide.
C. Care of Fungus Strain
Store stock culture of fungus on glucose agar slants at 2—
5°. At intervals <3 months, transfer to fresh agar slants, in-
cubate 10 days at 25-30°, and store at 2-5° until next transfer
period. Do not use culture that has been kept at or above room
temp. >10 days as source of inoculum for culture. (Cultures
may be kept at room temp, to preserve strain and to inoculate
cultures if transferred at intervals <10 days.)
D. Preparation of Conidial Suspension
Prep, petri dish cultures by planting inoculum at center of
agar plate and incubating culture at 25-30° for >10, but <15
days. Remove mycelial mats from surface of 5 agar plate cul-
tures, using sterile spatula or heavy flattened wire. Transfer to
heat-sterilized glass tissue grinder, 966.04B(e), and macerate
with 25 mL sterile physiological NaCl soln (0.85% NaCl), or
to heat- sterilized erlenmeyer contg 25 mL sterile saline with
glass beads, and shake thoroly. Filter suspension thru sterile
absorbent cotton to remove hyphal elements. Est. density of
conidial suspension by counting in hemacytometer and store
at 2-10° as stock spore suspension (125-155 X 10 6 conidia/
mL) for <4 weeks for use in prepg test suspensions of conidia.
Stdze test conidial suspensions as needed by dilg stock spore
suspension with physiological NaCl soln so that it contains 5
x 10 6 conidia/mL.
E. Operating Technic
Prep, dilns of fungicide. (Tests are similar to those de-
scribed in 955. 11C.) Place 5 mL of each fungicide soln and
of phenol control solns in 25 X 150 mm test-culture tubes,
arrange in order of ascending dilns, place tubes in 20° H 2
bath, and let come to temp. With graduated pipet, place 0.5
mL spore suspension in first tube of fungicidal soln, shake,
and immediately replace in H 2 bath; 30 sec later add 0.5 mL
conidial suspension to second tube. Repeat at 30 sec intervals
for each fungicidal diln. If more convenient, run test at 20 sec
intervals. After 5, 10, and 15 min exposure to fungicide, re-
move sample from each conidia-fungicide mixt. with 4 mm
loop and place in 10 mL glucose broth, 955. 17B. To eliminate
risk of faulty results due to possible fungistatic action, make
subtransfers from the initial glucose broth subculture tubes to
fresh tubes of glucose broth, using the 4 mm loop before in-
cubation, or make initial subcultures in glucose broth contg
either 0.05% Na thioglycolate, 1.5% isooctylphenoxy-poly-
ethoxy-ethanol, or mixt. of 0.07% lecithin (Asolectin, Asso-
ciated Concentrates, Inc., 32-34 61st St, Woodside, NY 11377)
and 0.5% polysorbate 80 (Tween 80), whichever gives lowest
result. Incubate inoculated tubes at 25-30°. Read final results
after 10 days, altho indicative reading can be made in 4 days.
Note: Highest diln that kills spores within 10 min is com-
monly considered as highest diln that could be expected to
disinfect inanimate surfaces contaminated with pathogenic fungi.
Refs.: Arch. Dermatol. Syphilol. 28, 15(1933). J. Bacterid.
42, 225(1941); 47, 102(1944). JAOAC 37, 616(1954);
38, 274(1955); 56, 308(1973).
960.09 Germicidal and Detergent
Sanitizing Action of Disinfectants
Final Action
(Suitable for detg min. concn of chem. that can be permitted
for use in sanitizing prec leaned, nonporous food contact sur-
faces. Min. recommended starting concn is 2-4 x this concn.
Test also dets max. water hardness for claimed conens. As
control, check accuracy of hard-water tolerance results with
pure C14 alkyl dimethyl benzyl NH 4 chloride at 700 and 900
ppm hardness, and pure C 16 alkyl dimethyl benzyl NH 4 chlo-
ride (Cetalkonium Chloride, at 400 and 550 ppm hardness,
expressed as CaC0 3 .)
A. Reagents
(a) Culture media. — (/) Nutrient agar A. — Boil 3 g beef
ext, 5 g peptone (Bacto, from Difco or equiv.; special grades
must not be used), and 15 g salt- free agar in 1 L H 2 0. Do not
use premixed, dehydrated media. Tube, and autoclave 20 min
at 121°. Use for daily transfer of test culture. (2) Nutrient agar
B. — Prep, as above but use 30 g agar. Use for growing test
cultures in French square bottles. (3) Nutrient agar {AOAC), —
See 955.11A(c). Use for prepg stock culture slants.
(b) Subculture media. — (J) Use tryptone glucose ext agar
(Difco), adding 25 mL stock neutralize^ (c)/L. (2) Tryptone
glucose ext agar (Difco).
AOAC Official Methods of Analysis (1990)
Sanitizing Action
139
Table 960.09A Percent Light Transmission at Various Wavelengths Corresponding to Bacterial Concentrations
%
Light Transmission with Filters,
nm
Av. Bacteria]
370
420
490
530
550
580
650
Count/mL
7.0
4.0
6.0
6.0
6.0
7.0
8.0
13.0 x 10 9
8.0
5.0
7.0
7.0
7.0
8.0
9.0
11.5
9.0
6.0
8.0
8.0
8.0
9.0
10.0
10.2
10.0
7.0
9.0
9.0
9.0
11.0
11.0
8.6
11.0
8.0
10.0
10.0
10.0
12.0
13.0
7.7
13.0
9.0
12.0
12.0
12.0
13.0
15.0
6.7
(c) Neutralizer stock soln. —Mix 40 g Asolectin (Associ-
ated Concentrates, 32-34 61st St, Woodside, NY 11377), 280
mL polysorbate 80, and 1.25 mL phosphate buffer, (e); dil.
with H 2 to 1 L and adjust to pH 7.2. Dispense in 100 mL
portions and autoclave 20 min at 121°.
(d) Neutralizer blanks. — For use with <200 ppm quater-
nary NH 4 compd. Mix 100 mL neutralizer stock soln, (c), 25
mL 0.25M phosphate buffer stock soln, (e), and 1675 mL H 2 CX
Dispense 9 mL portions into 20 x 150 mm tubes. Autoclave
20 min at 121°.
(e) Phosphate buffer stock soln. — 0.25M. Dissolve 34.0 g
KH 2 P0 4 in 500 m'L H 2 0, adjust to pH 7.2 with IN NaOH,
and dil. to 1 L.
(f) Phosphate buffer dilution water. — Add 1 .25 ml 0.25M
phosphate buffer stock soln, (e), to 1 L H 2 and dispense in
99 mL portions. Autoclave 20 min at 121°.
(g) Test organisms. — Use Escherichia coll ATCC No. 1 1229
or Staphylococcus aureus ATCC 6538. Incubate 24 and 48 hr,
resp. Maintain stock cultures on nutrient agar (AOAC), (a)(5),
at refrigerator temp.
B. Resistance to Phenol of Test Cultures
Det. resistance to phenol at least every 3 months by 955.11.
Resistance of E. coli should be equiv. to that specified for S.
typhi in 955.1 ID and that for Staph, aureus equiv. to that
specified for this organism in 955.12.
C. Apparatus
(a) Glassware. — 250 mL wide-mouth erlenmeyers; 100 mL
graduate; Mohr, serological, and/or bacteriological (APHA
specification) pipets; 20 x 150 mm test tubes. Sterilize at 180°
in hot air oven ^2 hr.
(b) Petri dishes. — Sterile.
(c) French square bottles. — 175 mL, borosilicate (Thomas
Scientific). Use of other containers will give variable results.
(d) Water bath.— Controlled at 25°.
D. Preparation of Culture Suspension
From stock culture inoculate tube of nutrient agar A,
960.09A(a)(/), and make >3 consecutive daily transfers (<30),
incubating transfers 20-24 hr at 35-37°. Do not use transfers
>30 days. If only 1 daily transfer has been missed, no special
procedures are required; if 2 daily transfers are missed, repeat
with 3 daily transfers.
Prep. 175 mL French square culture bottles contg 20 mL
nutrient agarB, 960.09 A(a)(2), autoclave 20 min at 121°, and
let solidify with bottle in horizontal position. Inoculate culture
bottles by washing growth from slant with 5 mL phosphate
buffer diln H 2 0, 960.09A(f ), into 99 mL phosphate buffer diln
H 2 0, and adding 2 mL of this suspension to each culture bot-
tle, tilting back and forth to distribute suspension; then drain
excess liq. Incubate 18-24 hr at 35-37°, agar side down. Re-
move culture from agar surface of 4 or more bottles, using 3
mL phosphate buffer diln H 2 and glass beads in each bottle
to suspend growth. Filter suspension thru Whatman No. 2 pa-
per pre wet with 1 mL sterile phosphate buffer, and collect in
sterile tube. (To hasten filtration, rub paper gently with sterile
policeman.) Stdze suspension to give av. of 10 x 10 9 organ-
isms/mL by diln with sterile phosphate buffer diln H 2 0,
960.09A(f).
If Lumetron colorimeter is used, dil. suspension in sterile
Lumetron tube to give % T according to Table 960. 09 A.
If McFarland nephelometer and BaS0 4 stds are used, select
7 tubes of same id as that contg test culture suspension. Place
10 mL of each suspension of BaS0 4 , prepd as indicated in
Table 960.09B, in each tube and seal tube. Stdze suspension
to correspond to No. 4 std.
E. Synthetic Hard Water
Prep. Soln J by dissolving 31.74 g MgCl 2 (or equiv. of hy-
drates) and 73.99 g CaCl 2 in boiled distd H 2 and dilg to 1
L. Prep. Soln 2 by dissolving 56.03 g NaHC0 3 in boiled distd
H 2 and dilg to 1 L. Soln 1 may be heat sterilized; Soln 2
must be sterilized by filtration. Place required amt Soln 1 in
sterile 1 L flask and add >600 mL sterile distd H 2 0; then add
4 mL Soln 2 and dil. to 1 L with sterile distd H 2 0. Each mL
Soln 1 will give a water equiv. to ca 100 ppm of hardness
calcd as CaC0 3 by formula:
Total hardness as ppm CaC0 3
= 2.495 x ppm Ca + 4.115 x ppm Mg
pH of all test waters <2000 ppm hardness should be 7.6-
8.0. Check prepd synthetic waters chemically for hardness at
time of tests, using following method or other methods de-
scribed in 14th ed. of Standard Methods for the Examination
of Water, Sewage, and Industrial Wastes.
F. Hardness Method
(a) EDTA std soln.— Dissolve 4.0 g Na 2 H 2 EDTA.2H 2 and
0.10 g MgCl 2 .6H 2 in 800 mL H 2 and adjust by subsequent
diln so that 1 mL of soln is equiv. to 1 mg CaC0 3 when titrd
as in (c). Check EDTA soln after prepn or, if com. purchased,
against CaC0 3 std at least every 2 months.
(b) Calcium std soln. — 1 mL = 1 mg CaC0 3 . Weigh 1.00
g CaC0 3 , dried overnight or longer at 105°, into 500 mL er-
lenmeyer and add dil. HC1 thru funnel until CaCO^ is dis-
solved. Add 200 mL H 2 0, boil to expel C0 2 , and cool. Add
few drops Me red indicator and adjust color to intermediate
orange with dil. NH 4 0H or HC1 as required. Transfer quant,
to 1 L vol. flask and dil. to vol.
(c) Determination. — Dil. 5-25 mL sample (depending on
hardness) to 50 mL with H 2 in erlenmeyer or casserole. Add
1 mL buffer soln (67.5 g NH 4 C1 and 570 mL NH 4 OH dild to
Table 960. 09B Preparation of BaS0 4 Suspensions Corre-
sponding to Bacterial Concentrations
2% BaCI 2
1% H 2 S0 4
Av. Bacterial
Std No.
Soln, mL
(v/v) Soln, mL
Count/mL
1
4.0
96.0
5.0 x 10 9
2
5.0
95.0
7.5
3
6.0
94.0
8.5
4
7.0
93.0
10.0
5
8.0
92.0
12.0
6
10.0
90.0
13.5
7
12.0
88.0
15.0
140
Disinfectants
AOAC Official Methods of Analysis (1990)
1 L with H 2 0), 1 mL inhibitor (5.0 g Na 2 S.9H 2 or 3.7 g
Na 2 S.5H 2 dissolved in 100 mL H 2 0), and 1 or 2 drops in-
dicator soln (0.5 g Chrome Black T in 100 mL 60-80% al-
cohol). Titr. with EDTA std soln slowly, stirring continuously,
until last reddish tinge disappears from soln, adding last few
drops at 3-5 sec intervals.
Hardness as mg CaC0 3 /L
= (mL std soln x 1000)/mL sample
G. Preparation of Samples
Use composition declared or detd as guide to sample wt re-
quired for vol. sterile H 2 used to prep. 20,000 ppm soln.
From this stock diln, transfer 1 mL into 99 mL of the water
to be used in test to give concn of 200 ppm. In making trans-
fer, fill 1 mL pipet and drain back into stock soln; then refill,
to correct for adsorption on glass. After mixing, discard 1 mL
to provide 99 mL of the test water in 960.09H.
H. Operating Technic
Measure 99 mL water to be used in test ? contg bactericide
at concn to be tested, into chem. clean, sterile, 250 mL wide-
mouth erlenmeyer and place in const temp, bath until it reaches
25°, or >20 min. Prep, duplicate flasks for each germicide to
be tested. Also prep, similar flask contg 99 mL sterile phos-
phate buffer diln H 2 0, 960.09A(f), as "initial numbers" con-
trol.
Add 1 mL culture suspension to each test flask as follows:
Whirl flask, stopping just before suspension is added, creating
enough residual motion of liq. to prevent pooling of suspen-
sion at point of contact with test water. Add suspension mid-
way between center and edge of surface with tip of pipet slightly
immersed in test soln. Avoid touching pipet to neck or side of
flask during addn. Transfer 1 mL portions of this exposed cul-
ture to neutralizer blanks exactly 30 and 60 sec after addn of
suspension. Mix well immediately after transfer.
For "numbers control" transfer, add 1 mL culture suspen-
sion to 99 mL sterile phosphate diln H 2 in same manner. In
case of numbers control, plants need be made only immedi-
ately after adding and mixing thoroly <30 sec. (It is advan-
tageous to use milk pipets to add culture and withdraw sam-
ples.)
Plate from neutralizer tube to agar, using subculture medium
960.09A(b)(7) for quaternary NH 4 compds and 960.09A(b)(2)
with numbers control. Where 0.1 mL portions are planted, use
1 mL pipet graduated in 0.1 mL intervals. For dilns to give
countable plates, use phosphate buffer diln H 2 0, 960.09A(f).
For numbers control, use following diln procedure: Transfer 1
mL exposed culture (1 mL culture suspension transferred to
99 mL phosphate buffer diln H 2 in H 2 bath) to 99 mL phos-
phate buffer diln H 2 0, 960.09A(f), (diln J). Shake thoroly and
transfer 1 mL diln 1 to 99 mL phosphate buffer diln H 2 0,
960.09A(f), (diln 2). Shake thoroly and transfer 1 mL diln 2
to 99 mL phosphate buffer diln H 2 (diln 3). Shake thoroly
and transfer four 1 mL and four 0. 1 mL aliquots from diln 3
to individual sterile petri dishes.
For test samples, use following diln procedure: Transfer 1
mL exposed culture into 9 mL neutralizer, 960.09A(d). Shake
and transfer four 1 mL and four 0. 1 mL aliquots to individual
sterile petri dishes. For numbers control, use subculture me-
dium 960.09 A(b)(2); for tests with quaternary NH 4 compds,
use medium 960.09A(b)(7). Cool agar to solidify, and then
invert and incubate 48 hr at 35° before counting.
/. Results
To be considered valid, results must meet std effectiveness:
99.999% reduction in count of number of organisms within 30
sec. Report results according to actual count and % reduction
over numbers control. Counts on numbers control for germi-
cide test mixt. should fall between 75 and 125 X 10 6 /mL for
% reductions to be considered valid.
J. Sterility Controls
(a) Neutralizer. — Plate 1 mL from previously unopened tube.
(b) Water. — Plate 1 mL from each type of water used.
(c) Sterile distilled water. —Plate 1 mL.
After counting plates, confirm that surviving organisms are
E. coli by transfer to brilliant green bile broth fermentation
tubes or lactose broth and EMB agar; confirm Staph, aureus
by microscopic examination.
Refs.: Am. J. Public Health 38, 1405(1948). J. Milk Food
Technol. 19, 183(1956). Fed. Regist. 21, 7020(1956).
JAOAC 41, 541(1958); 56, 308(1973).
961.02 Germicidal Spray Products
as Disinfectants
First Action 1961
Final Action 1964
(Suitable for detg effectiveness of sprays and pressurized spray
products as spot disinfectants for contaminated surfaces)
A. Reagents
Use culture media and reagents specified in 955. 11 A,
955.11B(e) and (f), and 955. 14A except that test organism
Salmonella typhi is not used.
Use as test organisms Trichophyton mentagrophytes ATCC
No. 9533, prepd as in 955.17D, to which has been added 0.02
mL octyl-phenoxy-polyethoxy-ethanol (Triton X 100, Rohm &
Haas Co.)/ 10 mL suspension to facilitate spreading, Salmo-
nella choleraesuis ATCC No. 10708, 955.14A(b), Staphylo-
coccus aureus ATCC No. 6538, maintained as in 955.14A(b),
and Pseudomonas aeruginosa. ATCC No. 15442, maintained
as in 964.02.
B. Apparatus
Use app. specified in 955. 11B and 955. 14B, and in addn:
(a) Capillary pipets. — 0.1 mL, graduated to deliver 0.01
mL. Sterilize in air oven 2 hr at 180°.
(b) Microscope slides. — Non-corrosive, 25 x 25 mm (1 x
1"), or 18 X 36 mm glass slide. Sterilize by placing individual
slides in petri dish matted with 2 pieces 9 cm filter paper
(Whatman No. 2, or equiv.) in air oven 2 hr at 180°.
(c) Bacteriological culture tubes. — Pyrex, 32 X 200 mm.
(d) Metal forceps. — Sharp points, straight, 115 mm long.
C. Operating Technic
Thoroly shake 48 hr nutrient broth cultures of S. choler-
aesuis and Staph, aureus and let settle 10 min. With sterile
capillary pipet or sterile 4.0 mm loop, transfer 0.01 mL culture
onto 1 sq in. sterile test slide in petri dish and immediately
spread uniformly over entire area. Cover dish immediately and
repeat operation until 12 slides have been prepd for each or-
ganism. (Use 2 slides as control.) Dry all slides 30-40 min at
37°.
Spray 10 slides for specified time and distance. Hold each
slide 10 min, drain off excess liq., and transfer slide to indi-
vidual 32 X 200 mm tube contg 20 mL appropriate subculture
medium, 955.11A(d), with flamed forceps. Shake culture tho-
roly. If broth appears cloudy after 30 min, make subculture to
fresh individual tubes of subculture broth. Transfer 2 un-
sprayed slides, as viability controls, to individual subculture
tubes in same manner.
Incubate all tubes used for primary and secondary transfers
AOAC Official Methods of Analysis (1990)
Sporicidal Activity
141
48 hr at 37°. Read as + (growth) or - (no growth). Killing
of test organisms in 10 of 10 trials is presumptive evidence of
disinfecting action.
Det. resistance of S. choleraesuis as in 955. 11C; with S.
aureus as in 955.12; with Ps. aeruginosa as in 955.13; and
with T. mentagrophytes as in 955. 17 A.
If there is reason to believe that lack of growth in subtransfer
tubes is due to bacteriostasis, inoculate all incubated subcul-
ture tubes with loop needle inoculation of respective test cul-
ture and reincubate. Growth of these inocula eliminates bac-
teriostasis as cause of lack of growth. If there is question as
to possibility of contamination as source of growth in subcul-
ture tubes, make gram stains and/or subculture for identifi-
cation, according to respective test culture.
If fungicidal activity as well as germicidal activity is in-
volved, use test suspension of 7\ mentagrophytes spores,
955. 17D, and prep. 1.2 slides, using 0.01 mL std spore sus-
pension, spraying and subculturing exactly as above. Make
subcultures in glucose broth, 955. 17B, incubating 7 days at
25-30°.
Refs.: JAOAC 44, 422(1961); 50, 763(1967). Soap Chem.
Spec. 38(2), 69(1962); 61, 400(1978).
966.04 Sporicidal Activity of Disinfectants
First Action 1966
Final Action 1967
(Suitable for detg sporicidal activity of liq. and gaseous chems.
Applicable to germicides for detg presence or absence of spo-
ricidal activity against specified spore-forming bacteria in var-
ious situations and potential efficacy as sterilizing agent.)
A. Reagents
(a) Culture media. — (7) Soil extract nutrient broth. — Ext
1 lb garden soil in 1 L H 2 0, filter several times thru S&S No.
588 paper, and dil. to vol. (pH should be >5.2). Add 5 g beef
ext. (Difco), 5 g NaCl, and 10 g peptone (Anatone,
955.11 A(a)). Boil 20 min, dil. to vol., adjust with 17V NaOH
to pH 6.9, and filter thru paper. Dispense in 10 mL portions
into 25 x 150 mm tubes, and autoclave 20 min at 121°. Use
this broth to propagate test culture of Bacilli.
(2) Nutrient agar. — See 955.11A(c). Use slants of this me-
dium to maintain stock culture of Bacilli.
(3) Modified fluid thioglycolate medium USP. — Prep, as in
955.11A(d)(2), except add 20 mL 17V NaOH to each L before
dispensing for sterilization. Use this medium to subculture spores
exposed to 2.5N HC1. For spores exposed to unknown ger-
micides, use fluid thioglycolate medium, 955.11A(d)(2).
(4) Soil extract-egg-meat medium.. — Add 1.5 g Bacto Egg-
Meat Medium dehydrated (Difco No. 0042-17) to 25 X 150
mm tube; then add 15 mL garden soil ext, (7), and sterilize
20 min at 121°. Use this medium to propagate test cultures of
Clostridia and maintain stock cultures of species of this genus.
(b) Test organisms. — Use Bacillus subtilis, ATCC No.
19659, or Clostridium sporo genes y ATCC No. 3584, for rou-
tine evaluation. Method is also applicable for use with strains
of B. anthracis, CI. tetani, or other spore forming species.
(c) Dilute hydrochloric acid. — 2.57V. Use to det. resistance
of dried spores. Stdze and adjust to 2.57V as in 936. 15B.
B. Apparatus
(a) Glassware. — Bacteriological culture tubes, unflared, 25
x 150 mm; 100 mL g-s cylinders graduated in 1 mL divisions;
65 mm id funnels; supply of 15 x 110 mm petri dishes matted
with 2 sheets 9 cm S&S No. 597 or Whatman No. 2 filter
paper. Sterilize all glassware and matted petri dishes 2 hr in
air oven at 180°.
(b) Water bath.— See 955.11B(b).
(c) Racks.— See 955.116(c).
(d) Transfer loop, hook, and forceps. — See 955.14B(c).
(e) Tissue grinder. — Thomas Scientific, No. 3431-E20, Size
B, or equiv.
(f ) Suture loop carrier. — From spool of size 3 surgical silk
suture (silk black braid A-59, USP, Ethicon, Inc., Rte 22,
Sommerville, NJ 08876), prep, std loops by wrapping the silk
around ordinary pencil 3 times, slipping coil so formed off end
of pencil, and holding it firmly with thumb and index finger
of left hand while passing another piece of suture through coil,
knotting, and tying securely. Then shear off end of coil and
knotted suture to within 2 mm. This should provide overall
length of ca 65 mm of suture in 2-loop coil that can be con-
veniently handled in ordinary aseptic transfer procedure.
Ext loops in groups of 100-200 in Soxhlet extn app., using
CHC1 3 , for 24 h. Air-dry 12-18 h at room temp, in hood.
Place 100 loops in 100 mL 0.5N HC1 for 10 min or until all
loops are completely submerged in soln. Decant, and rinse re-
peatedly with distd H 2 for 15 min. Check rinse H 2 for ab-
sence of HO, using litmus paper. Air-dry on filter paper mats
under ambient conditions or in incubator.
(g) Cylinder carriers . — " Penicy finders , M porcel ain , 8 ± 1 mm
od, 6±1 mm id, 10± I mm long. (Available from Fisher Sci-
entific Co., No. 7-907.) Sterilize 2 hr in 180° air oven. Wash
used Penicylinders with Triton X-100 and rinse with H 2 4
times.
C. Operating Technic
Grow all Bacilli in soil ext nutrient broth and all Clostridia
in soil ext- meat-egg medium. Inoculate 3 tubes, using 1 loop
stock culture, and incubate 72 hr at 37°. Place supply of suture
loops and cylinder carriers in sep. petri dishes matted with
filter paper, and sterilize 20 min at 121°. Use new loops for
each test. Penicylinders must be free from chips or cracks.
Filter CI. sporogenes thru funnel contg 2x5x5 cm sq piece
of moist cotton or glass wool into sterile 25 x 150 mm test
tubes, using same funnel. In prepg B. subtilis culture, pour
tube of 72 hr culture into tissue grinder and macerate to break
up pellicle. Filter thru sterile funnel contg moist cotton or glass
wool into sterile 25 x 150 mm tube, repeating operation for
other 2 tubes. Place 10 sterile suture loops or Penicylinders
into each of 3 tubes contg 10 mL filtrate from 72 hr culture
of CI. sporogenes, agitate, and let stand 10-15 min. Using
this technic, contaminate 35 loops or cylinders. Place contam-
inated suture loops and /or cylinders into petri dish matted with
2 layers of filter paper. Drain. Proceed similarly for B. sub-
tilis .
Place the 35 suture loops or cylinders contaminated with CI.
sporogenes or B. subtilis in vac. desiccator contg CaCl 2 and
draw vac. of 69 cm (27") Hg for 20 min. Dry 24 hr under vac.
(Spores dried and held under these conditions will retain re-
sistance >1 days.)
Transfer 10 mL 2.5AT HC1, 966.04A(c), into sterile 25 x
150 mm tube. Place tube in 20° const temp. H 2 bath and let
come to temp. Rapidly transfer 4 dried, contaminated loop or
cylinder carriers to acid tube. Transfer remaining dried, con-
taminated suture loop or cylinder carriers to tube of thiogly-
colate subculture medium, 966.04A(a)(J), as viability control.
After 2, 5, 10, and 20 min, withdraw individual loops or cyl-
inders from acid and transfer to individual tubes of subculture
medium. Rotate each tube vigorously 20 sec and resubtransfer.
Incubate 21 days at 37°. Test spores should resist HC1 ^2 min,
and many may resist HC1 for full 20 min.
When testing sporicidal or sterilizing activity of gas, place
142
Disinfectants
AOAC Official Methods of Analysis (1990)
carriers in polyethylene bags or in petri dishes with lids ajar.
Certain gases may require rehydration of spores before ex-
posure to gas. Rehydrate spores on carriers by 1 hr immersion
in H 2 0, using <20 mL H 2 0/6 carriers. Drain carriers 20 min
on petri dishes matted with filter paper. After exposure to gas,
remove carriers, using aseptic technic to subculture media as
specified in next par.
For aq. sporicides and sterilizers, place 10 mL product at
diln recommended for use or under investigation into each of
six 25 x 150 mm tubes. Place tubes in 20° H 2 bath and let
come to temp. Using flamed forceps, place 5 suture loops or
cylinders, contaminated with CI. sporogenes or 5. subtilis and
dried 24 hr under vac, into each of the 6 tubes contg disin-
fectant, using 2-min intervals for seeding each tube. Five su-
ture loops or cylinders can be placed into each tube within 5
sec. This seeding operation will take 10 min. After contact
period specified for disinfectant has been achieved, remove
suture loops or cylinders, using sterilized needle hook, from
each tube of disinfectant to subculture medium or other sub-
culture medium specified in 955. 11 A(d) (select medium contg
most suitable neutralizer), placing 1 suture loop or cylinder per
tube. Five cylinders can be removed within each 2 min inter-
val. Flame transfer needle hook after each carrier has been
transferred to subculture medium. After completing transfer,
resubtransfer each suture loop or cylinder to fresh tube of
thioglycolate medium and incubate 21 days at 37°. If no growth
is observed after 21 days, heat-shock tubes 20 min at 80° and
reincubate 72 hr at 37°. Report results as + (growth) or — (no
growth) values.
Killing in 59 of 60 replicates on 1 carrier at diln and time
specified is considered evidence of sporicidal efficacy against
1 test spore and for confidence level of 95%. Tests with both
B. subtilis and CI. sporogenes, using 30 replicates with each
of 2 carriers specified to provide min. of 120 carriers, are re-
quired to presumptively support unqualified sporicidal claim
or for presumptive evidence of sterilizing activity at concn,
time, and conditions specified. For sporicidal claims, no more
than 2 failures can be tolerated in this 120 carrier trial. For
sterilizing claims, no failures can be tolerated.
Refs.: JAOAC 36, 480(1953); 39, 480(1956); 40, 759(1957);
49, 721(1966); 50, 194(1967); 56, 308(1973); 61,
371(1978); 68, 279(1985).
965.12 Tuberculocidal Activity
of Disinfectants
First Action 1965
Final Action 1967
Repealed First Action 1988
(Suitable for detg max. tuberculocidal diln of disinfectants used
on inanimate surfaces. This method has not been validated for
glutaraldehyde-based products)
/. Presumptive In Vitro Screening Test Using
Mycobacterium smegmatis
A. Reagents
(a) Test organism. — Mycobacterium smegmatis (PRD No.
1) (available from Microbiology Lab., U.S. Environmental
Protection Agency, Benefits & Use Div., Bldg 306, BARC-
East, Beltsville, MD 20705). Maintain on nutrient agar slants
by monthly transfers. Incubate new stock transfer 2 days at
37°; then store at 2-5°. From stock culture inoculate tubes of
Proskauer-Beck broth, (b)(7), incubate 48 hr in slanting po-
sition, carry 30 days, using 48 hr transfers, and use these 48
hr cultures to start test cultures. Inoculate 1 or 2 tubes of Pros-
kauer-Beck broth. Incubate 6-7 days at 37°. Incubate tubes 48
hr in slanting position to provide max. surface aeration and
then in upright position 4-5 days. Add 1.5 mL sterile 2.0%
Bacto-Gelatin soln and homogenize culture with sterilized glass
tissue grinder, 966.046(e). Adjust to 20% T at 650 nm with
sterile Proskauer-Beck broth for use in testing.
(b) Culture media. — (7) Modified Proskauer-Beck broth. —
Dissolve 2.5 g KH 2 P0 4 , 5.0 g asparagine, 0.6 g MgS0 4 .7H 2 0,
2.5 g Mg citrate, 20.0 mL glycerol, 0.0046 g FeCl 3 , and 0.001
g ZnS0 4 .7H 2 in 1 L H 2 0. Adjust to pH 7.2-7.4 with IN
NaOH. Filter thru paper, place 10 mL portions in sep. 20 x
150 mm tubes, and sterilize 20 min at 121°, Use for propa-
gating 48 hr test starter cultures and 6-7 day test cultures.
(2) Subculture media. — Use (7) with addn of suitable neutzg
agents such as purified lecithin (Azolectin) or Na thioglyco-
late, where necessary.
(3) Nutrient agar. — Prep, as in 955.11A(c). Use to main-
tain stock culture.
(4) Sterile distilled water.— See 955.14A(d).
B. Apparatus
(a) Glassware, water bath, transfer loops and needles, and
petri dishes. —See 955.14B(a), (b), (c), and (e).
(b) Carriers. —See 966.04B(g).
C. Operating Technic
Transfer 20 sterile Penicylinder carriers, using flamed ni-
chrome wire hook, into 20 mL 6-7 day homogenized stdzd
broth culture, 965.12A(a), in sterile 25 x 150 mm medicant
tube. After 15 min contact, remove cylinders and place on end
in vertical position in sterile petri dish matted with filter paper,
955.14B(e). Cover and place in incubator at 37° and let dry
>20 min but <60 min. This will provide dried test carriers in
groups of 20 in individual petri dishes. With each group of 20
carriers, add 1 dried cylinder at 30 sec intervals to each of 20
tubes contg 10 mL diln of germicide to be tested (at 20° in
FLO bath). Flame lips of medicant and subculture tubes. Im-
mediately after placing carrier in medicant tube, swirl tube 3
times before placing it back in H 2 bath. (Thus, by time 20
tubes have been seeded, 9 min and 30 sec have elapsed, leav-
ing 30 sec interval prior to subculturing series at 10 min ex-
posure for each carrier. The 30 sec interval between transfers
allows adequate time for flaming and cooling transfer hook
and making transfer in manner so as to drain all excess med-
icant from carrier.) Transfer carrier to 10 mL subculture me-
dia, 965.12A(b)(2). Shake all subculture tubes thoroly and in-
cubate 12 days at 37°. Report results as + (growth) or — (no
growth). Where there is reason to suspect that results may be
affected by bacteriostatic action of medicant carried over in
subculture tubes, use suitable neutralizer in subculture media.
Make >30 carrier exposures at each of 3 relatively widely
spaced dilns of germicide under test between no response and
total response diln levels. Calc. % of carriers on which or-
ganism is killed at each diln. Using log % probit paper (3 cycle
logarithmic normal No. Y3 213 HG, Codex Book Co., Inc.,
74 Broadway, Norwood, MA 02062), locate % kill points on
diln lines employed (log scale). Draw best fitting straight line
thru these 3 points and extend to intercept 99% kill line. Read
diln line (log scale) at point of intercept. This is presumed 95%
confidence end point for product. (Do not use presumptive test
organism for checking validity of this presumptive end point.)
//. Confirmative In Vitro Test for Determining
Tuberculocidal Activity — First Action 1965
D. Reagents
(a) Culture media. — (7) Modified Proskauer-Beck me-
dium.. — Prep, as in 966. 12 A (b)(7), and in addn, place 20 mL
AOAC Official Methods of Analysis (1990)
TUBERCULOCIDAL ACTIVITY
143
portions in 25 x 150 mm tubes. Use 10 mL portions for daily
transfers of test cultures and 20 mL portions for subculturing
porcelain cylinders.
(2) Middlebrook 7H9 Broth Difco A.— Dissolve 4.7 g in
900 mL H 2 contg 2 mL glycerol and 15.0 g agar. Heat to
bp to dissolve completely. Distribute in 180 mL portions and
autoclave 15 min at 121°, To each 180 mL sterile medium at
45°, add 20 mL Middlebrook ADC Enrichment (Difco) under
aseptic conditions and distribute in 10 mL portions in sterile
20 X 150 mm tubes. Slant. Use to maintain test culture.
(3) Middlebrook 7H9 Broth Difco B.— Dissolve 4.7 g in
900 mL H 2 contg 2 mL glycerol and 1.0 g agar. Heat to bp
to dissolve completely. Distribute in 18 mL portions in 25 x
150 mm tubes, and autoclave 15 min at 121°. To each 18 mL
sterile medium at 45° add 2 mL Middlebrook ADC Enrichment
under aseptic conditions. Use to subculture for survival.
(4) Kirchners Medium Difco. — Dissolve 13.1 g in 1 L H 2
contg 20 mL glycerol and heat to bp to dissolve completely.
Distribute in 18 mL portions in 25 x 150 mm tubes and au-
toclave 15 min at 121°. If commercial medium is not available,
add 5 g asparagine (Difco), 2.5 g Na citrate, 0.6 g MgS0 4 ,
2.5 g monopotassium phosphate, 1.5 g dipotassium phosphate,
1 g Bacto agar in 1 L H 2 0, and add 20 mL glycerol. To each
1 8 mL sterile medium at 50-55° add 2 mL Middlebrook ADC
Enrichment under aseptic conditions. Use to subculture for
survival.
(5) TB Broth Base Difco {without Polysorbate 80). — Dis-
solve 11.6 g in I L H 2 contg 50 mL glycerol and 1.0 g agar.
Heat to bp to dissolve completely. Distribute in 18 mL por-
tions in 25 X 150 mm tubes, and autoclave 15 min at 121°.
To each 18 mL sterile medium at 50° add 2 mL Dubos Me-
dium Serum (Difco) under aseptic conditions. Use to subcul-
ture for survival.
(b) Test organism. — Mycobacterium bovis (BCG) (Bionet-
ics Research, Inc., 115 S Sangamon St, Chicago, IL 60607).
Maintain stock cultures on culture medium (a)(2) by monthly
or 6 weeks transfer. Incubate new stock transfer 15-20 days
at 37° until sufficient growth is indicated; then store at 2-5°.
From stock culture, inoculate tube of culture medium (a)(7)
and incubate 21-25 days at 37°. Allow to remain quiescent
until 21 -25th day. Make daily transfers from 21 day cultures.
Transfer culture to heat-sterilized glass tissue grinder, add 1.0
mL 0.1% Tween 80 in saline soln (Difco), grind, and dil. with
culture medium (a)(7) to give 20% T at 650 nm. Use to in-
oculate porcelain cylinders used in test. Tests will be satisfac-
tory only when organism is killed on all 10 carriers by aq.
phenol (1 +50) and shows survival after exposure to aq. phenol
(1 + 75) control. Prep, dilns from 5% std phenol soln,
955.116(f).
(c) Sterile distilled water. — See 955.14A(d).
(d) Normal horse serum. — Difco Laboratories.
E. Apparatus
(a) Glassware, water bath, transfer loops and needles, and
petri dishes.— See 955.14B(a), (b), (c), and (e).
(b) Carriers.— See 966.04B(g) o
F. Operating Technic
Soak ring carriers overnight in \N NaOH; rinse with tap
H 2 and then with distd H 2 until distd H 2 is neut. to phthln;
then rinse twice with distd H 2 0. Place clean ring carriers in
multiple of 10 or 20 in capped erlenmeyer or 20 x 150 mm
tubes. Autoclave 20 min at 121°, cool, and hold at room temp.
Transfer 10 sterile ring carriers, using flamed wire hook, into
enough (ca 15-20 mL) 21-25 day stdzd test culture,
965.12D(b), in 25 X 150 mm medication tube. After 15 min
contact period, remove cylinders, using flamed wire hook, and
place on end in vertical position in sterile petri dish matted
with filter paper, 955.14B(e). Cover, place in incubator at 37°,
and let dry 30 min.
Let 10 tubes contg 10 mL use-diln germicide sample to be
tested come to 20° (or desired temp., if germicide use is rec-
ommended for temp, other than room temp.) in H 2 bath and
add 1 contaminated cylinder carrier at either 30 sec or 1 min
intervals to each tube. (Thus, by time 10 tubes have been seeded,
9 min will have elapsed, plus 1 min interval before transfer of
first carrier in series to individual tube of 10 mL neutralizer
appropriate for germicide tested, or 10 mL neutralizer blank,
960.09A(d), if 1 min intervals are used. This interval is con-
stant for each tube with prescribed exposure period of 10 min.
Interval between transfers allows adequate time for flaming
and cooling wire hook and making transfer in manner so as to
drain all excess medication from carrier.) Transfer carrier to
10 mL neutralizer appropriate for germicide tested, after ex-
actly 10 min contact. Shake tube contg carrier in neutralizer
thoroly and place carrier in tube contg 20 mL broth,
965.12D(a)(7). From same tube, take 2 mL portions serum
and place in any 2 of the subculture media, 965.12D(a)(J),
(4), (5). Repeat this with each of the 10 carriers. Incubate 1
tube of each subculture medium with 2 mL sterile serum as
control. Where there is reason to suspect that germicide to be
tested may possess bacteriostatic action, use suitable neutral-
izer in lieu of serum. Shake each subculture tube thoroly, in-
cubate 60 days at 37°, and report results as + (growth) or -
(no growth). If no growth or only occasional growth is ob-
served in subculture, incubate addnl 30 days before making
final reading. Max. diln of germicide which kills test organism
on the 10 carriers, and no growth in each of the 2 mL aliquots
for 2 extra media, represents max. safe use-diln for practical
tuberculocidal disinfection.
Refs.: JAOAC 48, 635(1965); 50, 767(1967); 53, 860(1970);
70, 318(1987).
972.04 Bacteriostatic Activity of Laundry
Additive Disinfectants
First Action 1972
(Applicable to antimicrobial products, recommended for use
during laundering operations, which are intended to provide
residual bacteriostatic treatment to laundered fabric. Method
includes treatment of fabric with product and subsequent bac-
teriostatic testing of treated fabric.)
A. Reagents
(a) Culture media. — (/) Nutrient broth. — See 955.11A(a).
(2) Nutrient agar A. — See 955.11A(c). Use for monthly
transfer of stock cultures.
(3) Nutrient agar B. — Boil 3 g beef ext, 5 g peptone (An-
atone), 8 g NaCl, and 10 g agar (Difco) in 1 L H 2 6. Transfer
100 mL portions to erlenmeyers, and autoclave 20 min at 121°.
Use for agar plate tests to evaluate bacteriostatic activity of
treated fabric. See also (c).
(b) Test organisms. — Use Staphylococcus aureus ATCC No.
6538 and Klebsiella pneumoniae, aberrant ATCC No. 4352
(formerly Escherichia coli), and maintain as in 955.11B(e).
(c) 2,3,5-Triphenyl tetrazolium chloride. — Use as optional
biological indicator. With S. aureus, use 0.15% soln; with K.
pneumoniae, aberrant, use 0.25% soln. Autoclave each 20 min
at 121°. Apply as in 972.04D.
(d) Alkaline nonionic wetting agent. — Prep. aq. soln contg
0.5% alkyl phenol poly glycol ether wetting agent and 0.5%'
Na 2 C0 3 . Use to scour test fabric.
144
Disinfectants
AOAC Official Methods of Analysis (1990)
B. Apparatus
(a) Test fabric. — 80 x 80 thread s/sq in. plain weave cotton
print cloth, completely desized, bleached, and without bluing
or optical brighteners. Scour before use by boiling ca 300 g 1
hr in 3 L H 2 contg 1.5 g nonionic wetting agent and 1.5 g
Na 2 C0 3 . Then rinse fabric, first in boiling H 2 and then in
cold H 2 0, until all visual traces of wetting agent are removed.
Air-dry and cut into long strip 5 cm (2") wide and weighing
exactly 15 g.
(b) Stainless steel spindle. -™ Fabricate from single contin-
uous piece of stainless steel wire Vie" diam. and bent to contain
3 horizontal extensions 5 cm (2") long connected by 2 vertical
sections ca 5 cm (2") long. Shape so that vertical sections form
150° angle, and sharpen free ends of 2 outer horizontal exten-
sions to point (see Fig. 972.04). Use as carrier for test fabric.
Primary objective of spindle is to prevent wadding or lodging
of test fabric during agitation in exposures to test chem. solns.
(c) Exposure chamber. — Clean, dry 1 pt Mason jar with
rubber washer or gasket and metal screw cap.
(d) Agitator. — Device to rotate Mason jar thru 360° vertical
orbit of 10-20 cm (4-8") diam. at 45-60 rpm for 5 min. Laun-
derometer or Tumble Jar described in AATCC70 B-1967, 43,
B154, B155, or ASTM D583-63 is adequate.
(e) Water bath. — Thermostatically controlled at 25°.
(f) Petri dishes. — Sterile, 100 X 15 mm.
(g) Glassware.— See 955.11B(a).
(h) Transfer loops and needles. — See 955.11B(d).
C. Preparation of Fabric
(a) Fabric mounting. — Pierce one end of prescoured, 15 g
test fabric strip and secure onto an outer horizontal extension
of test spindle; then wind strip around 3 horizontal extensions
with enough tension to obtain 12 (but not 13) entire laps. Se-
cure final end of test fabric strip to previous laps with stainless
steel safety pin.
(b) Fabric treatment with product. — Dil. product as di-
rected to 75 mL (most frequently, use directions are based on
dry wt of laundry fabric equiv. to 15 g test fabric), add to
Mason jar (exposure chamber), and maintain in H 2 bath at
25°. Add addnl materials to Mason jar as required by use di-
rections for product. These are:
(J) Product recommended as final rinse additive in indus-
trial laundering operation. — Add no addnl materials; 5:1
(v/w) treatment product soln to dry fabric ratio is represen-
tative of industrial laundering operations.
Outer Extension
zz*
Horizontal
Extensions
5/16"
2~
o
Side
View
Horizontal
Extensions
Outer Extension
:>* _L
* Points
pencil
sharp or
canted
FIG. 972.04 — Stainless steel spindle for winding test fabric
(2) Product recommended as final rinse additive in home
or coin-operated laundering operations. — Add 150 mL H 2
to Mason jar. Resultant 10:1 (v/w) treatment product soln to
dry fabric ratio is representative of home and coin -operated
laundering operations.
(3) Product recommended as final rinse additive in both in-
dustrial and home laundering operations. — Prep. 2 jars contg
product soln according to (/) and (2) so that 2 test fabric strips
may be treated at different treatment product soln to dry fabric
ratios (5:1 and 10:1 (v/w)).
(4) Product recommended as final rinse additive and de-
scribed as compatible with adjunct chemicals which may be
used in this cycle (sours, bleaches, optical brighteners, sof-
teners, etc). — Prep, so that required vol. of product treatment
soln contains adjunct chemicals according to description and
amts specified on product label or advertising literature.
D. Operating Technic
Place test spindle with test fabric in Mason jar contg product
soln. Secure rubber gasket and Mason jar cap, remove from
H 2 bath, place jar in agitator, and rotate 5 min. Addnl ma-
nipulation with test spindle is required if use directions do not
specify addn of product in final rinse phase of laundry cycle.
In this instance, to det. durability of antimicrobial agent in
fabric, execute 3 rinse operations as follows: Immediately after
end of initial 5 min agitation, drain treatment soln from Mason
jar and replace with 100 mL H 2 0. Secure Mason jar contg test
spindle, return to agitator, and rotate 2 min. Repeat operation
twice more.
Following all required fabric treatment operations, remove
test spindle from Mason jar and unwind test fabric strip from
spindle. Let test fabric strip air dry with long axis of strip in
horizontal position.
When test strip is dry, remove 1 sq in. bacteriological test
samples. Five test samples are required for single bacterio-
static test against 1 test organism. In each such instance, at
least 2 test samples must be removed from middle 20% of
length of test strip.
Perform bacteriostatic agar plate tests as follows: Prep. 5
replicate plates in each test for each organism. Sep. inoculate
flasks contg 100 mL sterile, liq. (<40°) nutrient agar B with
1 mL 24 hr nutrient broth culture of S. aureus and K. pneu-
moniae, aberrant. Immediately thereafter, if desired, add 1 mL
appropriate soln of 2,3,5-triphenyl tetrazolium chloride to in-
oculated nutrient agar B . Vigorously swirl contents of erlen-
meyers to ensure complete mixing. Add 10 mL portions of
inoculated agar to 100 mm sterile petri dishes, distribute evenly,
and let cool and harden. As soon as plates harden, implant
single 1 sq in. treated fabric test sample on center of 1 test
agar plate surface. Using blunt forceps, press each fabric test
sample onto agar surface to ensure complete and uniform con-
tact. Incubate test plates 48 hr at 37°. If desired, test plates
may be refrigerated 18-20 hr before incubation. Following in-
cubation, examine test plates to det. presence or absence of
zones of inhibition along each side of test fabric sample.
E. interpretation
Use clear zone of inhibition adjacent to each side of test
fabric sample as index of bacteriostatic activity. Size of zone
is not considered important, but zone is required to extend along
entire edge to be acceptable. Score zone of inhibition along
single side of sq test fabric samples as 1 , so that for 5-replicate
plate test, a score of 20 shows that bacterio stasis occurs along
all 4 sides of each sample. Total score of 18/20 sides dem-
onstrating bacteriostasis is required for effective demonstration
of residual bacteriostatic activity of laundry fabric treated with
antimicrobial laundry additive product during laundering op-
eration. Unless qualified residual bacteriostatic claim is made,
AOAC Official Methods of Analysis (1990)
Pool Disinfectants
145
residual treatment must be bacteriostatic against both S. aureus
and K. pneumoniae, aberrant.
Ref.: JAOAC52, 836(1969).
965.13 Disinfectants (Water)
for Swimming Pools
First Action 1965
Final Action 1970
(Suitable for presumptive evidence of acceptability of products
for disinfecting swimming pool water)
A. Test Culture Media
(a) Nutrient Agar A. —See 960.09A(a)(7).
(b) Nutrient Agar B (Trypticase Soy Agar, BBL). — See
965.13B(b).
(c) Nutrient Agar C. — Prep, as in 955.11A(c).
B. Subculture Media
(a) Tryptone glucose extract agar (Difco). — Dissolve 24 g
in 1 L freshly distd H 2 and heat to bp to dissolve completely.
Autoclave 15 min at 121°. Use for plate counts of E. coli sur-
vivors.
(b) Trypticase soy agar (BBL). — Suspend 40 g powder in
1 L H 2 0. Let stand 5 min and mix thoroly. Heat gently with
occasional agitation and boil ca 1 min or until soln is com-
plete. Autoclave 15 min at 121°. Let cool and reautoclave 15
min at 121°. Use for plate counts of S. faecalis survivors.
(c) Fluid thioglycolate medium (Difco). — See 955.11A(d)(2).
(d) Lactose broth (Difco). — Dissolve 19 g in 1 L H 2 t). Dis-
pense 10 mL portions into tubes with fermentation vials. Au-
toclave 15 min at 121°. Use for detg presence of E. coli sur-
vivors.
(e) Eosin methylene blue agar (Difco). — Suspend 36 g in
1 L H 2 and heat to bp to dissolve completely. Autoclave 15
min at 121°. Use for confirming E. coli survivors.
(f) S-F agar (Difco).— Dissolve 36 g in 1 L H 2 0. Add 15
g agar and heat to bp to dissolve completely. Autoclave 15
min at 121°. Use for confirming S. faecalis survivors.
C. Neutralizer Stock Solns
(a) Sodium thiosulfate soln. — Dissolve 1 g Na 2 S 2 3 in 1 L
H 2 0. Dispense in 100 mL portions and autoclave 20 min at
15 lb.
(b) Azolectin soln.— See 960.09A(c).
(c) Other preparations . — Prepns found to be suitable and
necessary, depending upon nature of germicidal prepns to be
tested.
D. Neutralizer Blanks
(a) With 0.6 ppm residual chlorine or less. — Dil. 10 mL
neutralizer stock soln, 965.13C(a), with 90 mL sterile H 2 0.
Dispense aseptically in 9 mL portions into sterile 25 x 150
mm tubes.
(b) With quaternary ammonium compounds and phenolic
derivatives. — Mix 10 mL neutralizer stock soln, 965.13C(b),
2.5 mL 0.25M phosphate buffer stock soln, 965.13E(a), and
167.5 mL H 2 0. Dispense in 9 mL portions into 20 X 150 mm
tubes. Autoclave 20 min at 121°.
(c) Other preparations. — Use dims of 965.13C(c) as suit-
able.
E. Reagents
(a) Phosphate buffer stock soln.—0.25M. See 960.09A(e).
(b) Phosphate buffer dilution water. — See 960.09A(f).
(c) Sodium thiosulfate std solns. — (J) 0.1N. Dissolve ex-
actly 24.820 g Na 2 S 2 3 .5H 2 in H 2 and dil. to 1 L. Stdze
as in 942.27B.
(2) 0.001N.—DW. 10 mL soln (7) to 1 L with H 2 0.
(d) Starch indicator soln. — Mix ca 2 g finely powd. potato
starch with cold H 2 to thin paste; add ca 200 mL boiling
H 2 0, stirring constantly, and immediately discontinue heating.
Add a few drops of CHC1 3 as preservative.
(e) Sterile phosphate buffer stock solns. — (7) Dissolve 11.61
g anhyd. K 2 HP0 4 in 1 L H 2 and autoclave 20 min at 121°.
(2) Dissolve 9.08 g anhyd. KH 2 P0 4 in 1 L H 2 and autoclave
20 min at 121°.
(f) NaOCl stock soln. — Approx. 5%. Store NaOCl stock
soln in tightly closed bottle in refrigerator and det. exact avail-
able CI at frequent intervals by As 2 3 titrn, 955.16A(c).
(g) Test organism. — Use Escherichia coli ATCC 11229 and
Streptococcus faecalis ATCC No, 6569 (American Type Cul-
tures Collection, 12301 Parklawn Dr, Rockville, MD 20852).
Maintain, by monthly transfer, stock cultures of E. coli on
Nutrient Agar C, 955.11A(c), and 5 '. faecalis on Nutrient Agar
B, 965.13B(b); store at 4-5°.
F. Apparatus
(a) Glassware. — 500 mL wide-mouth erlenmeyers; 100 mL
graduates; Mohr pipets; milk pipets; 20 x 150 mm tubes; Board
of Health tubes (6mm x 50mm); 200, 500, and 1000 mL vol.
flasks. Wash in strong, fresh chromic acid cleaning soln, and
fill and drain with H 2 >3 times. Heat >2 hr at 180° in hot
air oven.
(b) Petri dishes. — Sterile.
(c) Water bath. — Controlled at 20 or 25°.
G. Preparation of Culture Suspension
From stock culture, inoculate tube Nutrient Agar A for E.
coli and Nutrient Agar B for S. faecalis; make ^3 consecutive
daily transfers (<30), incubating transfer 20-24 hr at 35-37°.
Do not transfer >30 days. If only 1 daily transfer has been
missed, no special procedures are required; if 2 daily transfers
are missed, repeat with 3 daily transfers. Remove culture from
agar surface, using 5 mL phosphate buffer diln H 2 0,
965.13E(b). Transfer culture suspension to sterile centrf. tube
and centrf. 1-2 min at speed necessary to settle agar particles.
Transfer supernate to another sterile centrf. tube and centrf. to
obtain complete sepn of cells. Discard supernate and resus-
pend cells in 5 mL buffer diln H 2 0. With S. faecalis, centrf.,
discard supernate, and resuspend cells in 5 mL buffer diln H 2
2 addnl times. Finally, stdze suspension to give av. of 2.0 X
10 8 organisms/mL by diln with sterile phosphate diln H 2 0.
If Lumetron is used, dil. suspension in sterile Lumetron tube
to give % T according to Table 965.13. Make serial diln plate
count of each culture suspension before use, using phosphate
buffer diln H 2 0, 965.13E(b), and subculture medium,
965.13B(a), with E. coli, and (b) with S. faecalis. Incubate
diln plates in inverted position 48 hr at 35-37°. Use Quebec
Colony Counting Chamber and report results in terms of num-
ber of bacteria/mL suspension. Count of 2.0 X 10 8 is desired
so that 1 mL test culture suspension + 199 mL test soln will
provide soln contg 1 x 10 6 organisms/mL. Permitted variation
Table 965.13 Percent Light Transmission at Various Wave-
lengths Corresponding to Bacterial Concentra-
tions
%
Light Transmission
with Filter, nm
Av.
Bacterial
Bacteria
370
420
490 530 580
650
Count/mL
E. coli
S. faecalis
90
86
88
82
89 88 91
85 85 87
92
89
2.0 x 10 8
2.0 x 10 8
146
Disinfectants
AOAC Official Methods of Analysis (1990)
in test culture suspension is +500,000 and — 100,000/mL of
200 mL test soln. Use actual count for calcg zero time count
in later tests.
H. Determining Chlorine Demand of Freshly Distilled Test
Water
Place 200 mL H 2 in each of five 500 mL erlenmeyers. To
flasks 1-5, resp., add 0.025, 0.05, 0.075, 0.1, and 0.15 mL
of 200 ppm available CI prepd from NaOCl soln, 965.13E(f).
Shake each flask, and let stand several min. Add crystal KI
and 1 mL HOAc, and swirl. Add 1 mL starch soln, 965.13E(d).
Flask showing perceptible blue indicates CI demand has been
satisfied.
/. Operating Technic
Place ca 600 mL freshly sterilized distd H 2 in 1 L vol.
flask. Add ca 1.5-3.0 mL K,HP0 4 buffer, 965.13E(e)(7), and
0.5 mL KH 2 P0 4 , 965.13E(e)(2), and dil. to 900 mL. Add
enough NaOCl from suitable diluent of std stock soln,
965.13E(f), to satisfy CI demand of 1 L test H 2 0, 965.13H,
and to provide ca 0.6 ppm residual available CI. Dil. to vol.
(Example: If CI demand of H 2 is 0.1 ppm, add 3.5 mL of
200 ppm soln of available CI made from std stock NaOCl soln ,
965.13E(f), and dil. to vol. This should provide soln with ca
0.6 ppm residual available CI at pH 7. 5 ±0.1.) Transfer 199
mL of this test soln to each of three 500 mL erlenmeyers and
place in H 2 bath at either 20 or 25°. Let come to temp.
To first flask, add 1 mL boiled distd H 2 and det. residual
available CI as follows: Add small crystal KI and 1 mL HOAc;
then add 1 mL starch soln, 965.13E(d). Blue soln indicates
presence of CI. Titr. with 0.00W Na 2 S 2 3 , 965.13E(c)(2),
until color disappears; mL O.OOIA' Na 2 S 2 3 X 0.1773 - ppm
residual available CI. This represents available CI at time in
test. Result should be ^0.58 but <0.62.
To each of remaining flasks add 1 mL test culture suspen-
sion, 965. 13G, as follows: Swirl flask, stopping just before
suspension is added, to create enough centrifugal motion to
prevent pooling of suspension at point of contact with test H 2 0.
Add suspension midway between center and edge of liq. sur-
face, immersing tip of pipet slightly below surface of H 2 0.
Avoid touching pipet to neck or side of test flask during op-
eration.
From one of these 2 flasks transfer 1 mL aliquots to neu-
tralizer blanks, 965.13D(a), after intervals of 0.5, 1, 2, 3, 4,
5, and 10 min. Shake neutralizer blank thoroly immediately
after adding sample. Prep, serial diln plate counts from neu-
tralizer blanks, using phosphate buffer diln H 2 0, 965.13E(b),
and subculture medium, 965.13B(a) for E. coli, and (b) for
S. faecalis.
After prepg diln plate counts, inoculate 5 lactose broth tubes,
965.13B(d), with 1.0 mL aliquots from each neutralizer blank
tube for each time interval when E. coli is used as the test
organism, and 5 thioglycolate broth tubes, 965.13B(c), with
1 .0 mL aliquots from each neutralizer blank tube for each time
interval when S. faecalis is test organism.
Incubate all diln plates in inverted position and subculture
tubes 48 hr at 37°. Use Quebec Colony Counting Chamber in
reading diln plates and report results in terms of number of
surviving bacteria/mL test H 2 0. Absence of colony growth on
diln plates and absence of growth in all 5 lactose or thiogly-
colate tubes, as case may be, is necessary to show complete
kill of test organism.
Immediately after transferring 10 min interval sample from
second flask to neutralizer blank tube, remove third flask from
H 2 bath and det. residual available CI exactly as specified
for first flask. Results should represent residual available CI
present at 10 min exposure interval. To be acceptable, concn
of available CI in this flask should be >0.4 ppm. Results in
CI control test described above should show complete kill of
E. coli and S. faecalis within 0.5 min.
With unknown sample, prep. 2 flasks contg 199 mL each
of soln at concn recommended or to be studied, using CI de-
mand-free, unbuffered, freshly distd H 2 previously prepd in
1 L vol. flask where CI demand, as detd above, has been sat-
isfied by addn of NaOCl soln. Place flasks in H 2 bath at 20
or 25°; let come to temp. Inoculate 1 flask with 1 mL std test
culture suspension of E. coli and other with 1 mL std test cul-
ture suspension of S '. faecalis . Subculture at exactly same time
intervals and in same manner used with NaOCl control except
vary composition of neutralizer blank depending upon nature
of chem. or mixt. of chems under investigation. For example,
mixt. of Cl-contg chem. and quaternary NH 4 compd would
require special neutralizer blank prepd by using both neutral-
izer stock solns, 965.13C(a) and (b).
Where no concn of chem. under study has been recom-
mended and objective of study is to det. concn of unknown
necessary to provide result equiv. to that obtained with CI con-
trol std, use series of three or four 500 mL flasks contg 199
mL of various concns of chem. and 1 mL stdzd culture sus-
pension with each test organism. Report results as log (number
of survivors) at each time interval both for CI controls and
various concns of unknown under test.
Lowest concn of unknown germicide or germicidal mixt.
providing results equiv. to those obtained with NaOCl as CI
std is considered lowest concn which could be expected to pro-
vide acceptable disinfecting activity in swimming pool water.
Refs.: JAOAC 47, 540(1964); 48, 640(1965).
7. Pesticide Formulations
Associate Chapter Editors:
Peter D. Bland
ICI Americas, Inc.
G. Marshals Gentry
Florida Dept of Agriculture and Consumer Services
Edwin R. Jackson
Mississippi State Chemical Laboratory
James E. Launer
Oregon Dept of Agriculture
David F. Tomksns
Monsanto Co.
(Chemical names for pesticides mentioned in this chapter are
given at end of chapter.)
(Pesticide ref. stds may be available from the following: All-
tech Associates, 2051 Waukegan Rd, Deerfield, IL 60015;
Analabs, Inc.; Applied Science; Chem Service Inc., PO Box
194, West Chester, PA 19380; RFR Corp., 1 Main St, Hope,
RI 02831; and Supelco.)
984.03
Fertilizer-Pesticide
Formulation Mixtures
Sampling Methods
First Action 1984
See 929.01 and 969.01.
920.11
GENERAL METHODS
935.06 Sampling
of Pesticide Formulations
Procedure
(Caution: See safety notes on pesticides.)
Examine shipping cases closely for code numbers, different
labels, and other pertinent information. Give special attention
to products subject to deterioration.
Caution: Use care in sampling and transporting toxic ma-
terials to avoid personal injury and contamination of trans-
portation facilities in case of breakage. Avoid mutual contam-
ination with other products during transportation.
Mark each sample container according to laboratory require-
ments.
(a) Small package retail units. — Take one unopened unit
(1 lb if dry, 1 pt if liq.), except take min. of 2 units of small
baits in cake form. Size of sample is governed by composition
of material and anal, methods.
(b) Large package dry products (25 lb or more). — Sample
unopened containers, using trier long enough to reach bottom
of container by inserting into container at one edge or corner
and probing diagonally toward opposite edge or corner. Take
cores by code or batch number. Analyze cores from same code
or batch number as composite or individually. Clean trier mo-
ral y after sampling each batch.
Store samples in air-tight glass, metal, plastic, or cardboard
containers.
(c) Large package liquid products (5 gallons or more). —
Use glass, plastic tubing, or stainless steel trier with plunger,
or rubber tubing for certain materials. Store samples in glass
or containers of other noncorrosive material with screw top
caps lined with Teflon or other- inert material. Plastic con-
tainers may be used only for carefully selected products.
Pesticide Formulations
Preparation of Sample
Final Action
Thoroly mix all samples before analysis. Det. H 2 0-sol. As
on samples as received, without further pulverization or drying.
In case of lye, NaCN, or KCN, weigh large amts in weighing
bottles and analyze aliquots of their aq. solns.
920.12 Moisture in Pesticide Formulations
Final Action
(Applicable to Paris green, powd Pb arsenate, Ca arsenate, Mg
arsenate, Zn arsenite, powd Bordeaux mixt., and Bordeaux
mixt. with arsenicals)
Dry 2 g to const wt at 105-110° and report loss in wt as
moisture.
922.03 Arsenic (Total)
in Pesticide Formulations
Hydrazine Sulfate Distillation Method
Final Action
(Nitrates do not interfere. Applicable to detn of total As in
Paris green, Pb arsenate, Ca arsenate, Zn arsenite, Mg arse-
nate, and Bordeaux mixt. with arsenicals)
A. Reagents
(a) Arsenious oxide std soln. — 0.1 or 0.05/V. See 939.12.
(b) Iodine std soln.— -0. 1 or 0.05/V. See 939.13.
(c) Bromate std soln. — 0.1 or 0.05iV. Dissolve ca 2.8 or
1.4 g KBr0 3 in boiled H 2 and dil. to 1 L. Stdze as follows:
Pipet 25 mL aliquots As 2 3 soln, (a), into 500 mL erlenmey-
147
148
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
ers. Add 15 mL HC1, dil. to 100 mL, heat to 90°, and titr.
with the KBr0 3 soln, using 10 drops Me orange, (g). Do not
add indicator until near end of titrn, and agitate soln contin-
uously to avoid local excess of KBr0 3 soln. Add KBr0 3 soln
very slowly near end point; at end point soln changes from red
to colorless.
(d) Hydrazine sulfate- sodium bromide soln. — Dissolve 20
g N 2 H 4 .H 2 S0 4 and 20 g NaBr in 1 L HC1 (1+4).
(e) Sodium hydroxide soln. — Dissolve 400 g NaOH in H 2
and dil. to 1 L.
(f) Starch indicator. — Mix ca 2 g finely powd. potato starch
with cold H 2 to thin paste; add ca 200 mL boiling H 2 0, stir-
ring constantly, and immediately discontinue heating. Add ca
1 mL Hg, shake, and let soln stand over the Hg.
(g) Methyl orange indicator. — 0.05%. Dissolve 0.5 g Me
orange in H 2 and dil. to 1 L.
B. Apparatus
See Fig. 922.03. Set 500 mL distn flask on metal gauze that
fits over circular hole in heavy sheet of asbestos board, which
in turn extends out far enough to protect sides of flask from
direct flame of burner. First receiving flask holds 500 mL and
contains 40 mL H 2 0; second holds 500 mL and contains 100
mL H 2 0. Vol. in first flask should be <40 mL, otherwise compd
of As may sep. that is difficult to dissolve without danger of
loss of AsCJ 3 . Keep both flasks cool by placing in pan of cir-
culating H 2 0, or contg H 2 and ice.
C. Determination
(Caution: See safety notes on pesticides and arsenic trioxide.)
Weigh sample contg ^0.4 g As and transfer to distg flask.
Add 50 mL N 2 H 4 .H 2 S0 4 -NaBr soln, close flask with stopper
that carries funnel tube, and connect side tube with condenser.
Boil 2-3 min, add 100 mL HC1 from dropping funnel, and
distil until vol. in distg flask is reduced to ca 40 mL; add 50
mL more HC1 and continue distn until vol. is again reduced
to ca 40 mL. Wash down condenser, transfer contents of re-
ceiving flasks to 1 L vol. flask, dil. to vol., mix thoroly, and
proceed as in (a) or (b):
(a) Pipet 200 mL aliquot into erlenmeyer and nearly neutze
with NaOH soln, using few drops phthln, and keeping soln
well cooled. If neut. point is passed, add HO until again slightly
acid. Neutze with NaHC0 3 , add 4-5 g excess, and add std I
soln from buret, shaking flask continuously until yellow color
disappears slowly from soln. Add 5 mL starch indicator and
keep adding std I soln dropwise to permanent blue.
(b) Pipet 200 mL aliquot into erlenmeyer and titr. with KBr0 3
soln, (c), beginning "... heat to 90° ..."
Calc. % As. Report as As 2 3 or As 2 5 , according to whether
As is present in trivalent or pentavalent form. If condition of
arsenic is unknown, report as As.
Refs.: Ind. Eng. Chem. 14, 207(1922). JAOAC 5, 33,
402(1922); 6, 313(1923); 48, 564(1965).
CAS-7440-38-2 (arsenic)
924.04 Arsenic (Total)
in Pesticide Formulations
lodometric Method
Final Action
(Applicable in presence of sulfides, sulfites, thiosulfates , and
large amts of S or org, matter)
A. Reagent
Sodium thiosulfate soln. — Dissolve 13 g crystd Na 2 S 2 3 .5H 2
in H 2 and dil. to 1 L.
See 922. 03 A for other reagents and solns and 922. 03B for
app.
B. Determination
(Caution: See safety notes on pesticides and arsenic tri-
oxides.)
Weigh sample contg <0.4g As and transfer to distg flask.
Add 50 mL N 2 H 4 .H 2 S0 4 -NaBr soln, 922.03A(d), and distil as
in 922.03C. Dil. distillate to vol. in 1 L vol. flask, mix tho-
roly, and transfer 200 mL aliquot to 400 mL Pyrex beaker or
porcelain casserole. Add 10 mL HN0 3 and 5 mL H 2 S0 4 , evap.
to sirupy consistency on steam bath, and then heat on hot plate
to white fumes of H 2 S0 4 . Cool, and wash into 500 mL erlen-
meyer. If vol. H 2 S0 4 is appreciably lessened by fuming, add
enough H 2 S0 4 to make total vol. ca 5 mL. Dil. to 100-150
mL, add 1.5 g KI, and boil until vol. is reduced to ca 40 mL.
Cool under running H 2 0, dil. to 100-150 mL, and add Na 2 S 2 3
soln, 924. 04A, dropwise until I color just disappears. Nearly
neutze H 2 S0 4 with NaOH soln, 922.03A(e), finish neutzn with
NaHC0 3 , add 4-5 g excess, and titr. with std I soln as in
FIG. 922.03 — Apparatus for distilling arsenious chloride
AOAC Official Methods of Analysis (1990)
General Methods
149
922.03C(a). From mL std soln used, calc. % As in sample.
Report as As 2 3 , As 2 O s , or As as in 922. 03C.
Ref.: JAOAC 7, 313(1924).
CAS-7440-38-2 (arsenic)
963.06 Arsenic (Total)
in Pesticide Formulations
Bon Exchange Method
First Action 1963
Final Action 1968
(Applicable to inorg. arsenates and arsenites)
A. Apparatus
Ion exchange column. — Use All inn filter tube 10 X 2.7 cm
od with coarse filter disk. Attach piece of rubber tubing to
bottom of filter tube and regulate flow with Hoffman clamp.
To tube add aq. slurry of Dowex 50W-X8, 50-100 mesh, us-
ing resin bed vol. of 12 mL, and place 500 mL separator above
tube.
Regenerate resin bed before each run by first back-washing
column few min with H 2 0; then elute with 350 mL IN HC1
followed by 200 mL H 2 6 at 20 mL/min.
B. Preparation of Sample
(Caution: See safety notes on pesticides.)
Weigh 200 mg sample (100 mg if As content is >30%) into
150 mL beaker, add 7 mL HN0 3 , and bring to bp. Add 3 mL
2N KBr0 3 and evap. to dryness, avoiding spattering. Back-
wash and regenerate resin during this evapn. Dissolve cooled
residue in 2 mL 67V HC1 without heat and add 8 mL H 2 0. Filter
into separator, and wash filter with three 10 mL portions H 2 0.
(If residue dissolves completely in 2 mL 6/V HO, omit filtra-
tion, and dil. directly to 40 mL.) Pass soln thru resin column
at 20 mL/min and collect eluate in 250 mL erlenmeyer. Wash
separator and column with 20 and 40 mL portions H 2 into
same erlenmeyer.
C. Determination
Add 50 mL HC1 to eluate to make 4/V. Add 1 g NaHC0 3 ,
0.2 g at time, swirling constantly. Add 1 g KI, stopper, and
swirl until all KI dissolves. After 5 min, titr., without starch
indicator, with 0.05N Na 2 S 2 3 , 942.27, to disappearance of I.
(Recognition of end point is facilitated by titrg on porcelain
stand. In presence of starch, reaction between I and Na 2 S 2 3
is retarded, so appreciable amt of Na 2 S 2 3 reacts with acid.
End point becomes indistinct if >30 mL Na 2 S 2 3 is used in
titrn.) 1 mL 0.05A^ Na 2 S 2 3 = 1.873 mg As.
Refs.: Anal. Chem. 22, 1066(1950). JAOAC 46, 672(1963).
CAS-7440-38-2 (arsenic)
925.02 Arsenic (Water-Soluble) in
Pesticide Formulations
Titrimetric Method
Final Action
to 32°. Stopper flask and place in constant temp. H 2 bath at
32°. Digest 24 hr, shaking hourly 8 hr during this period. Filter
thru dry filter. If filtrate is not clear, refilter thru buchner contg
paper and enough Filter-Cel coating to give clear soln. Discard
first 50 mL.
Transfer 250-500 mL clear filtrate to erlenmeyer, add 3 mL
H 2 S0 4 , and evap. to ca 100 mL on hot plate. Add 1 g KI, and
continue boiling until vol. is ca 40 mL. Cool, dil. to ca 200
mL, and add Na 2 S 2 3 soln, 924. 04A, dropwise, until I color
is exactly removed. (Avoid use of starch indicator at this point.)
Neutze with NaHC0 3 , add 4-5 g excess, titr. with std I soln,
shaking flask continuously, until yellow disappears slowly, add
5 mL starch indicator, 922.03A(f), and continue titrn to per-
manent blue. Correct for amt std I soln necessary to produce
same color, using same reagents and vol. From mL std I soln
used, calc. % H 2 0-sol. As in sample.
CAS-7440-38-2 (arsenic)
922.04 Lead in Pesticide Formulations
Gravimetric Method
Final Action
(Applicable to such prepns as Bordeaux-Pb arsenate, Bor-
deaux-Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca
arsenate)
(Caution: See safety notes on nitric acid, fuming acids, pes-
ticides, hydrogen sulfide, and arsenic trioxide.)
Weigh 1 g powd sample and transfer to beaker. Add 5 mL
HBr (ca 1.38 sp gr) and 15 mL HO, and evap. to dryness to
remove As. Repeat treatment; add 20 mL HO, and again evap.
to dryness. Add 25 mL 2N HO to residue, heat to bp, filter
immediately to remove Si0 2 , and wash with boiling H 2 to
vol. of 125 mL. See that all PbCl 2 is in soln before filtering;
if it will not dissolve completely in 25 mL IN acid, add 25
mL more and dil. filtrate to 250 mL. Pass in H 2 S until pptn
is complete. Filter, and wash ppt thoroly with 0.5 A" HO satd
with H 2 S. Save filtrate and washings for Zn detn.
Transfer paper with sulfides of Pb and Cu to 400 mL Pyrex
beaker and completely oxidize all org. matter by heating on
steam bath with 4 mL H 2 S0 4 and ca 20 mL fuming HNO3 in
covered beaker. Evap. on steam bath, and then completely re-
move HN0 3 by heating on hot plate to copious white fumes
of H 2 S0 4 . Cool, add 2-3 mL H 2 0, and again heat to fuming.
Cool, add 50 mL H 2 and 100 mL alcohol, and let stand sev-
eral hr (preferably overnight). Filter thru gooch, previously
washed with H 2 0, then with acidified alcohol (100 parts H 2 0,
200 parts alcohol, and 3 parts H 2 S0 4 ), and finally with alco-
hol, and dried at 200°. Wash ppt of PbS0 4 in crucible ca 10
times with acidified alcohol, and then with alcohol, to remove
H 2 S0 4 . Retain filtrate and washings for Cu detn, if desired.
Dry at 200° to const wt, keeping crucible covered to prevent
loss from spattering. From wt PbS0 4 , calc. % Pb in sample,
using factor 0.6832.
Ref.: JAOAC 5, 398(1922).
CAS-7439-92-1 (lead)
(Applicable to detn of H 2 0-sol. arsenic in Pb arsenate, Ca ar-
senate, Zn arsenite, Mg arsenate, and Bordeaux mixt. with
arsenicals)
(Caution: See safety notes on pesticides.)
To 2 g original sample if powder, or 4 g if paste, in 1 L
Florence flask, add 1 L recently boiled H 2 that has been cooled
922.05 Copper in Pesticide Formulations
Final Action
(Applicable to such prepns as Bordeaux-Pb arsenate, Bor-
deaux-Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca
arsenate)
150
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
A. Electrolytic Method
Evap. filtrate and washings from PbS0 4 pptn, 922.04, to
fuming; add few mL fuming HN0 3 to destroy org. matter, and
continue evapn to ca 3 mL. Take up with ca 150 mL H 2 0,
add 5 mL HN0 3 , and filter if necessary. Wash into 250 mL
beaker, adjust vol. to 200 mL, and electrolyze, using rotating
anode and weighed gauze cathode with current of 2-3 amp.
After all Cu has apparently deposited (ca 30 min), add 15-20
mL H 2 to electrolyte and continue electrolysis few min. If
no further deposition occurs on newly exposed surface of elec-
trode, wash with H 2 without breaking current either by si-
phoning or quickly replacing beaker with electrolyte succes-
sively with 2 beakers of H 2 0. Interrupt current, rinse cathode
with alcohol, dry few moments in oven, and weigh. Calc. %
Cu in sample.
B. Volumetric Thiosulfate Method
Proceed as in 922. 05A to point at which filtrate and wash-
ings from PbS0 4 pptn are treated with fuming HN0 3 and evapd
to vol. of ca 3 ml, Take up in ca 50 mL H 2 0, add NH 4 OH
in excess, and boil to expel excess NH 3 , as shown by color
change in liq. and partial pptn. Add 3-4 mL HOAc (4+1),
boil 1-2 min, cool, add 10 mL 30% KI soln, and titr. with
std Na 2 S 2 3 soln until brown color becomes faint. Add starch
indicator, 922.03 A(f), and continue titrn cautiously until blue
color due to free I entirely disappears. From mL std Na 2 S 2 3
soln used, calc. % Cu in sample.
Thiosulfate std soln. — Prep, soln contg 39 g Na 2 S 2 3 .5H 2 0/
L. Accurately weigh 0.2-0.4 g pure electrolytic Cu and trans-
fer to 250 mL erlenmeyer roughly marked at 20 mL intervals.
Dissolve Cu in 5 mL HN0 3 (1 + 1), dil. to 20 or 30 mL, boil
to expel red fumes, add slight excess satd Br-H 2 0, and boil
until Br is completely removed. Cool, and add 10 mL NaOAc
soln (574 g trihydrate/L). Prep. 42 g/100 mL KI soln made
very slightly aJk. to avoid formation and oxidn of HI. Add 10
mL of the KI soln and titr. with Na 2 S 2 3 soln to light yellow.
Add enough starch indicator, 922. 03 A (f), to produce marked
blue. As end point nears, add 2 g KSCN and stir until com-
pletely dissolved. Continue titrn until ppt is perfectly white. 1
mL Na 2 S 2 3 soln = ca 10 mg Cu.
It is essential for Na 2 S 2 3 titrn that concn of KI in soln be
carefully regulated. If soln contains <320 mg Cu, at comple-
tion of titrn 4.2-5 g KI should have been added for each 100
mL total soln. If greater amts of Cu are present, add KL soln
slowly from buret with const agitation in amts proportionately
greater.
Ref.: JAOAC 5, 398(1922).
CAS-7440-50-8 (copper)
918.01 Zinc in Pesticide Formulations
Gravimetric Method
Final Action
(Applicable to such prepns as Bordeaux-Pb arsenate, Zn ar-
senite, Bordeaux-Zn arsenite, Bordeaux-Paris green, and Bor-
deaux-Ca arsenate)
A. Reagent
Mercury-thiocyanate soln. — {Caution: See safety notes on
mercury salts.) Dissolve 27 g HgCl 2 and 30 g NH 4 SCN in H 2
and dil. to 1 L.
B. Determination
Cone, filtrate and washings from sulfide pptn, 922.04, by
gentle boiling to ca 50 mL; then evap. on steam bath to dry-
ness. Dissolve residue in 100 mL H 2 contg 5 mL HC1, and
add 35-40 mL Hg-thiocyanate soln with vigorous stirring. Let
stand ^1 hr with occasional stirring. Filter thru weighed gooch,
wash with H 2 contg 20 mL Hg-thiocyanate soln/L, and dry
to const wt at 105°. Calc. to % Zn, using factor 0.1312.
Note: Some Fe is usually present and during Zn detn should
be in ferrous condition. In pptg sulfides pass H 2 S into soln
long enough to reduce Fe as well as to ppt Cu and Pb.
ZnHg(SCN) 4 ppt normally is white, and occluded Fe(SCN) 3
should not give more than faint pink color.
Refs.: J. Am. Chem. Soc. 40, 1036(1918). JAOAC 5,
398(1922).
CAS -7440-66-6 (zinc)
929.04 Fluorine (Total)
in Pesticide Formulations
Lead Chlorofluoride Method
Final Action
A. Reagents
(a) Fusion mixture. — Mix 30 g anhyd. Na 2 C0 3 with 40 g
anhyd. K 2 C0 3 .
(b) Lead chlorofluoride wash soln. — Dissolve 10 g Pb(N0 3 ) 2
in 200 mL H 2 0, dissolve 1 g NaF in 100 mL H 2 and add 2
mL HO, and mix these 2 solns. Let ppt settle and decant.
Wash ppt 4 or 5 times with 200 mL H 2 by decanting; then
add ca 1 L cold H 2 to ppt and let stand >1 hr, with oc-
casional stirring. Filter and use clear filtrate. (Prep, more
wash soln as needed by adding more H 2 to ppt of PbCIF
and stirring.)
(c) Silver nitrate std soln. — 0. 1 or 0.2yV. Stdze as in 941. 18E.
(d) Potassium or ammonium thiocyanate std soln. — 0.1 jV.
Stdze against std AgN0 3 soln under same conditions as in detn.
(e) Ferric indicator. — To cold satd Cl-free
FeNH4(SO4)2.12H 2 soln add enough colorless H.NO3 to bleach
brown color.
(f) Bromophenol blue indicator. — Grind 0. 1 g powder with
1.5 mL 0.17V NaOH and dil. to 25 mL.
B. Determination
(a) Samples difficult to decompose such as cryolite , and
others that contain aluminum or appreciable amounts of sili-
ceous material. — Mix 0.5 g sample (or less if necessary to
contain 0.01-0.10 g F) with 5 g fusion mixt. and 0.2-0.3 g
powd. Si0 2 in Pt dish, cover with 1 g fusion mixt., and heat
to fusion over Bunsen burner. (Use of blast lamp is unnec-
essary since it is preferable not to heat much beyond melting
temp. If much Al is present, uniform, clear, liq. melt cannot
be obtained; particles of white solid will sep. in melt. Cooled
melt should be colorless, or at least should not have more than
gray color.)
Leach cooled melt with hot H 2 and when disintegration is
complete, filter into 400 mL beaker. Return insol. residue to
Pt dish with jet of H 2 0, add 1 g Na 2 C0 3 , dil. to 30-50 mL,
and boil few min, disintegrating any lumps with flat-end rod.
Filter thru same paper, wash thoroly with hot H 2 0, and adjust
vol. of filtrate and washings to ca 200 mL. Add 1 g ZnO dis-
solved in 20 mL HN0 3 (1+9), boil 2 min, stirring constantly,
filter, and wash thoroly with hot H 2 0. During this washing
return gelatinous mass to beaker 3 times and thoroly disinte-
grate in wash soln because proper washing of this ppt on filter
is difficult. (Mass can easily be returned to beaker by rotating
funnel above beaker while cutting ppt loose from paper with
jet of wash soln.)
AOAC Official Methods of Analysis (1990)
General Methods
151
Add 2 drops bromophenol blue to filtrate, and with cover
glass almost entirely over beaker, add HN0 3 (1+4) until color
just changes to yellow. Make soln slightly alk. with 10% NaOH
soln, and with cover glass on beaker, boil gently to expel C0 2 .
Remove from burner; add the HN0 3 until color just changes
to yellow and then 10% NaOH until color just changes to blue;
then add 3 ml 10% NaCl soln. (Vol. of soln at this point
should be ca 250 mL.)
Add 2 mL HCI (1 + 1) and 5 g Pb(N0 3 ) 2 and heat on steam
bath. As soon as Pb(N0 3 ) 2 is in soln, add 5 g NaOAc, stir
vigorously, and digest on steam bath 30 min with occasional
stirring. Let stand overnight, filter, and wash ppt, beaker, and
paper once with cold H 2 0, then 4 or 5 times with PbCIF wash
soln, and then once more with cold H 2 0.
Transfer ppt and paper to beaker in which pptn was made,
stir paper to pulp, add 100 mL HN0 3 (5 + 95), and heat on
steam bath until ppt dissolves. (5 min is ample to dissolve ppt.
If sample contains appreciable amt of sulfates, ppt will contain
PbS0 4 , which will not dissolve. In such case heat 5-10 min
with stirring and consider PbCIF to be dissolved.) Add slight
excess 0.LV or 0.27V AgN0 3 , digest on steam bath 30 min,
and cool to room temp., protecting from light; filter, wash
with cold H 2 0, and det. AgN0 3 in filtrate by titrn with std
thiocyanate soln, using 10 mL ferric indicator. Subtract amt
of AgN0 3 found in filtrate from that originally added. Differ-
ence is amt required to combine with CI in the PbCIF; from
this difference calc. % F in sample. 1 mL O.liV AgN0 3 —
0.00190 g F.
(b) Water-soluble fluorides in presence of organic mat-
ter. — In presence of <50% org. matter such as flour, pyreth-
rum, tobacco powder, and derris or cube powders, which readily
decompose without addn of powd Si0 2 and contain little or no
sulfates, Al, or siliceous compds, mix 0.5 g sample (or less
if necessary to contain 0.01-0.1 g F) with 5 g fusion mixt.,
cover with 1 g fusion mixt,, and heat to fusion over Bunsen
burner. Leach cooled melt with hot H 2 0, and when disinte-
gration is complete, filter into 600 mL beaker. Wash thoroly
with hot H 2 and proceed as in (a), third par.
In presence of >50% org. matter or org. matter that is im-
practical to free without preliminary ashing, such as apple peel
and pulp, transfer enough sample to Pt crucible to be repre-
sentative of mixt. and to contain 0.01-0.1 g F. Add 15 mL
H 2 and enough F-free CaO (0.3-0.4 g) to make mixt. dis-
tinctly alk. to phthln, mix with glass rod, and evap. to dryness
on steam bath and in oven at 105°. Ignite at low heat, pref-
erably in furnace (<600°), until org. matter is thoroly charred.
Pulverize, with glass rod, any lumps present in charred ash,
mix with 5 g of the fusion mixt., and proceed as in (a), first
par., beginning "... cover with 1 g fusion mixt., ..."
(c) Water-soluble samples in absence of organic matter and
appreciable quantities of sulfates or aluminum salts. — In ab-
sence of org. matter or other interfering substances, fusion may
be omitted and detn made on aliquot of aq. soln contg 0.01-
0.1 g F, as in (a), third par.
In presence of Al, as in samples contg Na 2 SiF 6 and
KAI(S0 4 ) 2 1 2H 2 0, transfer sample to 400 mL beaker, dissolve
in 150 mL hot H 2 0, add 6 g fusion mixt., and boil. Add 1 g
ZnO dissolved in 20 mL HNO3 (1 + 9), boil 2 min with const
stirring, filter into 500 mL vol. flask, and wash thoroly with
hot H 2 0. Cool to room temp, and dil. to vol. Transfer 200
mL aliquot contg 0.01-0.10 g F to 600 mL beaker and pro-
ceed as in (a), third par.
(d) Sodium and magnesium fluosilicates, or samples con-
taining more than 5% sulfates in absence of aluminum and
boron, with or without moderate amounts of organic mat-
ter. — With large amts of Na 2 SiF 6 and some other more volatile
fluosilicates, e.g., MgSiF 6 , where there is possibility of some
F being evolved as SiF 4 before fusion is effected, or in samples
contg appreciable amts of sulfates, distil F as in 933. 03B, and
det. F in distillate as follows: Add several drops bromophenol
blue, make alk. with NaOH, and adjust vol. to ca 250 mL by
gently boiling down vol. from 400 to 250 mL. Proceed as in
(a), third par., beginning "Remove from burner; ..."
Notes: These methods give accurate results for 0.01-0.10 g
F. Below 0.01 g, results tend to be slightly low, and above
0.10 g, slightly high. Convenient sample to fuse is one contg
0.07-0.08 g F; too large sample may result in incomplete fu-
sion. Large amts of B compds and alkali salts retard or prevent
complete pptn of PbCIF. B has greater effect when amt of F
is large than when it is small . In methods described B has little
effect, and it may be disregarded in analysis of insecticides if
amt of F to be pptd is <0.03 g. With some prepns contg Na 2 B 4 ?
or H 3 B0 3 , where it is difficult to obtain representative mixt.
when extremely small sample (0.1 g) is used for analysis, take
larger sample and ppt PbCIF from aliquot of fusion soln. Amt
of alkali carbonates specified in fusion and in washing of insol.
residue is not large enough to cause low results. If sample
contains S, remove it with CS 2 and det. F on air-dried residue,
allowing in calcns for % S removed. {Caution: See safety notes
on flammable solvents, toxic solvents, and carbon disulfide.)
Refs.: J. Res. Natl. Bur. Standards 3, 581(1929). JAOAC 25,
670(1942); 27, 74(1944); 28, 72(1945).
CAS-7782-41-4 (fluorine)
921.04 Fluorine (Total)
in Pesticide Formulations
Modified Travers Method
Final Action
(Applicable in absence of B, Al, and large amts of pyrethrum
powder)
(Caution: See safety notes on asbestos.)
A. Reagents
(a) Alcoholic potassium chloride soln. — Dissolve 60 g KCI
in 400 mL H 2 0, add 400 mL alcohol, and test with phthln; if
soln is not neut., adjust to exact neutrality with NaOH or HCI
soln.
(b) Sodium hydroxide std soln. — 0.2/V. Prep, and stdze as
in 936.16.
B. Determination
Treat 0.5 g sample in small beaker with 20-25 mL H 2 0.
Add 0.3 g finely divided pptd Si0 2 and few drops Me orange.
Add HCI dropwise until soln assumes apparently permanent
pink; then add 2 mL excess, cover beaker with watch glass,
and boil 1 min. Cool to room temp., add 4 g KCI, and stir
until KCI dissolves. Add 25 mL alcohol and let stand 1 hr,
stirring frequently. Filter thru gooch contg disk of filter paper
covered with medium pad of asbestos. Wash ppt with ale. KCI
soln until one washing does not destroy color made by 1 drop
0.2N NaOH and phthln (usually 3-4 washings). Transfer cru-
cible and contents to 400 mL beaker, add 100 mL recently
boiled H 2 and 1-2 mL phthln, heat, and titr. with std NaOH
soln. Finish titrn with the F soln actively boiling. Calc. % F.
1 mL 0.2N NaOH = 0.0057 g F.
Refs.: Compt. rend. 173, 714, 836(1921). JAOAC 14,
253(1931).
CAS-7782-41-4 (fluorine)
152
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
933.03 Fluorine (Total)
in Pesticide Formulations
Distillation Method
Final Action
(Applicable to H 2 0-sol. or FLO-insol. insecticides
in absence of gelatinous Si0 2 , B, and Al)
A. Reagents
(a) Sodium alizarin sulfonate indicator.— Dissolve 0.1 g Na
alizarin sulfonate in 200 mL H 2 0.
(b) Thorium nitrate soln. — Approx. 0.057V. Stdze in terms
of g F/mL by titrg F obtained by distn from std NaF as in
933. 03B. In stdzg for use with 933.036(b), add 5 mL satd
KMn0 4 soln in addn to other reagents in distn flask.
B. Determination
(a) In absence of organic matter. — Weigh sample contg ca
0.09 g F, and with aid of little H 2 transfer to 250 mL Claisen
distn flask (B) contg 12 glass beads. Adjust to ca 30 mL and
close flask with 2-hole rubber stopper, thru which pass ther-
mometer (D) and 4 mm glass tube, both of which extend into
soln. (The 4 mm glass tube extends ca 5 cm above rubber
stopper and by means of rubber tube, E, connects still with 1
L Florence flask (A) contg H 2 for steam generation. Flask is
equipped with steam discharge, H, and pressure tube, G. See
Fig. 933.03.)
Bring H 2 in steam generating flask to boil with pinchcock,
F, in release tube open. Connect distg flask to condenser, C,
and add 25 mL H 2 S0 4 thru top of 4 mm tube, using pipet or
special funnel. With pinchcock, F, open, connect rubber tub-
ing to 4 mm tube. Light burner under Claisen flask. Regulate
flow of steam by adjusting burner flames and pinchcock, F,
so that vol. of soln is held const and temp, in flask, B, is kept
at 145-150°. Continue distn until 400 mL distillate collects.
Dil. to 500 mL in vol. flask, transfer 50 mL aliquot to tall-
form 150 mL beaker, and add 5 drops indicator, 933.03A(a).
Adjust acidity with 1% NaOH soln and HC1 (1+249) until
pink just disappears. Add 2 mL of the HCl, and titr. with 0.057V
FIG. 933.03— Apparatus for determining fluorine
Th(N0 3 ) 4 to permanent pink, using buret graduated in 0.05
mL.
(b) In presence of organic matter. — (Caution: See safety
notes on permanganates.) In presence of moderate amts of org.
matter, transfer sample contg ca 0.09 g F and contg ^0.2 g
org. matter, with aid of little H 2 0, to 250 mL Claisen distn
flask contg 12 glass beads. Add 5 mL satd KMn0 4 soln, adjust
to ca 30 mL, and proceed as in (a), beginning "... close flask
with 2-hole rubber stopper, ..."
In presence of large amts of org. matter, transfer sample to
medium-size Pt dish, add 15 mL H 2 and enough F-free CaO
to make mixt. distinctly alk. to phthln, mix with glass rod,
and evap. to dryness on steam bath and in oven at 105°. Ignite
at low heat, preferably in furnace (<600°), until org. matter
is thoroly charred. Pulverize any lumps present in charred ash
with glass rod, transfer to 250 mL Claisen distn flask by brush-
ing, and finally wash out dish with 30 mL H. 2 S0 4 (1+9). Ex-
cept to add 22 mL instead of 25 mL H 2 S0 4 , proceed as in (a),
par. 2.
Note: If coating of pptd Si0 2 forms on inside of distn flask,
remove by treatment with hot coned alkali soln, as it is capable
of retaining F during distn of some samples and giving it up,
at least in part, in later distns.
Refs.: J. Am. Chem. Soc. 55, 1741(1933). Ind. Eng. Chem.
Anal. Ed. 5, 7(1933); 9, 551(1937); 11, 21(1939).
JAOAC 21, 459(1938); 53, 378(1970).
CAS-7782-41-4 (fluorine)
945.05 Fluorine Present
as Sodium Fluosilicate
in Pesticide Formulations
Final Action
(B, CaO, and alum absent)
A. Reagents
Alcoholic potassium chloride and sodium carbonate soln. —
Dissolve 1 .0 g Na 2 C0 3 in 100 mL ale. KC1 reagent, 921.04A(a).
For other reagent see 921. 04A.
B. Determination
Weigh 1 g sample into Pt dish, and add rapidly, with con-
tinuous stirring, 50 mL of the ale. KC1 -Na 2 C03 reagent. Do
not let soln become acid, and if necessary, use more reagent
to insure alky.
Continue stirring until all sol. portions of sample dissolve.
Proceed as in 921. 04B, beginning: "Filter thru gooch ..."
Calc. % Na 2 SiF 6 (1 mL 0.2N NaOH = 0.009403 g Na 2 SiF 6 ).
CAS-16893-85-9 (sodium fluosilicate)
972.05 Organochlorine Pesticide
Contamination of Pesticide Formulations
Thin Layer Chromatographic Method
First Action 1972
Final Action 1977
AOAC-CIPAC Method
(Applicable to detection of contamination by 0.01% chlori-
nated hydrocarbons such as aldrin, DDT, dieldrin, and endrin,
and 0.05-0.10% of chlordane, strobane, and toxaphene)
A. Apparatus
(a) Thin layer chromatographic apparatus. — See 970. 52F.
(b) Ultraviolet apparatus. — Sterilamp G-15T8, or equiv.
AOAC Official Methods of Analysis (1 990)
Inorganics and Organometallics
153
B. Reagents
(a) Adsorbent. — Aluminum oxide G, Type E (Brinkmann
Instruments, Inc.).
(b) Mobile solvents.— (1) n-Hexane, (2) n - he xane- acetone
(98 + 2), and (3) n-hexane-alcohol (98+2).
(c) Pesticide std solns. — 1 (Jtg/fxL EtOAc, acetone, or any
convenient solv.
C. Preparation of Sample
Ext 8 g sample with 20 mL acetone in 250 mL erlenmeyer
by shaking intermittently 5 min. Let solids settle. If soln is
turbid, filter or centrf. to obtain clear supernate for spotting.
For samples contg large amts S, use 8 g sample and 20 mL
pet ether.
D. Preparation of Piates
Dissolve 0.1-0.15 g AgN0 3 in 1-2 mL H 2 in 100 mL
beaker, add 58 mL MeOH, and mix. Weigh 40 g adsorbent,
(a), in 250 mL flask, add AgN0 3 -MeOH soln, and shake vig-
orously 20 sec. Apply slurry as 0.25 mm thick layer to five
20 X 20 cm (8 x 8") plates positioned on plastic mounting
board. After plates appear dry, store in desiccator over des-
iccant. When plate is dry, scrape 1 cm strip from side edges
to ensure even solv. front. Use plate immediately after re-
moval from desiccator.
E. Detection
Pour tt-hexane into glass chromatgc tank to depth of 10-20
mm. Place 2 paper blotters (ca 7.5 X 22 cm) on each side of
tank or large blotter covering back of tank and let equilibrate
>2 hr before use.
Spot 10 jxL sample ext on plate with 100 uJL syringe. Do
not disturb adsorbent layer. Also spot std solns of pesticides
declared as part of formulation. Spots should be ^6 mm diam.
and placed <30 mm from bottom of plate. Place plate in chro-
matgc tank, and let plate develop >10 cm. Remove plate and
expose to shortwave UV, 972.05A(b). (Caution: See safety
notes on hazardous radiations.) Chlorinated org. pesticides
should be visible as dark spots against white or light gray
background. Expose plates >1 hr. Longer exposure will not
harm plates.
To confirm identification of pesticide, repeat TLC step with
different mobile solv., 972.058(b)(2) or (J).
Ref.: J AOAC 55, 851(1972).
960.10 Herbicide Formulations
(Ester Forms of Hormone-Type)
Volatility Determination
First Action 1960
Final Action 1961
A. Material
(a) Paper bags. — No. 20 to open with flat bottom. Close
with paper clips.
(b) Filter paper. — 7 cm diam.
(c) Bacteriological loop. — 0.01 mL. Wash with acetone af-
ter each application or heat to cherry red in flame.
(d) Test plants. — Actively growing tomato seedlings 65—
75 mm high in 3-4" pots.
(e) Formulation to be tested. — Use 0.01 mL aliquot of 4
lb/gal. formulation or equiv. vol. of other concns.
(f) High and low volatile ester stds. — Use Bu ester of 2,4-D
as high volatile ester and tetrahydrofurfural ester of 2,4-D as
low volatile ester with same wt of acid /gal. as formulations
to be tested.
B. Operating Technic
Open bags with flat bottom and place plant toward one side
on bottom of bag. Apply 0.01 mL of formulation to middle
of filter paper by means of bacteriological loop, and for con-
trols, apply 0.01 mL solv. only. Place treated paper in bottom
of bag. Do not touch treated part of paper against plant, sides
of bag, or pot. Close bag by folding top, secure with clips,
and let stand 24 hr at 85-1 10°F (29-43°C).
Use 3 plants per treatment and 3 for controls. Repeat test
on another day.
Remove plants from bag, let stand 24 hr, and read curvature
(stem bending, epinasty) response. (Fold and discard used bags
to prevent contamination.) Rate plants according to scale as
follows:
(1) Normal growth of untreated check- no apparent re-
sponse.
(2) Epinasty 1-20° compared to normal- no curling.
(3) Epinasty 21-40° compared to normal-slight curling.
(4) Epinasty 41-60° compared to normal-moderate curling.
(5) Epinasty 61-80° compared to normal-moderate curling.
(6) Epinasty 81 to >90° compared to normal-severe form-
ative effects.
Mean response of 1 to 2.4 for all tests indicates low vola-
tility. Mean response of 2.5 to 6 indicates volatile formulation.
To detect small differences between low volatile esters, or
differences between 2,4-D and 2,4,5-T types, hold plants 7
days after treatment to allow time for modified leaves or stem
lesions to develop. Absence of such responses indicates that
formulation was a low volatile 2,4,5-T ester.
Ref.: J AOAC 43, 367(1960).
INORGANIC AND ORGANOMETALL1C
PESTICIDES AND ADJUVANTS
920.13
Paris Green Pesticide
Formulations
Final Action
A. Moisture
See 920.12.
B. Totai Arsenic
See 922.03.
C. Water-Soluble Arsenious Oxide*
—Surplus 1965
See 4.031, 10th ed.
920.14* Paris Green Pesticide
Formulations
Total Arsenious Oxide
Final Action
Surplus 1965
(Following methods det. only As present in trivalent form
(As 2 3 ) and Sb present in trivalent form (Sb 2 3 ) in absence
of ferrous and cuprous salts.)
A. Method I
See 4.028-4.029, 10th ed.
B. Method II
See 4.030, 10th ed.
154
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
920.15* Paris Green Pesticide
Formulations
Total Copper
Final Action
Surplus 1965
A. Electrolytic Method
See 4.032, 10th ed.
B. Volumetric Thiosulfate Method
See 4.033, 10th ed.
920.16 Lead Arsenate
Pesticide Formulations
Moisture
Final Action
{Caution: See safety notes on pesticides and arsenic trioxide.)
(a) Powder. — Dry 2 g to const wt at 105-110°. Report loss
in wt as H 2 0.
(b) Paste. — Proceed as in (a), using 50 g. Grind dry sam-
ple to fine powder, mix well, transfer small portion to sample
bottle, and again dry 1-2 hr at 105-110°. Use this anhyd.
material to det. total Pb and total As.
920.17 Lead Arsenate
Pesticide Formulations
Total Arsenic
Final Action
A. Method I
See 922.03C.
B. Method II
(Not applicable in presence of Sb)
Dissolve 1 g powd sample with HN0 3 (1+4) in porcelain
casserole or evapg dish, add 5 mL H 2 S0 4 , and heat on hot
plate to copious evolution of white fumes. Cool, add little H 2 0,
and again evap. until white fumes appear, to assure removal
of last trace of HN0 3 . Wash into 200 mL vol. flask with H 2 0,
cool, dil. to vol., and filter thru dry filter. Transfer 100 mL
filtrate to erJenmeyer and proceed as in 925.02, beginning w . . .
add 1 g KI, ..." From mL std 1 soln used, calc. % total As
as As 2 5 .
Ref.: USDA Bur. Chem. Bull. 105, p. 167.
CAS-7440-38-2 (arsenic)
920.18 Lead Arsenate
Pesticide Formulations
Total Arsenious Oxide
Final Action
Weigh 2 g powd sample and transfer to 200 mL vol. flask,
add 100 mL H 2 S0 4 (1 + 6), and boil 30 min. Cool, dil. to vol.,
shake thoroly, and filter thru dry filter. Nearly neutze 100 mL
filtrate with NaOH soln, 922.03A(e), using few drops phthln.
If neut. point is passed, make acid again with the dil. H 2 S0 4 .
Continue as in 925.02, beginning "Neutze with NaHC0 3 , ..."
From mL std I soln used, calc. % As 2 3 .
Ref.: JAOAC 3, 332(1920).
920.19 Lead Arsenate
Pesticide Formulations
Total Arsenic Oxide
Final Action
A. Reagents
(a) Potassium iodide soln. — Dissolve 20 g Kl in H 2 and
dil. to 100 mL.
(b) Thiosulfate std soln.— 0.05N. Prep, daily by dilg O.liV
soln, 942.27. 1. mL 0.057V Na 2 S 2 3 = 2.873 mg As 2 O s .
B. Determination
Weigh 0.5 g powd sample and transfer to erlenmeyer. Add
25-30 mL HC1 and evap. to dryness on steam bath. Add 50
mL HO and if necessary to effect soln, heat on steam bath,
keeping flask covered with watch glass to prevent evapn of
acid. Cool to 20-25°, add 10 mL of the KI soln and 50 mL
(or more if necessary to produce clear soln) 25% NH 4 Cl soln,
and immediately titr. liberated I with std Na 2 S 2 3 soln. When
color becomes faint yellow, dil. with ca 150 mL H 2 and con-
tinue titrn carefully, dropwise, until colorless, using starch in-
dicator, 922.03A(f), near end point. From mL Na 2 S 2 3 soln
used, calc. % As 2 5 .
Ref.: JAOAC 3, 333(1920).
920.20 Lead Arsenate
Pesticide Formulations
Water-Soluble Arsenic
Final Action
Proceed as in 925.02, and calc. results as As 2 5 .
920.21 Lead Arsenate
Pesticide Formulations
Total Lead
Final Action
In 600 mL beaker on hot plate heat 0.5 g powd sample and
ca 25 mL HN0 3 (1+4). Filter to remove any insol. residue.
Dil. to >400 mL, heat nearly to bp, and add NH 4 OH to slight
pptn, then HN0 3 (1 + 9) to redissolve ppt, adding 1-2 mL ex-
cess. Into this soln, kept almost boiling, pipet 50 mL hot 10%
K 2 Cr0 4 soln, stirring constantly. Decant while hot thru weighed
gooch, previously heated to 140-150°, and wash ppt several
times by decanting and then on filter with boiling H 2 until
washings are colorless. Dry PbCr0 4 at 140-150° to const wt.
From wt PbCr0 4 , calc. % Pb, using factor 0.641 1. (PbCr0 4
ppt may contain small amt PbHAs0 4 , which will cause slightly
high results, but this error rarely is >0. 1.-0.2%.)
Refs.: USDA Bur. Chem. Bull. 137, p. 40; 152, p. 68.
CAS-7439-92-1 (lead)
921.05
Calcium Arsenate
Pesticide Formulations
Final Action
{Caution: See safety notes on pesticides.)
A. Moisture
See 920.12.
B. Total Arsenic
See 922.03.
AOAC Official Methods of Analysis (1990)
Inorganics and Organometallics 1 55
C. Total Arsenious Oxide
(a) Not applicable in presence of nitrates. — Weigh 1 g
sample, transfer to 500 mL erlenmeyer, and dissolve in 100
mL HC1 (.1+3). Heat to 90° and titr. with std KBr0 3 soln,
922.03A(c), using 10 drops Me orange, 922.03A(g). From mL
std KBr0 3 soln used, calc. % As 2 3 .
(b) Applicable in presence of small amounts of nitrates. —
Proceed as in (a) except to titr. at room temp.
Refs.: JAOAC 5, 33(1921); 6, 392(1922).
D. Water-Soluble Arsenic
Proceed as in 925.02, and calc. results as As 2 5 . (In testing
Ca arsenate by this method, low value for H 2 0-sol. As is not
assurance against plant injury when using this product.)
921.06 Calcium Arsenate
Pesticides Formulations
Total Calcium
Final Action
A. Reagents
(a) Ammonium oxalate soln. — Dissolve 40 g (NH^CoCV
H 2 in 1 L H 2 0.
(b) Potassium permanganate std soln. — 0.17V. Prep, and
stdze as in 940.35.
R Method l
Dissolve 2 g sample in 80 mL HOAc (1 +3), transfer to 200
mL vol. flask, dil. to vol., and filter thru dry filter. Transfer
50 mL aliquot to beaker, dil. to ca 200 mL, heat to bp, and
ppt Ca with (NH 4 ) 2 C 2 4 soln. Let beaker stand 3 fir on steam
bath, filter, and wash ppt with hot H 2 0. Dissolve ppt in 200
mL H 2 contg 25 mL H 2 S0 4 (1 +4), heat to ca 70°, and titr.
with std KMn0 4 soln. From mL KMn0 4 soln used, calc. %
Ca.
C. Method It
(Not applicable in presence of Pb. Caution: See safety notes
on arsenic trioxide.)
Weigh 2 g sample, transfer to beaker, add 5 mL HBr (ca
1.38 sp gr) and 15 mL HO, and evap. to dryness under hood
to remove As. Repeat treatment, add 20 mL HC1, and again
evap. to dryness. Take up with H 2 and little HO, filter into
200 mL vol. flask, wash, and dil. to vol. Transfer 50 mL ali-
quot to beaker, add 10 mL HO and few drops HN0 3 , boil,
and make slightly alk. with NH 4 OH. Let stand few min and
filter. Dissolve ppt in HO (1 +4), reppt, filter thru same paper,
and wash with hot H 2 0. To combined filtrates and washings
add 20 mL HOAc (1 + 3) and adjust to ca 200 mL. Heat to bp,
ppt with (NH 4 ) 2 C 2 4 soln, and let stand 3 hr on steam bath.
Filter, and wash with hot H 2 0. Ignite at 950°, and weigh as
CaO; or dissolve and titr. as in 921. 06B. From wt CaO or mL
KMn0 4 soln used, calc. % Ca.
Refs.: JAOAC 5, 33(1921); 6, 392(1922).
CAS-7440-70-2 (calcium)
920.22
A. Moisture
See 920.12.
Zinc Arsenite
Pesticide Formulations
Final Action
B. Total Arsenic
Proceed as in 922.03 and calc. as As 2 3 .
C. Water-Soluble Arsenic
Proceed as in 925.02, and calc. results as As 2 3 .
D. Total Zinc*
Surplus 1965
See 4.054, 10th ed.
920.23* Zinc Arsenite
Pesticide Formulations
Total Arsenious Oxide
Final Action
Surplus 1965
A. Method I
See 4.051, 10th ed.
S. Method tl
See 4.052, 10th ed.
920.24* Copper in Copper Carbonate
Pesticide Formulations
Final Action
Surplus 1965
A. Electrolytic Method
See 4.055, 10th ed.
B. Volumetric Thiosulfate Method
5<?<? 4.056, 10th ed.
964.03 Copper in Copper Nap ht hen ate
Pesticide Formulations
First Action 1964
Final Action 1982
(Caution: See safety notes on pesticides.)
A. Titrimetric Method
Accurately weigh sample contg ca 0.2 g Cu into dry g-s
flask. Add 5 mL pet ether to coned products. Add 100 mL
H 2 0, 1.5 g NH 4 HF 2 , and 5-10 g KI. Stopper and shake vig-
orously until reaction is complete (usually ca 2 min). Wash
stopper and sides of flask with H 2 and titr. with std O.IjV
Na 2 S 2 3 (stdzd against Cu) to light brown. Add starch indi-
cator, 922.03A(f), titr. almost to end point, add 2 g KSCN,
shake to dissolve, and complete titrn to starch end point.
a Electrolytic Method
Accurately weigh sample contg ca 0.2 g Cu into 200 mL
separator. Add 50 mL pet ether and 25 mL HN0 3 (1+4), and
shake 2 min. Drain aq. phase into 250 mL beaker and save.
Wash pet ether with 15 and 10 mL HN0 3 (1+4), and combine
acid exts. Neutze with NH 4 OH, acidify with 6 mL H 2 S0 4 and
4 mL HN0 3 , and proceed as in 922. 05 A, beginning ". . . ad-
just vol. to 200 mL, ..." using ca 0.5 amp during first 10
min and 1 .5-2.0 amp for ca 20 min.
Ref.: JAOAC 47, 253(1964).
CAS-7440-50-8 (copper)
156
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
981.01 Copper (Water-Soluble)
in Water-insoluble Copper Fungicides
First Action 1981
CIPAC-AOAC Method
(Caution: See safety notes on atomic absorption spectropho-
tometer and chloroform.)
A. Principie
Water-soluble Cu is isolated by dispersing sample in H 2 0,
shaking, centrifg, and filtering. Cu in filtrate is detd either by
spectrophtric bathocuproine method or by atomic absorption.
B. Apparatus
(a) Filter paper. — 7 cm. (Whatman No. 1 chromatgc or
filter paper, S&S No. 2043b, and Albet No. 305 have been
satisfactory; the following papers have not been suitable:
Whatman Nos 32, 40, 42, and Albet No. 240. If other papers
are used, check adsorption of Cu before use.)
(b) Filter.— 13 mm Millipore, HA.WP.013-porosity 0.45
|xm, Sartorius, or equiv.
(c) Atomic absorption spectrophotometer. — With oxidizing
air-C 2 H 2 flame.
C. Reagents
(a) Deionized water. — For prepg all aq. solns and rinsing
glassware and app.
(b) Sodium acetate-acetic acid. — 0.05M, buffered at pH 6.5.
Dissolve 3.4 g NaOAc.3H 2 in H 2 0, add 1.8 mL 0.2M HOAc,
adjust to pH 6.5, and dil. to 500 mL with H 2 0.
(c) Cupric sulfate.— CuS0 4 .5H 2 0, contg 25.4% Cu. Check
Cu content, e.g., by electrolysis.
(d) Copper std stock so In. — 25 |mg Cu/mL. Dissolve 98.3
mg CuS0 4 .5H 2 in H 2 and dil. to 1 L with H 2 0.
(e) Ascorbic acid soln. — Dissolve 10 g ascorbic acid in H 2
and dil. to 100 mL with H 2 0.
(f) Sodium acetate soln. — Dissolve 40 g NaOAc.3H 2 in
H 2 and dil. to 100 mL with H 2 0.
(g) Chloroform. — Redistd.
(h) Bathocuproine soln. — Dissolve 0.050 g in 500 mL re-
distd CHC1 3 and store in dark glass bottles.
D. Isolation of Water-Soiuble Copper
Accurately weigh ca 0.350 g sample (w) and transfer to 250
mL conical flask. Add 100 mL H?0, stopper tightly, and shake
1 min. Let stand l.h in 20° H 2 bath, shaking suspension
every 15 min. Centrf. ca 50 mL suspension 20 min at 3000
rpm, taking care to avoid increase in temp. Filter ca 40 mL
supernate thru 13 mm filter.
Bathocuproine Method
E. Preparation of Standard Curve
Pipet 25.0 mL Cu std stock soln, (d), into 500 mL vol. flask
and dil. to vol. with NaOAc-HOAc buffer soln (1.25 (Jig Cu/
mL). Transfer 0, 4, 6, 8, 10, 12, 14, and 16 mL aliquots of
intermediate soln to 100 mL separators and dil. each to 20 mL
with NaOAc-HOAc buffer soln. Add 1 mL ascorbic acid soln
to each separator and shake 1 min. Add 2.5 mL NaOAc soln
and shake 1 min. Develop color by pipetting in 10.0 mL
bathocuproine soln. Ext. complex by vigorously shaking 1 min
(vigorous extn is necessary to ext all complex). Let layers sep.,
filter CHCI3 ext thru 7 cm paper into clean, dry tube, and stop-
per tube. Exactly 15 min after extn, measure A at 465 nm,
using CHCI3 as ref. Plot A vs jxg Cu. (Curve is linear <20 |xg
Cu, but does not necessarily pass thru origin.)
F. Determination
Pipet 10.0 mL sample filtrate into 100 mL vol. flask, dil.
to vol. with NaOAc-HOAc buffer soln, and mix.
Transfer 20.0 mL aliquot to 100 mL separator and continue
as in Preparation of Standard Curve , starting with "Add 1 mL
ascorbic acid soln ..."
If A of sample is higher than A of highest point of calibration
curve, diL sample soln with buffer soln so A falls on calibra-
tion curve. Read u,g Cu (0 corresponding to A found.
% Soluble Cu (w/w) - 2/200w
If soln has been dild, correct formula accordingly. Carry out
blank detn on reagents, using 20 mL NaOAc-HOAc buffer
soln. A must be of same order as that for A of point on
calibration curve.
Atomic Absorption Spectrophotometry Method
G. Preparation of Standard Curve
Transfer 0, 4.0, 8.0, 12.0, 16.0, and 20.0 mL aliquots of
Cu std stock soln (25 u>g Cu/mL) to 100 mL vol. flasks. Add
2 mL HNO3 to each, diL to vol. with H 2 0, and mix. (These
solns contain 0, 1, 2, 3, 4, and 5 u,g Cu/mL.)
Det. A at 324.7 nm of solns in oxidizing air-C 2 H 2 flame,
and plot calibration curve.
H. Determination
Pipet 25.0 mL sample filtrate into 50 mL vol. flask, add 1
mL HNO3, dil. to vol. with H 2 0, and mix. Det. A at 324.7
nm as in Preparation of Standard Curve. Obtain fig Cu/mL
from std curve,
% Soluble Cu (w/w) - (pig Cu/mL)/50w
If A of sample is higher than highest point of calibration curve,
dil. sample with 2% HNO3 and correct calcn for diln factor.
Ref.: JAOAC 64, 75(1981); corr. 233.
CAS-7440-50-8 (copper)
979.02 Fentin in Pesticide Formulations
Potentiometric Titration Method
First Action 1979
Final Action 1982
CIPAC-AOAC Method
A. Principle
Org. Sn compds are extd with acetone, diphenyltin compds
are quant, converted to insol. oxide with alk. alumina and fil-
tered, and acetone soln is titrd potentiometric ally.
B. Apparatus
(a) Filtration apparatus. — Glass bell with neck and remov-
able plate to permit glass buchner with fine porosity fritted
disk and long stem to drain into beaker under vac. (Fig. 979.02).
(b) Potentiometric titration apparatus. — pH meter with glass
and satd calomel electrodes is satisfactory.
C. Reagents
(a) Alkaline alumina. — Mix 150 g neutral A1 2 3 (Woeim
4649, or equiv.) with 150 mL alcohol contg 15 g KOH in 1
L r-b flask. Reflux 30 min, cool, and filter with suction thru
buchner. Dry powder in vac. 1 hr at 100° and 3-3.5 hr at 130°.
Pour warm powder into bottle and stopper tightly. Com. alk.
A1 2 3 is not satisfactory.
(b) Cellulose powder. — Whatman CF 11, or equiv.
AOAC Official Methods of Analysis (1990)
Inorganics and Organometallics
157
FIG. 979.02— Filtration apparatus
D. Determination
Accurately weigh into 100 mL glass beaker sample contg
ca 0.30 g triphenyltin compd. Add 2 g alk. A1 2 3 and 25 mL
acetone and stir with mag. stirrer 10 min. Prep, and process
blank of 2 g alk. A1 2 3 and 25 mL acetone in same manner.
Place 1 g cellulose powder and 1 g alk. A1 2 3 in funnel and
mix thoroly. Assemble filtration app. contg 250 mL beaker
and filter suspension thru funnel. Wash beaker and funnel with
four 20 mL portions acetone. Titr. filtrate potentiometrically
with stdzdO.UVHCl.
% Triphenyltin compd = (S - B) X N X (M/W) X 10
where S = mL HC1 used for sample, B = mL HC1 used for
blank, N = normality of HC1, M = mol. wt of compd (367.0
for triphenyltin hydroxide and 409.0 for the acetate), and W
— g sample.
Ref.: JAOAC61, 1504(1978).
984.04 Fentin in Fentin-Maneb
Pesticide Formulations
Gas Chromatographic Method
First Action 1984
CIPAC-AOAC Method
A. Principie
Fentin is converted with n-butylmagnesium chloride to bu-
tyltriphenyltin, which is extd with toluene and detd by GC
with docosane as internal std.
B. Reagents and Apparatus
(a) Dilute acetic acid. — Weigh 0.750 g HO Ac in 250 mL
vol. flask and dil. to vol. with toluene.
(b) n-Butylmagnesium chloride. — 2.5M in tetrahydrofuran
(Alfa Products, Div. of Morton Thiokol, Inc., 152 Andover
St, Danvers, MA 01923), or equiv.
(c) Butyltriphenyltin. — Prep, as follows: Purify triphenyltin
chloride by repeated crystn from isopropanol until diphenyltin
dichloride and/or tetraphenyltin are absent by GC or TLC.
(For TLC, use silica gel F254 (Merck) plates and develop in
CH 2 Cl 2 -acetone-HOAc-H 2 (80 + 5 + 2.5 + 0.5) or CH 2 C1 2 -
n-hexane-HOAc (30 + 20 + 2.5). Expose developed plates to
UV radiation (254 nm) or I vapors and spray with 0.1% pyro-
catechol violet soln in alcohol.) Dry salt at 50° under vac. to
remove isopropanol. Flush 250 mL 3 -necked flask equipped
with stirrer, reflux condenser, and dropping funnel with N.
Dissolve 20 g triphenyltin chloride in 80 mL toluene, add 40
mL n-butylmagnesium chloride, and let react 15 min under N,
Carefully destroy excess Grignard reagent (cool in H 2 bath)
by adding 50 mL 5% H 2 S0 4 and transfer mixt to sep. funnel.
Discard lower (aq.) layer. Wash org. layer with 20 mL 5%
H 2 S0 4 and then with H 2 until acid-free. Dry org. layer with
anhyd. Na 2 S0 4 . Filter soln into 500 mL r-b flask and evap.
solv. in rotary vac. evaporator at 50°. Dissolve residue in 400
mL hot isopropanol, cool, and filter butyltriphenyltin in buch-
ner funnel; wash with 25 mL cold isopropanol and dry at 50°
under vac. Check purity by GC and mp (corrected mp 60.0-
60.5°).
(d) Internal std soln. — Accurately weigh 2.25 g pure do-
cosane into 250 mL vol. flask, and dissolve and dil. to vol.
with toluene.
(e) Calibration soln. — Accurately weigh ca 0.20 g pure bu-
tyltriphenyltin into g-s flask, pipet in 10.0 mL internal std soln,
and add 40 mL toluene.
(f ) Gas chromato graph. — With on-column injection, flame
ionization detector, and 1.5 m X 3 mm (id) glass column packed
with 5% SE-30, or equiv. on 100-200 mesh Chromosorb W
(HP) or Gas-Chrom Q. Operating conditions: temps (°): col-
umn 220, injection port 250, detector 260; flow rates (mL/
min): N carrier gas 30, air 400, H 40. Alternatively, thermal
conductivity detector may be used with He carrier gas, bridge
current at 150 mamp, and detector at 300°.
C. Preparation of Sample
(a) Fentin acetate -rnaneb mixtures. — Accurately weigh
sample contg 205 ± 5 mg fentin acetate into 100 mL erlen-
meyer. Pipet in 10.0 mL internal std soln and 15 mL toluene
and stir 5 min on mag. stirrer. Add 8 mL n-butylmagnesium
chloride and let react 15 min under N. Destroy excess Grig-
nard reagent by careful and dropwise addn of 1.5 mL H 2 by
syringe or microburet. Dry dispersion with 2 g anhyd. Na 2 S0 4 .
Add 25 mL toluene (omit if thermal conductivity detector is
used) and filter.
(b) Fentin hydroxide -maneb mixtures. — Accurately weigh
sample contg 185 ± 5 mg fentin hydroxide into 100 mL er-
lenmeyer. Pipet in 10.0 mL internal std soln and 1.0 mL dil.
HO Ac. Add 5 mL toluene and stir 5 min on mag stirrer. Con-
tinue as described in (a).
D. Determination
Inject 1 |xL portions of calibration soln until peak area ratio
(area butyltriphenyltin peak/area docosane peak) varies <1%
for successive injections. Inject, in duplicate, 1 |xL portions
of sample solution followed by 1 jxL portions of calibration
soln. (For thermal conductivity detection, inject 10-20 |xL.)
Measure peak areas by tri angulation (peak ht by retention time)
or by electronic integration. Retention times: internal std 7 min;
butyltriphenyltin 14 min.
E. Calculations
Fentin acetate, % = R/R' x W /W x 100.5
Fentin hydroxide, % - R/R' x W'/W x 90.2
where R and R' — av. peak area ratios for sample and cali-
bration solns, resp.; W and W — mg sample and butyl-
triphenyltin, resp.; 100.5 = 100 x mol. wt fentin acetate/
mol. wt butyltriphenyltin = 100 X 409/407; 90.2 - 100 x
mol. wt fentin hydroxide /mol. wt butyltriphenyltin = 100 X
367/407.
Refs.: JAOAC 61, 1507(1978); 67, 479(1984).
CAS-900-95-8 (fentin acetate)
CAS-76-87-9 (fentin hydroxide)
158
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
920.25 Moisture in Bordeaux Mixture
Pesticide Formulations
Final Action
(Caution: See safety notes on pesticides.)
(a) Powder.— See 920.16(a).
(b) Paste.— Heat ca 100 g in oven at 90-100° until dry
enough to powder readily and note loss in wt. Powder this
partially dried sample and det. remaining H 2 in 2 g as in (a).
Det. C0 2 as in 920. 26B, both in original paste and in partially
dried sample. Calc. total H 2 by following formula:
, (100-fl)(6 + c) _
M = a + d
100
where M = % total H 2 in original paste; a = % loss in wt
of original paste during first drying; b = % loss in wt of par-
tially dried paste during second drying; c — % C0 2 remaining
in partially dried paste after first drying; and d = % total C0 2
in original paste.
920.26 Carbon Dioxide
in Bordeaux Mixture
Pesticide Formulations
Final Action
A. Apparatus
Use 200 ml erlenmeyer with 2-hole stopper; in one hole fit
dropping funnel with stem extending almost to bottom of flask,
and thru other hole pass outlet of condenser that is inclined
upward at 30° angle from horizontal. Connect upper end of
condenser with CaCl 2 tube, which in turn connects with double
U-tube filled in middle with pumice fragments, previously satd
with 20% CuS0 4 .5H 2 soln and subsequently dehydrated, and
with CaCl 2 at either end. Connect 2 weighed U-tubes to absorb
C0 2 , first filled with porous soda-lime, and second, l /a with
soda-lime and 2 / 3 with CaC1 2 , placing the CaCl 2 at exit end of
train. Attach Geissler bulb, partly filled with H 2 S0 4 , to last
U-tube to show rate of gas flow, and connect aspirator with
Geissler bulb to draw air thru app. Connect absorption tower
filled with soda-lime to mouth of dropping funnel to remove
C0 2 from air entering app.
B. Determination
Weigh 2 g powder or 10 g paste into the erlenmeyer and
add ca 20 mL H 2 0. Attach flask to app. , omitting the 2 weighed
U-tubes, and draw C0 2 -free air thru app. until it displaces
original air. Attach weighed U-tubes as in 920. 26A, close
stopcock of dropping funnel, pour into it 50 mL HC1 (1+4),
reconnect with soda-lime tower, and let acid flow into erlen-
meyer, slowly if there is much C0 2 , rapidly if there is little.
When effervescence diminishes, place low Bunsen flame un-
der flask and start flow of H 2 thru condenser, letting slow
current of air flow thru app. at same time. Maintain steady but
quiet boil and slow air current thru app. Boil few min after
H 2 begins to condense, remove flame, and continue air flow
at ca 2 bubbles/sec until app. is cool. Disconnect weighed
absorption tubes, cool in balance case, and weigh. Increase in
wt = C0 2 .
920.27 Copper in Bordeaux Mixture
Pesticide Formulations
Refs.: Fresenius, "Quantitative Chemical Analysis,'
6th German Ed., 1906, amplified and revised,
1180. U.S. Geol. Survey Bull. 700, p. 218.
CAS- 124-38-9 (carbon dioxide)
Trans.
Vol. 2,
Final Action
A. Electrolytic Method
(Also applicable to CuC0 3 and CuS0 4 )
Dissolve powd sample contg 0.2-0.25 g Cu in 45 mL HNO3
(1+4). Filter if necessary, dil. to 200 mL, and electrolyze as
in 922.05A.
B. Volumetric Thiosulfate Method
Dissolve 2 g powd sample in ca 25 mL H.NO3 (1+4), dil.
to 50 mL, add NH 4 OH in excess, and heat. Without removing
ppt that has formed, boil off excess NH 3 , add 3-4 mL HO Ac,
cool, add 10 mL 30% Kl soln, and titr. as in 922.05B, be-
ginning "... titr. with std Na 2 S 2 3 soln, ..."
CAS-7440-50-8 (copper)
920.28
Bordeaux Mixture
with Paris Green
Final Action
(Caution: See safety notes on pesticides.)
A. Moisture
See 920.25.
B. Carbon Dioxide
See 920.26B.
C. Total Arsenic
Proceed as in 922.03, using 2 g sample, and calc. results
as As 2 3 .
D. Total Arsenious Oxide*
—Surplus 1965
See 4.067, 1 0th ed.
E. Water-Soluble Arsenious Oxide*
Surplus 1965
See 4.068, 10th ed.
Copper
F. Electrolytic Method I
See 922.05A.
G. Electrolytic Method II— (Short Method)*
—Surplus 1965
See 4.070, 10th ed.
H. Volumetric Thiosulfate Method
See 922.05B.
920.29
Bordeaux Mixture
with Lead Arsenate
Final Action
(Caution: See safety notes on pesticides.)
A. Moisture
See 920.25.
B. Carbon Dioxide
See 920.26B.
AOAC Official Methods of Analysis (1990)
Inorganics and Organometallics 1 59
C. Total Arsenic
Proceed as in 922.03, using 2 g sample, and calc. results
as As 2 5 .
D. Water-Soluble Arsenic
Proceed as in 925.02 and calc. results as As 2 5 .
E. Copper— Electrolytic Method
See 922.05A
F. Copper— Volumetric Thiosulfate Method
See 922.05B.
G. Lead
See 922.04.
H. Lead and Copper — Electrolytic Method*
—Surplus 1965
See 4.079-4.080, 10th ed.
925.03
Bordeaux Mixture
with Calcium Arsenate
Final Action
952.01 Potassium Cyanate
Pesticide Formulations
Final Action
(Caution: See safety notes on pesticides.)
A, Reagent
Wash soln. — Satd aq. soln of hydrazodicarbamide,
NH 2 CONHNHCONH 2 . Prep, by mixing KOCN and semicar-
bazide.HCl, NH 2 CONHNH 2 .HCl, in H 2 0, filter, and wash ppt
with H 2 0. Transfer ppt to flask, add small amt H 2 0, shake
vigorously, and filter. (Solubility of ppt in H 2 is ca 1 part in
6600.)
B. Determination
Weigh sample contg 0.2-0.5 g KOCN into 100 mL beaker,
add 20 mL wash soln and 1 g semicarbazide.HCl, and let stand
24 hr. Filter hydrazodicarbamide on gooch or fine fritted glass
crucible, wash with 10 mL wash soln, and dry at 100° to const
wt. KOCN = wt residue x 0.6868,
Ref.: JAOAC 35, 377(1952).
CAS-590-28-3 (potassium cyanate)
(Caution: See safety notes on pesticides.)
A. Moisture
See 920.25.
B. Carbon Dioxide
See 920.26B
C. Total Arsenic
Proceed as in 922.03, using 2 g sample, and calc. results
as As 2 O s .
D. Water-Soluble Arsenic
Proceed as in 925.02 and calc. results as As 2 5 .
Copper
E. Electrolytic Method I
See 922.05A.
F. Electrolytic Method //*
—Surplus 1965
See 4.070, 10th ed.
G. Volumetric Thiosulfate Method
See 922.05B.
930. 12* Calcium Cyanide
Pesticide Formulations
Final Action
Surplus 1965
A. Cyanide
See 4.093-4.094, 10th ed.
Chloride
B. Method I
See 4.095, 10th ed.
C. Method II
See 4.096, 10th ed.
920.30* Sodium and Potassium Cyanide
Pesticide Formulations
Final Action
Surplus 1965
A. Cyanide
See 4.088-4.089, 10th ed.
Chloride
B. Method I
See 4.090-4.091, 10th ed.
C. Method II
See 4.092, 10th ed.
920.31 Sulfur (Soluble) in Lime Sulfur
Solutions and Dry Lime Sulfur
Gravimetric Method
Final Action
(Use low S reagents.)
A. Preparation of Sample
(a) Solns. — Accurately weigh ca 10 g soln, transfer to 250
mL vol. flask, and immediately dil. to vol. with recently boiled
and cooled H 2 0. Mix thoroly and either take necessary ali-
quots in individual pipets in mi n. time for detns or transfer to
small bottles, filling them completely and avoiding contact of
soln with air as much as possible. Stopper bottles, seal with
paraffin, and store in dark, cool place.
(b) Dry lime-sulfur. — Thoroly stir 5 g sample with ca 50
mL H 2 in 250 mL beaker. Let settle and decant thru paper
into 250 mL vol. flask. Repeat extn with H 2 until filtrate is
colorless and ca 200 mL is obtained. Transfer residue to paper,
wash with hot H 2 0, cool to room temp., and dil, to vol. Dry
residue 1.5 hr at 105°, and reserve for free S and sulfite S
detns in residue, if desired. (Ext S from dry residue with CS 2
(Caution: See safety notes on flammable solvents, toxic sol-
vents, and carbon disulfide.), evap. on steam bath or in air
current, dry 15 min at 105°, weigh, and calc. % S.)
160
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
Prep, so In in min. time and keep beaker and funnel covered
as much as possible.
B. Determination
With clean, dry pipet transfer 10 mL prepd soln, 920.31A(a)
or (b), to 250 mL beaker. Partially cover with cover glass and
add 2-3 g Na 2 2 in small portions, with stirring, from tip of
spatula. Continue adding Na 2 2 until all S appears to be ox-
idized to sulfate (yellow color disappears). Add slight excess
Na 2 2 , completely cover beaker with cover glass, and heat on
steam bath, stirring occasionally, 15-20 min.
Wash off cover glass and sides of beaker, acidify with HC1
(1+4), and filter if necessary. Dil. to 150-200 mL, heat to
bp, and add 10% BaCl 2 soln (11 mL/1 g BaS0 4 ), with const
stirring, at such rate that ca 4 min is required to add necessary
amt. Let stand until clear and cool, filter thru quant, paper,
wash until CI- free, ignite carefully, and heat to const wt over
Bunsen burner. Calc. % S from wt BaS0 4 , using factor 0.1374.
Ref.: JAOAC 3, 353(1920).
CAS-7704-34-9 (sulfur)
920.32 Sulfur (ThiosuSfate)
in Lime Sulfur Solutions
and Dry Lime Sulfur
Titrimetric Method
Final Action
A. Reagent
Ammoniacal zinc chloride soln. — Dissolve 50 g ZnCl 2 in ca
500 mL H 2 0, add 125 mL NH 4 OH and 50 g NH 4 C1, and dil.
to 1 L.
B. Determination
To 50 mL H 2 in 200 mL vol. flask add 50 mL prepd soln,
920.31A(a) or (b). Add slight excess of the ammoniacal ZnCl 2
soln and dil. to vol. Complete detn as rapidly as possible. Shake
thoroly and filter thru dry filter. To 100 mL filtrate add few
drops Me orange, 922.03A(g), or Me red (1 g Me red in 200
mL alcohol), and exactly neutze with O.iyV HC1. Titr. neut.
soln with 0.057V 1, 922.03A(b), using few drops starch indi-
cator, 922. 03 A (f). From mL 1 soln used, calc. % thiosulfate
S present. (Factor of I soln in terms of As 2 3 x 1.296 = equiv.
in thiosulfate S.)
B. Indirect Method
Difference between sol. S and sum of thiosulfate S and sul-
fate S - sulfide S.
Ref.: JAOAC 3, 353(1920).
920.33 Sulfur (Sulfide)
in Lime Sulfur Solutions
and Dry Lime Sulfur
Final Action
A. Zinc Chloride Method
To 10-15 mL H 2 in small beaker add 10 mL aliquot prepd
soln, 920.31A(a) or (b). Calc. amt ammoniacal ZnCl 2 soln,
920. 32A, necessary to ppt all S in aliquot and add slight ex-
cess. Stir thoroly, filter, wash ppt twice with cold H 2 0, and
transfer paper and ppt to beaker in which pptn was made. Cover
with H 2 0, disintegrate paper with glass rod, and add ca 3 g
Na 2 2 , keeping beaker well covered with watch glass. Warm
on steam bath with frequent shaking until all S is oxidized to
sulfate, adding more Na 2 2 if necessary. Acidify slightly with
HC1 (1+4), filter to remove shreds of paper, wash thoroly with
hot H 2 0, and det. S in filtrate as in 920.31B.
Ref.: JAOAC 3, 353(1920).
920.34
Lime Sulfur Solutions
and Dry Lime Sulfur
Final Action
A. Sulfate Sulfur
Slightly acidify soln from 920.32B with HC1 (1+4) and heat
to bp. Add slowly, with const stirring, slight excess 10% BaCl 2
soln, boil 30 min, let stand overnight, and filter. Calc. S from
wt BaS0 4 , and report as % sulfate S.
B. Total Calcium
To 25 mL prepd soln, 920.31A(a) or (b), add 10 mL HC1,
evap. to dryness on steam bath, add H 2 and few mL HC1
(1+4), warm until all CaCl 2 dissolves, and filter to remove S
and any Si0 2 present. Dil. filtrate to 200-250 mL, heat to bp,
add few mL NH 4 OH in excess, and then add excess satd
{NH 4 ) 2 C 2 4 soln. Continue boiling until pptd CaC 2 4 assumes
well defined granular form, let stand 1 hr, filter, and wash few
times with hot H 2 0. Ignite at 950° in Pt crucible to const wt
(CaO) and calc. % Ca. CaO x 0.7147 - Ca.
CAS-7440-70-2 (calcium)
935.07 Sodium Hypochlorite Solution
Pesticide Formulations
Final Action
Sodium Hypochlorite
Arsenious Oxide Titration Method
QAN. Prep, as in 939.12.
as in 939. 13 A. Stdze against
A. Reagents
(a) Arsenious oxide std soln.
(b) Iodine std soln. — Prep
(a).
B. Determination
Transfer 20 mL sample to 1 L vol. flask and dil. to vol.
Pipet 50 mL aliquot of mixt. into 200 mL erlenmeyer. Add
excess As 2 3 soln and then decided excess NaHC0 3 . Titr. ex-
cess As 2 3 with std I soln, using starch soln, 922.03A(f), or
the I as its own indicator. Subtract vol. I soln, corrected to
O.liV, from vol. As 2 3 soln used, and from this value and sp
gr of soln, calc. % NaOCl.
1 mL 0. \N As 2 3 = 0.003722 g NaOCl
Refs.: JAOAC 18, 63(1935).
CAS-768 1-52-9 (sodium hypochlorite)
935.08 Sodium Hypochlorite Solution
Pesticide Formulations
Final Action
A. Chlorine (Available)
Calc. % available CI from titrn, 935.07B. 1 mL 0. \N As 2 3
- 0.003545 g available CI.
B. Chlorine (Chloride)
Pipet 50 mL aliquot prepd soln, 935. 07B, into 200 mL er-
lenmeyer and add slight excess As 2 3 soln, 935.07A(a), calcd
from NaOCl titrn; add slight excess HN0 3 , neutze with CaC0 3 ,
and titr. with 0.1/V AgN0 3 , 941.18, using K 2 Cr0 4 soln,
AOAC Official Methods of Analysis (1990)
Inorganics and Organometallics
161
941.18B(b), or the Ag 3 As0 4 formed in soln, as indicator. Det.
blank on reagents and correct for any CI found. From this cor-
rected titrn and sp gr of sample, calc. % CI. From this value
subtract V2 the % available CI. Difference - % chloride CI.
CAS-7782-50-5 (chlorine)
C. Sodium Hydroxide
Stdze pH meter equipped with calomel and glass electrodes,
using std pH 6.9 buffer soln, 964.24(d).
Place 50 mL 10% BaCl 2 .2H 2 soln and 30 mL 3% H 2 2
soln in 250 mL beaker. Neutze to pH 7.5 with ca 0.1/V NaOH,
using pH meter. Add 10 mL sample from pipet, stir vigorously
1 min, and titr. to pH 7.5 with stdzd 0.1/V HC1, using pH
meter.
% NaOH - (mL HC1 x normality x 4.0)/
(mL sample x sp gr)
Ref.: JAOAC 18, 63(1935); 43, 346(1960).
CAS-1310-73-2 (sodium hydroxide)
D. Carbon Dioxide*
—Surplus 1965
See 4.158-4.159, 10th ed.
B. Total Chlorine
Dissolve 0.5 g sample in 50 mL H 2 in erlenmeyer and add
slight excess std As 2 3 soln as calcd from active CI titrn,
935.10A. Add 5 mL HN0 3 (1+4), neutze with CaC0 3f and
titr. with std AgN0 3 , 941.18, using K 2 Cr0 4 , 941.186(b), as
indicator. Det. blank on reagents and correct for any CI found.
From corrected titrn, calc. % total CI in sample. 1 mL 0.1 AT
AgN0 3 = 0.003545 g CI. If total C\ exceeds active CI, NaCl
is indicated.
C. Sodium*
—Surplus 1974
See 6.183, 11th ed.
Refs.: JAOAC 18, 63(1935).
927.01
Unsulfonated Residue
of Mineral Oats (1)
Final Action
A. Reagent
(Caution: See safety notes on sulfuric acid and fuming acids.)
935.09 Chlorine (Available)
in Calcium Hypochlorite
and Bleaching Powder
Arsenious Oxide Titration Method
Final Action
Weigh 5-10 g thoroly mixed sample into porcelain mortar,
add 30-40 mL H 2 0, and triturate to smooth cream (high-test
Ca(OCl) 2 will dissolve readily and not form a cream). Add
more H 2 0, stir well with pestle, and let insol. residue settle
few moments. Pour mixt. off into 1 L vol. flask, add more
H 2 0, and thoroly triturate sample and pour off as before. Re-
peat operation until all material is transferred to flask. Rinse
mortar and pestle, catch wash H 2 in flask, dil. to vol., and
mix. Without letting material settle, pipet 25-50 mL aliquot
into 200 mL erlenmeyer. Add excess std As 2 3 soln,
935.07A(a), and then decided excess of NaHC0 3 . Titr. excess
As 2 3 with std 1 soln, 935.07A(b), using starch soln,
922.03A(f), or 1 as its own indicator. Subtract vol. I soln,
corrected to 0. IN, from vol. As 2 3 soln used, and calc. %
available CI. 1 mL QAN As 2 3 - 0.003545 g available CI.
Refs.: JAOAC 18, 65(1935).
CAS-7778-54-3 (calcium hypochlorite)
935.10
ChSoramsne T
Pesticide Formulations
Final Action
A. Active Chlorine
Transfer 0.5 g sample to 300—500 mL erlenmeyer, dissolve
in 50 mL H 2 0, and add excess std As 2 3 soln, 935.07A(a),
and 5 mL H 2 S0 4 (1 + 4). Add decided excess NaHC0 3 and titr.
excess As 2 3 with std I soln, 935.07A(b), using starch soln,
922.03A(f), or 1 as its own indicator. From this titrn, calc.
active CI in sample. 1 mL QAN As 2 3 = 0.001773 g active
CI. (To convert active CI to available CI, multiply active CI
by 2.)
Fuming 38N sulfuric acid. — In tared g-s bottle (2.5 L acid
bottle is convenient) mix fuming H 2 S0 4 (free from N oxides)
(jc) with H 2 S0 4 (y) to obtain mixed acid (z), contg slightly
>82.38% total S0 3 . Depending on strength of fuming acid
available, use following proportions of 2 acids: 100 parts jc
(15-20% free S0 3 ) to 50 parts y; 100 parts jc (20-30% free
S0 3 ) to 75 parts y; or 100 parts x (50% free S0 3 ) to 140 parts
y. Mix thoroly (considerable heat is generated), let cool, and
again weigh to det. amt mixed acid obtained. Det. exact strength
of mixed acid (2) and also of reserve supply of acid (y) as
follows:
Pour ca 50 mL into small beaker and fill ca 10 mL weighing
bulb or pipet by slight suction, wiping off outside of bulb with
moist, then with dry, cloth. Weigh on analytical balance and
let acid flow slowly down sides of neck of 1 L vol. flask into
ca 200 mL cold H 2 0. (These sizes of bulb and flask give final
soln ca 0.57V.) When bulb has drained, wash all traces of acid
into flask, taking precautions against loss of S0 3 fumes. Dil.
to vol. and titr. from buret with std alkali, using same indicator
as used in stdzg. Calc. S0 3 content of both acids, and add
calcd amt of reserve acid (y) to amt of mixed acid (2) on hand
to bring z to 82.38% total S0 3 (equiv. to 100.92% H 2 S0 4 ).
After adding required amt of y, again analyze mixed acid to
make certain it is of proper concn (±0.15% H 2 S0 4 ). Keep acid
in small bottles or in special dispenser bottle (2) to prevent
absorption of H 2 from air.
B. Determination
Pipet 5 mL sample into 6" Babcock cream bottle, 938.01 B(b),
either 9 g 50% or 18 g 30% type. To reduce viscosity of heavy
oils, warm pipet after initial drainage by passing it several times
thru flame; then drain thoroly. If greater accuracy is desired,
weigh measured sample and calc, exact vol. from wt and sp
gr. Slowly add 20 mL 3SN H 2 S0 4 , gently shaking or rotating
bottle and taking care that temp, does not rise above 60°. Cool
in ice-H 2 if necessary. When mixt. no longer develops heat
on shaking, agitate thoroly, place bottle in H 2 bath, and heat
10 min at 60-65°, keeping contents of bottle thoroly mixed
by shaking vigorously 20 sec at 2 min intervals. Remove bottle
from bath and add H 2 S0 4 until oil is in graduated neck. Centrf .
5 min (or longer if necessary to obtain const vol. of oil) at
1200-1500 rpm. Read vol. of unsulfonated residue from grad-
162
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
uations on neck of bottle and, to convert to mL, multiply read-
ing from 9 g 50% bottle by 0.1 and reading from 18 g 30%
bottle by 0.2. From result obtained, calc. % by vol. of un-
sulfonated residue.
Refs.: (1) JAOAC 10, 30, 124(1927); 11, 35(1928).
(2) USDA Bull. 898, p. 48.
D, Unsulfonated Residues
Using 5 mL of the recovered oil,
927.01B.
926.02B, proceed as in
926.01
Soaps
Final Action
A. Moisture
Weigh ca 20 g sample into 300-500 mL flask; add 50 mL
toluene (tech. grade is satisfactory); and, to prevent foaming,
add ca 10 g lump rosin (do not use powd). Distil into Dean
and Stark type distg tube receiver and continue distn until no
more H 2 collects in receiver. Cool contents of tube to room
temp., read vol. H 2 under toluene in tube, and calc. % H 2 0.
Ref.: JAOAC 9, 27(1926).
B. Sodium and Potassium*
—Surplus 1974
Se<? 6.094, 11th ed.
926.02 Mineral Oil-Soap Emulsions
Final Action
A. Water
Weigh ca 25 g sample and proceed as in 926. 01 A, except
use less rosin.
Ref.: JAOAC 9, 28(1926).
a Total Oil
Weigh ca 10 g sample into Babcock cream bottle,
920.111B(a). Dil. with ca 10 mL hot H 2 and add 5-10 mL
H 2 S0 4 (1 + 1). Heat in hot H 2 bath ca 5 min to hasten sepn
of oil, add enough satd NaCl soln to bring oil layer within
graduated neck of bottle, centrf. 5 min at 1200 rpm, and let
cool. Read vol. of oil layer, det. density, and from these val-
ues calc. wt and %. From this % value deduct % fatty acids
(and phenols if present), detd sep., to obtain % oil.
Ref.: USDA Bur. Chem. Bull. 105, p. 165.
C. Soap
(Error will result if apparent mol. wt of fatty acids varies ap-
preciably from that of oleic acid.)
Weigh 20 g sample into separator, add 60 mL pet ether, and
ext mixt. once with 20 mL and 4 times with 10 mL 50% al-
cohol. Break emulsion if necessary by letting 1 or 2 mL 20%
NaOH soln run down wall of separator. Then gently swirl sep-
arator and let stand few min. Drain ale. layers and wash suc-
cessively thru pet ether contained in 2 other separators. Com-
bine ale. exts in beaker and evap. on steam bath to remove
alcohol. Dissolve residue in ca 100 mL H 2 made alk. with
NaOH. Transfer to separator, acidify with HO or H 2 S0 4 , ext
3 times with Et ether, and wash ether exts twice with H 2 0.
Combine ether exts, evap. in weighed beaker on steam bath,
and weigh as fatty acids. From wt fatty acids, calc. % soap
in sample as Na or K oleate.
Ref.: JAOAC 9, 28(1926).
E. Ash
Evap. 10 g sample, or more if necessary, in Pt dish. Ignite,
and leach charred mass with H 2 0. Ignite residue, add teach-
ings, evap. to dryness, ignite, and weigh. From this wt, calc.
% ash. Test ash for Cu, Ca, CaF 2 , etc.
Ref.: JAOAC 9, 29(1926).
930.13* Mercury in Organic Mercurial
Seed Disinfectants
Volatilization Method
Final Action
Surplus 1965
See 4.150-4.151, 10th ed.
930.14* Mercury in Organic Mercurial
Seed Disinfectants
Precipitation Method
Final Action
Surplus 1974
See 6.173, 11th ed.
971.04 Mercury in Organic Mercurial
Seed Disinfectants
Titrimetric Method
First Action 1971
Final Action 1974
A. Principle
Sample is digested under H 2 0-cooled condenser with fum-
ing H 2 S0 4 -fuming HN0 3 . Hg is detd by titrn with std SCN
soln with ferric alum as indicator. Small amts of chloride are
oxidized to CI and expelled thru condenser. Not applicable in
presence of large amts of Cl-contg materials.
3. Reagents
(a) Ferric indicator. — Dissolve 8 g FeNH 4 (S0 4 ) 2 .12H 2 in
80 mL H 2 0. Add enough HN0 3 to destroy brown Fe color and
dil. to 100 mL with H 2 0.
(b) Ferrous sulfate soln. — Dissolve 1 g FeS0 4 .7H 2 in H 2 0,
add 1 mL H 2 S0 4 , and dil. to 100 mL with H 2 0. Prep, fresh
for each detn.
C. Preparation of Sample
(a) Solns. — Mix thoroly and weigh, by difference, amt
sample (max. 10 g) contg preferably 0.07 g Hg into 500 mL
¥ erlenmeyer.
(b) Dusts. — Mix thoroly and, using glass weighing dish,
weigh amt sample as in (a). Transfer thru powder funnel into
500 mL ¥ erlenmeyer.
D. Determination
{Caution: Conduct detn in well ventilated hood. Method is
dangerous in presence of material which reacts violently with
H 2 S0 4 and/or HN0 3 , See safety notes on wet oxidation, nitric
acid, sulfuric acid, fuming acids, and mercury salts.)
AOAC Official Methods of Analysis (1990)
Inorganics and Organometalucs
163
Connect straight-tube, H 2 0-cooled condenser to erlenmeyer
contg sample. Place flask in cold H 2 bath. Carefully add 10
mL H 2 S0 4 thru top of condenser and mix by swirling. Add in
small portions, swirling after each addn, 30—40 mL fuming
H 2 S0 4 (20% free S0 3 ) thru top of condenser, followed by 10
mL red fuming HN0 3 (98% HN0 3 ). Remove from bath and
dry outside of flask. Heat with small flame to reflux at ca 30
drops /min with red fumes persisting in flask and condenser.
Heat 30 min; if small amt chloride is present, heat 2 hr with
occasional addn of fuming HN0 3 as required. Cool, and add
100 mL cold H 2 slowly thru top of condenser while cooling
flask in cold H 2 bath. Add 2 or 3 glass beads or boiling chips
and boil until N oxides have been expelled to top of condenser
(ca 2 min). Wash condenser with 50 mL cold H 2 0, disconnect
flask, and add satd KMn0 4 soln until color remains purple. (If
large amts insol. material are present, filter hot soln thru me-
dium tight asbestos mat in gooch before addn of KMn0 4 . Wash
flask and filter 5 times with hot H 2 0, and then add KMn0 4 .)
Cool flask, and destroy KMn0 4 with fresh 1% FeS0 4 soln.
Add 10 mL ferric indicator and titr. with 0.17V NH 4 SCN or
KSCN, 942.26, to appearance of first permanent faint orange.
1 mL 0AN NH 4 SCN or KSCN = 0.01003 g Hg.
Ref.: JAOAC 54, 685(1971).
CAS-7439-97-6 (mercury)
973.11 Mercury in Organic Mercurial
Seed Disinfectants
Gravimetric Method
First Action 1973
Final Action 1975
AOAC-CIPAC Method
(Applicable in presence of large amts CLcontg materials; not
applicable to chioro- or nitrophenols nor to materials not de-
composed by digestion mixt.)
A. Reagents
(a) Dilute sulfuric acid.— Add 30 mL H 2 S0 4 to H 2 in 100
mL vol. flask, cool, and dil. to vol. with H 2 0.
(b) Sodium sulfite soln. — 10%. Dissolve 10 g Na 2 S0 3 in
H 2 in 100 mL vol. flask and dil. to vol. with H 2 0.
(c) Ammonium citrate soln. — Should have sp gr of 1.09 at
20° and pH of 7.0 as detd potentiometrically.
Dissolve 370 g cryst. citric acid in 1.5 L H 2 and nearly
neutze by adding 345 mL NH 4 OH (28-29% NH 3 ). If concn
of NH 3 is 28%, add correspondingly larger vol. and dissolve
citric acid in correspondingly smaller vol. H 2 0. Cool, and check
pH. Adjust with NH 4 OH (1 + 7) or citric acid soln to pH 7.
Dil. soln, if necessary, to sp gr of 1.09 at 20°. (Vol. will be
ca 2 L.) Keep in tightly stoppered bottles and check pH from
time to time. If pH has changed from 7.0, readjust.
(d) Precipitating reagent. — Add 20 mL 1,2-propanedi-
amine (Eastman Kodak Co., P3170) to 100 mL \M CuS0 4
soln. Store in g-s container.
(e) Wash soln. — Add 1 g KI and 2 mL pptg reagent to 1
L H 2 0.
B. Preparation of Sample
(a) Solns. — Mix thoroly and weigh, by difference, sample
(max. 5 g) contg 0.02-0.08 g Hg into 125 mL ¥ erlenmeyer.
(b) Dusts. — Mix thoroly and, using glass weighing dish,
weigh sample as in (a). Transfer thru powder funnel into 125
mL ¥ erlenmeyer.
C. Determination
(Caution: Conduct detn in well ventilated hood.)
Add to sample in following order: 5 mL ethylene glycol,
swirling to thoroly suspend solids, 4 g KI, 10 mL dil. H 2 S0 4 ,
0.4 g I, and 2 glass beads. After thoro mixing, connect straight-
tube, H 2 0-cooled condenser and, with low flame, heat to slight
boil so that liq. condenses in lower portion of condenser. Swirl
occasionally, avoiding excessive heat and crystn of large amt
I in condenser. Reflux 1 hr and, while cooling flask in H 2
bath, immediately wash warm condenser with heavy stream of
ca 25 mL H 2 0. (If dye or I persists in condenser, loosen by
reheating flask contents, without H 2 in condenser, until liq.
refluxes slightly beyond adhering material. Wash condenser
again with ca 25 mL H 2 0, and cool flask.) Disconnect con-
denser and wash connections directly into flask. Add ca 2 mL
10% Na 2 S0 3 dropwise, with swirling, until I color slightly
lightens. (Excess I must be present.) Neutze soln with NH 4 OH,
using pH test paper, until very slightly alk. (pH 7.0-7.3). Cool,
and filter with vac. thru retentive paper (S&S Blue Ribbon,
or equiv.) in buchner into 400 mil beaker. Wash flask and
paper thoroly, keeping total filtrate <150 mL. Add 50 mL
NH 4 citrate soln, bring mixt. just to bp, and stir in 5 mL pptg
reagent. Cool and let stand >2 hr (preferably overnight); filter
thru medium porosity glass crucible, previously dried at 105°
and weighed. Transfer ppt with wash soln, and wash with same
soln several times. Rinse I from ppt with ca 25 mL alcohol in
5 mL portions (some samples may require up to 50 mL) until
filtrate is colorless. (Let alcohol stand few min with occasional
swirling after each addn before applying suction. Ppt should
be suspended in liq. each time.) Wash ppt with three 5 mL
portions CHC1 3 , suspending ppt each time as above until dye
and pesticides are completely removed. Finally wash with 5
mL alcohol, dry 30 min at 105°, cool, and weigh.
Ref.
Wt Hg = wt ppt X 0.218
JAOAC 56, 572(1973); 58, 309(1975).
CAS-7439-97-6 (mercury)
948.03* Alpha-Naphthylthiourea
in Rodenticide Formulations
First Action
Surplus 1965
See 4.132, 10th ed.
939.01 Thalious Sulfate
in Rodenticide Formulations
Final Action
(Caution: See safety notes on asbestos, wet oxidation, nitric
acid, fuming acids, and pesticides.)
Weigh sample contg 0.1-0.15 g T1 2 S0 4 (usually 10 g),
transfer to 800 mL Kjeldahl flask, and add 25 mL H 2 S0 4 fol-
lowed by 5-10 mL HN0 3 . After first violent reaction ceases,
heat until white fumes of H 2 S0 4 appear. Add few drops fuming
HN0 3 and continue heating and adding HNO3 until org. matter
is destroyed, as shown by colorless or light yellow soln. Cool,
add 10-15 mL H 2 0, again cool, and wash contents of flask
into 400 mL beaker, continuing washing until vol. is 60-70
mL. Boil several min to remove all HN0 3 , cool, and filter into
400 mL beaker. Wash with hot H 2 until vol. in beaker is 175
164
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
mL, neutze with NH 4 OH, and then slightly acidify with H 2 S0 4
(1+4). Add 1 gNaHS0 3 to ensure reduction of thallic to thal-
lous state. Heat to bp, add 50 mL 10% KI soln, stir, and let
stand overnight. Filter thru tight gooch contg 2 disks S&S 589
white ribbon paper covered by medium pad of asbestos. Wash
4 or 5 times with 10 mL portions 1% KI soln, and finally with
absolute alcohol. Dry to const wt at 105° (1-1.5 hr), and weigh
as TIL
% T1 2 S0 4 = (g Til x 0.7619 x 100)/g sample
Refs.: JAOAC 22, 411(1939); 25, 79(1942); 28, 72(1945).
CAS-7446-18-6 (thallous sulfate)
966.05 Fumigant Mixtures
Gas Chromatographic Method
First Action 1966
Final Action 1985
(Applicable to org. components of CS 2 , CC1 4 , (CH 2 )2C1 2 , and
(CH 2 )2Br 2 mixts. Precautions: Handle with care in hood or
well-ventilated area. Mixts are volatile, poisonous, and some-
times flammable and may be fatal if inhaled or swallowed.
They cause skin and eye irritation. In case of contact, im-
mediately remove contaminated clothing and flush affected area
with copious amts of H 2 0. Do not reuse clothing until free of
contamination. Do not use containers or equipment of Al, Mg,
or their alloys.)
A. Principle
Components are detd by GC. Peak area of each component
is measured and compared to stds of same fumigant mixt. Pre-
cision of method is ±0.6% for each component.
B. Sampling
Obtain representative 1 L sample from container. Sample
bulk containers by means of weighted bottle, lowered toward
bottom and raised at such rate that it is 3 / 4 full when with-
drawn. Sample drums or small containers with thief or thru
tap or valve located so that sample comes from well below
surface. Prevent contamination of product or sample.
Place sample in clean, dry, and sol v. vapor-tight glass bottle
of such size that it is nearly filled (not above shoulder) by
sample. Vapor-tight g-s bottles or screw-cap bottles with Sn
foil lined caps are satisfactory. Store samples at low temp.;
cool to <18° before opening for analysis.
C. Apparatus
(a) Gas chromato graph. — With flame ionization or thermal
conductivity detector. Typical operating conditions: Column
temp., 110°; injection port temp., 200°; flow rate, 80 mL He/
min.
(b) Recorder. — 0.05-1.05 mv, full scale response. Inte-
grator may be used.
(c) Syringe. — Hamilton Co. 10 jjlL No. 701N, or equiv.
(d) Column. — 1.2 m (4') stainless steel, 7 4 " od, 0.194" id,
packed with reagent 966.05D(a). Max. temp, is 160°. Other
columns can be used but chromatgc conditions and sample size
must be adjusted in accordance with column requirements. One
such column is: 3 m (10') stainless, 3 / J6 " od, 0.12" id, packed
with 20% by wt A^,A^~bis-(2-cyanoethyl) formamide on 80-100
mesh Chromosorb W, acid-washed. Columns are available from
com. suppliers. Criterion for use is emergence of each com-
ponent of mixt. of CS 2 , CC1 4 , (CH 2 ) 2 C1 2 , and (CH 2 ) 2 Br 2 as
sep. peak.
D. Reagents
(a) Column packing. — 30% by wt tricresyl phosphate on
Chromosorb P, 30-60 mesh.
(b) Carbon disulfide std. — ACS.
(c) Carbon tetrachloride std. — ACS.
(d) Ethylene dichloride std. — Purified 1,2-dichloroethane,
available from laboratory supply houses, or use center cut of
fractionation of com. product.
(e) Ethylene dibromide (1 ,2-dibromoethane) std. — Puri-
fied or distd as in (d).
E. Preparation of Standards
Prep, fresh stds just before analysis which approximate ex-
pected composition, by wt, of fumigant mixt. Place proper wt
of each component in 25 mL g-s vol . flask and mix well . Do
not prep, by vol. Cool CS 2 to prevent loss. Adjust wt stds to
detector response.
Carefully fill weighed 10 mL vol. flask to mark with prepd
std and weigh. Use this wt to det. g/5 |ulL values for each
component of std.
F. Determination
Purge column thoroly at 110° before use. Establish base-
line at full sensitivity. Inject 5 \xL std fumigant mixt. into
chromatograph. Attenuate successively so that each peak is at
max. % of chart scale, adjusting sample size and attenuation,
if necessary. Repeat injection. Detd area for each component,
corrected for any baseline drift, should differ by <1%. Order
of elution from column is: CS 2 , CC1 4 , (CH 2 ) 2 C] 2 , and (CH 2 ) 2 Br 2 .
Total analysis time is ca 21 min.
Inject 5 |iL sample into chromatograph. Det. corrected area
of each component from chromatogram, or note integrator
reading.
g Components X C/B, where S — wt component in std,
B — area for component in std, and C = area for component
in sample. Perform calcn for each component in sample.
% Component = g component in sample x 100/ sum of g
components in sample
Last equation is not applicable in presence of unmeasured
contaminants.
Refs.: JAOAC 48, 576(1965); 49, 207(1966).
CAS-75-15-0 (carbon disulfide)
CAS-56-23-5 (carbon tetrachloride)
CAS- 106-93-4 (ethylene dibromide)
CAS- 107-06-2 (ethylene dichloride)
PESTICIDES RELATED TO NATURAL
PRODUCTS AND THEIR SYNERGISTS
953.05 Allethrin (Technical)
and Pesticide Formulations
Titrimetric Method
First Action
{Caution: See safety notes on on pesticides.)
A. Principle
Allethrin reacts quant, with ethylenediamine to form chry-
santhemum monocarboxylic acid which is detd by titrn with
std NaOMe in pyridine. Chrysanthemum monocarboxylic acid,
anhydride, and acid chloride interfere quant, and are detd in-
dependently .
AOAC Official Methods of Analysis (1990)
Synthetics Related to Natural Products
165
B. Reagents
(a) Absolute alcohol. — SDF No. 2-B is satisfactory.
(b) Methanolic hydrochloric acid std soln. — 0. 17V. Dil. 17
ml HC1 (1 + 1) to 1 L with anhyd. MeOH. Stdze against std
OAN NaOH, using phthln. If used at temp,, T, different from
that at which stdzd, T (h calc. corrected normality = N[\~0.00\(T
- r )].
(c) Sodium methylate std soln. — 0.17V in pyridine. Transfer
50 mL 2N NaOMe (Caution: See safety notes on sodium bi-
phenyl, methylate, and ethyl ate.) to 1 L bottle contg 75 mL
anhyd. MeOH and dil. to 1 L with redistd pyridine. Stdze against
NBS benzoic acid, using pyridine as solv. and thymolphthal-
ein, (i), as indicator. Dispense from 50 mL automatic buret
with vents connected to Ascarite tubes. Stdze daily against std
methanolic HCl, (b).
(d) Methanolic potassium hydroxide std soln. — 0.027V.
Dissolve 112 g KOH in 1 L MeOH. Stdze as in 926.16D.
(e) Morpholine soln. — Transfer 8.7 mL redistd morpholine
to 1 L bottle and dil. to 1 L with anhyd. MeOH. Fit bottle
with 2-hole rubber stopper; thru 1 hole insert 20 mL pipet so
that tip extends below surface of liq. , and thru other hole insert
short piece of glass tubing to which is attached aspirator bulb.
(f) Ethylenediamine. — Redistd com. grade contg <3% H 2 0.
Dispense from automatic buret with vents connected to As-
carite tubes.
(g) Dimethyl yellow -methylene blue mixed indicator, — Dis-
solve 1 g dimethyl yellow (p-dimethylaminoazobenzene; cau-
tion', see safety notes on carcinogens) and 0.1 g methylene blue
in 125 mL anhyd. MeOH.
(h) a-Naphtholbenzein indicator. — 1% ale. soln.
(i) Thymolphthalein indicator. — 1% pyridine soln.
C. Determination of Chrysanthemum Monocarboxylic Acid
Chloride
Add 8-10 drops mixed indicator, (g), to ca 150 mL anhyd.
MeOH and add 0.17V HCl, (b), dropwise until soln appears
reddish brown by transmitted light. Add 0.027V KOH, (d),
dropwise until appearance of first green. Transfer 25 mL to
each of three 125 mL g-s erlenmeyers, reserving 1 flask as
ref. color for end point. Into each of other flasks add 1.5-2.5
g sample from weighing pipet, swirling flask while adding
sample. Within 5 min, titr. with 0.027V KOH, (d), to first green
end point, using blank as ref. color. Calc. milliequiv. chry-
santhemum monocarboxylic acid chloride/g sample, C — V
X 7V/g sample, where V= mL TV normal KOH required;
% Chrysanthemum monocarboxylic acid chloride — C x 18.67
D. Determination of Chrysanthemum Monocarboxylic Acid
Transfer 25 mL anhyd. alcohol to each of two 125 mL g-s
erlenmeyers, add 8-9 drops a-naphtholbenzein indicator, and
cool to 0° in ice bath. Neutze by adding 0.02/V NaOH dropwise
to bright green end point. To each flask add 1.5-2.5 g sample
from weighing pipet. Immediately titr. with 0.027V NaOH,
936. 16C, to first bright green end point. Calc. milliequiv.
chrysanthemum monocarboxylic acid and acid chloride/g
sample: D = X x 7V/g sample, where X = mL TV normal NaOH
required; (D - C) x 16.82 = % chrysanthemum monocar-
boxylic acid.
E. Determination of Chrysanthemum Monocarboxylic
Anhydride
Pipet 20 mL morpholine soln, (e), into each of four 250 mL
erlenmeyers, using same pipet. Fill pipet by exerting pressure
in bottle with aspirator bulb. Reserve 2 flasks for blanks; into
each of other flasks add 1 .5-2.5 g sample from weighing pi-
pet. Swirl flasks and let samples and blanks stand 5 min at
room temp. Add 4-5 drops mixed indicator, (g), to each flask
and titr. with OAN HCl, (b), until color changes from green
to faint red when viewed by transmitted light. Calc. millie-
quiv. chrysanthemum monocarboxylic anhydride /g sample: E
= (B - Y) x TV/g sample, where Y = mL TV normal HCl
required for sample, and B — mL TV normal HCl required for
blank; (E — 2C) x 31.84 = % chrysanthemum monocarbox-
ylic anhydride.
F. Determination of Allethrin
Add sample contg 0.8—1.1 g allethrin to each of two 250
mL erlenmeyers from weighing pipet. To each of 2 flasks as
blanks and to samples add 25 mL ethylenediamine, (f), with
swirling. Let samples and blanks stand 2 hr at 25±2°. Wash
down sides of flasks with 50 mL redistd pyridine. To each
flask add 6-10 drops thymolphthalein indicator, (i), and titr.
with OAN NaOMe, (c), to first permanent blue-green end point.
(With colorless samples, first blue end point may be used.)
Calc. milliequiv. allethrin /g sample: F — (Z - B) xTV/g sam-
ple, where Z = mL TV normal NaOMe required for sample,
and B = av. mL TV normal NaOMe required for blank; (F +
C - D - E) x 30.24 = % allethrin.
Refs.: Anal. Chem. 25, 1207(1953). JAOAC 40, 732(1957).
CAS-37-98-4 (allethrin)
973.1 2 d-frans-Allethrin
in Pesticide Formulations
Gas Chromatographic Method
First Action 1973
Final Action 1978
(Caution: See safety notes on pesticides.)
A. Principle
d-trans- Allethrin is dild in acetone contg dibutyl phthalate
as internal std. Ratios of GC peak hts of d-trans-allethrin and
dibutyl phthalate in sample and std are compared for quant,
detn. Method is applicable to both tech. d-trans-eulcthrin and
various formulations of it. Not applicable to formulations contg
large amt MGK Repellent 874 (2-hydroxyethyl-n-octyl sul-
fide).
B. Apparatus and Reagents
(a) Gas chromato graph. — Equipped with flame ionization
detector and 1.2 m (4') x 4 mm id glass column packed with
5% OV-1 (Analabs, Inc.) on 80-100 mesh Chromosorb W
(HP). Operating conditions: temps (°) — column 165, injection
port 230, detector 230; gas flows (mL/min) — N carrier gas
125, air 350-400, H 40-50; sensitivity— 10" 9 amp full scale,
attenuation 4x for tech. material, 10~ 9 amp full scale, atten-
uation 1 for formulations. Before use, condition column 2-3
hr at 275° with N flow 50 mL/min. If necessary, vary column
temp, or gas flow to attain retention times of ca 4 and 7 min
for internal std and d-trans- allethrin, resp. Also vary detector
sensitivity or injection vol. to attain >100 mm peak ht for each
compd (ca 16 |xg d-trans -allethrin). Theoretical plates /ft must
be >200.
Calc. theoretical plates/ft (N) as follows: N = 16L 2 /(M 2 x
F), where L = retention of GC peak in mm; M = peak baseline
produced by drawing tangents to points of inflection of peak;
and F = length of column (ft).
(b) Internal std soln. — 4.0 mg dibutyl phthalate /mL ace-
tone.
(c) d-trans-Allethrin std solns. — (J) Soln 1 . — Approx. 4 mg/
mL. Accurately weigh ca 1.0 g d-trans-a\lethrin (available from
McLaughlin Gormley King Co., 8810 Tenth Ave N, Minne-
166
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
apolis, MN 55427) into 50 mL vol. flask and dil. to vol. with
acetone. Pipet 20 mL this soln into 100 mL vol. flask, add 50
mL internal std soln by pipet, and dil. to vol. with acetone.
Use this soln for detn of tech. material. (2) Soln 2. — Approx.
1 mg/mL. Pipet 25 mL Soln J into 100 mL vol. flask and dil.
to vol. with acetone. Use this soln for detn of d-trans-ailethrin
in formulations.
C. Preparation of Sample
(a) Technical material. — Accurately weigh sample contg
ca 1.0 g d-trans-a\\ethrin into 50 mL vol. flask and dil. to vol.
with acetone. Pipet 20 mL aliquot into 100 mL vol. flask, add
50 mL internal std soln by pipet, and dil. to vol. with acetone.
(b) Formulations. — Accurately weigh sample contg ca 200
mgd-rratt.y-allethrin into 50 mL vol. flask, add 25 mL internal
std soln by pipet, and dil. to vol. with acetone. Pipet 25 mL
aliquot into 100 mL vol. flask and dil. to vol. with acetone.
D. Gas Chromatography
(a) Technical material. — Inject aliquots (ca 3 |xL) std Soln
J until ratio of d- trans -a\\othrin:dibuty\ phthalate peak hts var-
ies <1% for successive injections. Repeat with sample soln,
followed by duplicate injections of std soln. If peak ht ratios
differ >± 1% from previous std injections, repeat series of in-
jections.
(b) Formulations. — Proceed as in (a), using std Soln 2. Re-
peat std injections after each series of 3 sample injections. If
peak ht ratios differ >±1.5% from previous std injections,
repeat injections.
E. Calculations
(a) Technical material, — Calc. peak ht ratios for duplicate
std injections before and after sample injections and average
the 4 values. Calc. and average peak ht ratios for sample in-
jections.
% d-trans-Attethrin = (R/R r ) x (W /W) x P
where W and W = g std and sample, resp.; P = % purity of
std; and R' and R - peak ht ratios of std and sample, resp.
(b) Formulations . — Calc. av. for all std peak ht ratios and
for sample peak ht ratios.
% rf-fra/w-AUethrin = 2 (R/R r ) x (W /W) x (P/5)
Ref.: JAOAC 55, 907(1972).
CAS- 1 37-98-4 (allethrin)
985.03 Cypermethrin in Pesticide Formulations
Capillary Gas Chromatographic Method
First Action 1985
Final Action 1987
(Method is suitable for tech. and formulated cypermethrin.)
A. Principle
Sample is dissolved in CH 2 Cl 2 contg dicyclohexyl phthalate,
and 1.0 jjlL is injected into capillary GC in split mode, with
flame ionization detection. Peak areas are measured for each
cypermethrin isomer and dicyclohexyl phthalate and compared
with those from std injection.
B. Apparatus
(a) Capillary gas chromato graph. — With heated, glass-lined
split mode injection port, flame ionization detector, and au-
tomatic sample injector. Temps (°) — column 240, injection port
250, detector 250; gas flows (mL/min) — He carrier gas 2.75,
split vent 200 (split ratio 72.6:1), septum purge 0.5-1.0, He
auxiliary gas to detector 30, H 60, air 240; column head pres-
sure 15-20 psig; sample size 1.0 jjlL; retention times (min) —
cypermethrin isomers: cis A, 11.18, trans C, 11.55, cis B,
11.85, trans D, 12.02; internal std, 5.58. Adjust parameters
to assure sepn of 4 peaks and peak hts ca 60-80% full scale
on chart at quoted retention times. Sepn of isomer peaks is
critical to avoid inclusion of impurity peaks in active ingre-
dient calcn.
(b) Column. — 25 m x 0.32 mm (id) fused silica column
with thick film OV-1 phase (Hewlett-Packard Co., Avondale
Division, Cat. No. 19091-62025). Precondition 1 h at 260°
before use.
C. Reagents
(a) Dicyclohexyl phthalate internal std soln. — Weigh 0.9 g
dicyclohexyl phthalate (Pfaltz and Bauer, 172 E Aurora St,
Waterbury, CT 06708), dissolve in CH 2 C1 2 , and dil. to 500
mL. Check internal std soln for interfering components by in-
jecting 1.0 |xL into chromatography Store in tightly capped
bottles to avoid evapn.
(b) Cypermethrin std soln. — Accurately weigh ca 100 mg
cypermethrin std of known purity (ICI Americas, Inc.) into
scintillation vial. Pipet 10.0 mL internal std soln into vial, cap,
and shake to dissolve. Store in tightly capped bottles to avoid
evapn .
D. Determination
(a) Liquid and technical samples. — Accurately weigh amt
sample contg ca 100 mg cypermethrin into vial. Pipet 10.0 mL
internal std soln into vial, cap, and shake to dissolve,
(b) Powder formulations. — Accurately weigh amt sample
contg ca 100 mg cypermethrin into vial. Pipet 10.0 mL inter-
nal std soln into vial, cap, and shake on wrist- action shaker
10 min. Let insoluble inerts settle 10 min before analysis.
Inject 2 or more 1.0 jxL aliquots of std soln to optimize
instrument and integration parameters and to stabilize instru-
ment. Monitor response factor until results agree ±2%. Inject
4 aliquots of std soln and 2 aliquots of sample in succession.
Calc. response factor, R, for each injection; and take means
for std and sample for calcn:
R = total area of 4 cypermethrin
isomer peaks/area internal std peak
Cypermethrin, % = (R/R f ) X (W /W) x P
where R and R f — response factors for sample and std solns,
resp.; W and W = mg std and sample, resp.; and P = purity
(%) of std.
Ref.: JAOAC 68, 592(1985).
CAS-523 15-07-8 (cypermethrin)
986.02 Cypermethrin
in Pesticide Formulations
Gas Chromatographic Method
First Action 1986
CIPAC-AOAC Method
A. Principle
Sample is dissolved in methyl isobutyl ketone contg di-
(ethylhexyl) phthalate as internal std; 4 cypermethrin isomers,
isolated as one peak, are detd by gas chromatgy with flame
ionization detection.
0. Apparatus and Reagents
(a) Gas chromatograph with recorder and integrator. — With
flame ionization detector and 1.0 m X 4 mm (id) glass column
packed with 3% OV-101 on 100-120 or 80-100 mesh Chro-
AOAC Official Methods of Analysis (1990)
Synthetics Related to Natural Products 167
mosorb WHP, capable of on-column injection. Condition newly
packed column overnight at 260° with low N flow. Operating
conditions: temps— inlet 250°, column 230-240°, detector 250°;
carrier gas flow to elute internal std at ca 5.5 min and cyper-
methrin at ca 11.5 min with > 30 mm between intercepts of
tangents on baseline of std and internal std peaks; adjust H and
air for detector as recommended by manuf . ; adjust sensitivity
to give peak hts 75% full scale.
(b) Di-(2-ethythexyl) phthalate (DEHP; dioctyl phthal-
ate). — Fisher Scientific Reagent, or equiv.
(c) Methyl isobutyl ketone (MIBK),— GC quality (J.T. Baker
Inc., no. 9212, or equiv.)
C. Preparation of Standards
(a) Internal std soln. — 20 mg DEHP/mL. Weigh ca 10 g
DEHP into 500 mL vol. flask, dil. to vol. with MIBK, and
mix (soln I). Concn may be varied to accommodate column
and instrument differences. If necessary, adjust concn so that
peak ht or area of DEHP closely matches peak ht or area of
cypermethrin within 10%.
(b) Cypermethrin std soln. — 4.0 mg/mL. Warm sealed bot-
tle of cypermethrin std (ICI- Americas, Inc.) at 40-50° until
no crystals remain; shake bottle. Accurately weigh, in dupli-
cate, ca 0.2 g std into 50 mL vol. flask, and dissolve in 3-4
mL MIBK. Pipet 10.0 mL internal std soln into each flask,
dil. to vol. with MIBK, and mix (solns CA, CB). Similarly,
weigh ca 0.1 g cypermethrin std into 25 mL vol. flask, dil. to
vol. with MIBK, and mix (soln CO).
D. Preparation of Sample
(a) Technical formulations . — Proceed as above under cy-
permethrin std soln, using sample wt contg ca 0.2 g cyper-
methrin (solns SA, SB, SO).
(b) Wettable powders. — Accurately weigh, in duplicate,
sample contg 0.2 g cypermethrin into 50 mL vol. flask, pipet
in 10.0 mL internal std soln. and add sufficient MIBK to sus-
pend powder. Thoroly shake flask 10 min, dil. to vol. with
MIBK, and let powder settle or centrf. until clear. Similarly,
prep, soln without internal std, using sample contg ca 0.1 g
cypermethrin /25 mL MIBK.
(c) Ultra-low volume formulations . — Proceed as above un-
der technical formulations, beginning "Accurately weigh. . .".
(d) Emulsifiable concentrates. — Proceed as above under
wettable powders.
E. System Performance Check and Determination
Using instrument conditions listed under Apparatus and Re-
agents, inject 1.5 |ulL portions of solns I, CO, and SO onto
column and check for interfering peaks. On-column injection
is necessary. Inject std soln CA and adjust parameters to give
peak ht ca 75% full scale with peak quality and elution time
specified.
Inject 1.5 jxL portions of std solns CA and CB until response
ratio (area cypermethrin peak/area internal std peak) varies
<0.5% of mean. (Area measurements by digital electronic in-
tegration are preferred over other methods.) Carry out injec-
tions of std and sample solns in following sequence: CA, , SA 1?
SA 2 , CBj, CA 2 , SB 1; SB 2 , CB 2 . Average response ratios for
sample and stds that bracket each sample. Successive response
ratios should agree ± 5% of their mean. If not, repeat analysis.
Cypermethrin, % = (R/R') x (W'/W) x P
where R and R' = av. peak area ratios for sample and std,
resp.; W = g cypermethrin in std soln; W — g sample extd
for analysis; and P = % purity of std.
Ref.: JAOAC 70, 51(1987).
CAS-523 15-07-8 (cypermethrin)
986.03 Permethrin in Pesticide Formulations
Gas Chromatographic Method
First Action 1986
Final Action 1988
CIPAC-AOAC Method
A. Principle
Sample is dissolved in methyl isobutyl ketone contg n-oc-
tacosane as internal std, and permethrin is detd as total area
of 2 isomer peaks by gas chromatgy with flame ionization de-
tection.
B. Apparatus and Reagents
(a) Gas chromatograph with recorder and integrator. — With
flame ionization detector and 1.0 m X 4 mm (id) glass column
packed with 3% OV-210 on 100-120 or 80-100 mesh Chro-
mosorb WHP, capable of on-column injection. Condition newly
packed column overnight at 275° with low N flow. Operating
conditions: temps — inlet 260°, column 190-220°, detector 250°;
carrier gas flow to elute internal std in ca 4.0 min and trans-
permethrin at ca 9.5 min with >30 mm between intercepts of
tangents on baseline of internal std and std cis- and trans-iso-
mer peaks; adjust H and air for detector as recommended by
manuf.; adjust sensitivity to give peak hts 75% full scale.
(b) n-Octacosane std. — With no peaks at retention times of
permethrin isomers (Kodak Laboratory Chemicals, or equiv.).
(c) Methyl isobutyl ketone (MIBK). — GC quality (J.T. Baker
Inc., no. 9212, or equiv.).
C. Preparation of Standards
(a) Internal std soln. — 1 mg w-octacosane/mL. Weigh ca
0.5 g w-octacosane into 500 mL vol. flask, dissolve in 300 mL
MIBK, dil. to vol. with MIBK, and mix (soln I). Concn may
be varied to accommodate column and instrument differences.
If necessary, adjust concn so that peak ht of n-octacosane closely
matches peak ht of permethrin isomers.
(b) Permethrin std soln. — 4.0 mg/mL. Warm sealed bottle
of permethrin std (ICI Americas, Inc.) at 40-50° until no crys-
tals remain; shake bottle. Accurately weigh, in duplicate, ca
0.1 g std into 100 mL g-s erlenmeyer. Pipet 25.0 mL internal
std soln into each flask and shake until permethrin is dissolved
(solns CA, CB). Similarly, weigh ca 0.1 g permethrin std into
25 mL vol. flask, dissolve in 15 mL MIBK, dil. to vol. with
MIBK, and mix (soln CO).
D. Preparation of Sample
(a) Technical formulations . — Proceed as above under per-
methrin std soln, using sample wt contg ca 0.1 g permethrin
(solns SA, SB, SO).
(b) Wettable and dustable powders (suspendibility >50%). —
Accurately weigh, in duplicate, sample contg 0. 1 g permethrin
into 100 mL g-s erlenmeyer. Pipet 25.0 mL internal std soln
into flask, stopper, and shake thoroly 10 min. Let settle, filter
thru Whatman No. 54 paper into g-s flask, and use filtrate for
analysis. Similarly, prep, soln without internal std, using sam-
ple contg ca 0.1 g permethrin/25 mL MIBK.
(c) Emulsifiable concentrates. — Proceed as above under
wettable and dustable powders.
(d) Water -dispersible granules. — Grind ca 20 g sample to
fine powder and thoroly mix. Accurately weigh, in duplicate,
sample contg 0. 1 g permethrin into 100 mL g-s erlenmeyer.
Pipet 25 mL internal std soln into flask and place in ultrasonic
bath 10 min. Proceed as above under wettable and dustable
powders, beginning "Let settle. . .".
E. System Performance Check
Using instrument conditions listed under Apparatus and Re-
agents, inject 3 or more 1.5 fxL portions of soln CA onto col-
168
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
umn and adjust paramenters to give peak ht ca 75% full scale
with peak quality and elution time specified. On-column in-
jection is necessary. Inject 1.5 u,L solns I, CO, and SO and
check for interfering peaks.
F. Determination
Inject 1.5 jjlL std solns CA and CB until response ratio (total
area of cis- and /rtws-permethrin peaks /area internal std peak)
varies <0.5% of mean. (Area measurements by digital elec-
tronic integration are preferred over other methods.) Carry out
injection of std and sample solns in following sequence: CA],
SA 1; SA 2 , CBj, CA 2 , SB), SB 2 , CB 2 . Average response ratios
for sample and stds that bracket each sample. Successive re-
sponse ratios should agree ± 5% of their mean. If not, repeat
analysis.
Permethrin, % - {R/R') X (W /W) X P
where R and R' = av. peak area ratios for sample and std,
resp.; W = g permethrin in std soln; W = g sample extd for
analysis; and P = % purity of std.
Ref.: JAOAC 70, 53(1987).
CAS-52645-53-1 (permethrin)
938.01 Rotenone in Derris
and Cube Powder
Crystallization Method
Final Action
{Caution: See safety notes on carbon tetrachloride and toxic
dusts.)
A. Reagents
(a) Purified rotenone. — Dissolve rotenone in boiling CC1 4 ;
cool in refrigerator or ice bath at 0-10° until pptn of rotenone-
CC1 4 solvate stops. Filter thru buchner and wash once or twice
with ice-cold CC1 4 . Cone, filtrate, crystallize, and filter as be-
fore. Transfer cryst. residue to beaker, add ca twice their vol.
alcohol, and heat nearly to boiling. (Crystals need not dissolve
completely.) Cool to room temp., filter thru buchner, and draw
air thru cryst. residue until most alcohol is removed. Remove
rotenone from funnel, dry in air, and finally heat 1 hr at 105°.
Mp, detd in Pyrex, of purified material should be 163-164°.
(Mother liquors may be coned and rotenone-CCl 4 solvate al-
lowed to crystallize. Cryst. material may be used for further
purification, or kept for prepn of wash solns or for seeding to
induce crystn in detn.)
(b) Rotenone-CCl 4 solvate. — Ppt rotenone from CC1 4 soln,
filter by suction, and dry in air,
(c) Rotenone-CCl 4 wash soln. — Sat. CC1 4 at 0°, and keep
at 0° during use.
(d) Alcohol saturated with rotenone at room temp.
(e) Charcoal, activated. — Norit-A neutral, or equiv.
B. Preparation of Solution
(a) Weigh 30 g (if sample contains >7% rotenone, use amt
to give 1.0—1.5 g rotenone in 200 mL aliquot) finely powd
root and 10 g of the C, (e), into 500 mL g-s erlenmeyer. Add
300 mL CHC1 3 , measured at known room temp.; fasten stop-
per securely and place flask on shaking machine. Agitate vig-
orously ^4 hr, preferably interrupting shaking with overnight
rest (or flask may be shaken continuously overnight). Rapidly
filter mixt. into suitable flask, using fluted paper without suc-
tion and keeping funnel covered with watch glass to avoid evapn
loss. Stopper flask and adjust temp, of filtrate to that of orig-
inal CHCI3.
(b) Alternative extraction method. — If sample has ratio of
rotenone to total ext of >0.4, use amt contg 1.0-1.5 g rote-
none and successively ext 4 times with CHCI3, using 300 mL
CHCI3 and 4 hr agitation for first extn as in (a) and 200 mL
and 2 hr each for other extns. Filter after each extn and return
marc to flask for extn with fresh sol v. Finally combine exts,
evap. almost to dryness, and use entire ext to det. rotenone.
(c) Extraction method for formulations containing 0.75-1.0%
rotenone with or without sulfur and /or pyrethrins. — Weigh
two 50 g portions sample into sep. 500 mL g-s erlenmeyers.
Add 5 g of the C and 300 mL CHC1 3 , measured at known
room temp., to each. Stopper and continue as in (a).
C Determination
{Caution: See safety notes on distillation, pipets, acetone, car-
bon tetrachloride, and chloroform.)
Pipet 200 mL soln, 938. 01B (or entire soln if alternative
extn, (b), is used), into 500 mL Pyrex erlenmeyer and distil
until ca 25 mL remains. (For formulations, 938.01B(c): In ab-
sence of S , combine the 2 exts in one of the erlenmeyers . In
presence of S, remove all CHC1 3 on steam bath in air current,
avoiding prolonged heating. Add 35 mL acetone to each res-
idue and boil gently on steam bath to dissolve all resins. Re-
move from steam bath, stopper tightly, and hold 2 hr at 0-5°.
Filter both acetone solns thru same 15 mL, medium porosity,
fritted glass buchner into single 500 mL erlenmeyer. Rinse and
wash with acetone at 5°. Remove acetone as CHC1 3 was re-
moved above.)
Evap. almost to dryness on steam bath in current of air.
Remove remainder of solv. under reduced pressure, heating
cautiously on steam bath when necessary to hasten evapn.
(Suction may be applied directly to flask if stopper with vent
is used to release pressure, so that excessive vac, may be
avoided. Use flasks with slightly convex bottoms; do not use
flasks below av. wt,) Dissolve ext in 15 mL hot CC1 4 and
again, in similar manner, remove all solv. Repeat with another
10-15 mL portion hot CC1 4 . (This treatment removes all CHC1 3
from resins. CHC1 3 ext is usually completely sol. in CC1 4 ; if
small amts of insol. material are present, purification de-
scribed later will eliminate them.)
Dissolve residue in ca 10 mL CC1 4 and transfer quant, with
hot CC1 4 to 50 mL erlenmeyer marked at 25 mL. Adjust vol.
to 25 mL by evapg on steam bath or by adding CC1 4 . Cool
flask in ice bath several min, stopper flask, and swirl until
crystn is apparent. Seed with few crystals of rotenone-CCl 4
solvate if necessary to induce crystn. If at this stage only small
amt of cryst. material seps, add accurately weighed amt of
purified rotenone, 938.01A(a), estd to be enough to assure that
final result, expressed as pure rotenone, is ^1 g. Then warm
to dissolve completely, and again induce crystn. At same time
prep, satd soln of rotenone in CC1 4 , 938.01A(c), for washing.
Place flasks contg ext and washing soln in ice bath capable of
holding temp, at 0°, and let stand overnight. (Store ice bath
in refrigerator to keep ice from melting too rapidly.)
After 17-18 hr in ice bath, rapidly filter ext thru weighed
gooch fitted with filter paper disk, removing flask from ice
bath only long enough to pour each fraction of ext into cru-
cible. Rinse cryst. residue from flask and wash under suction
once with the ice-cold satd rotenone-CCl 4 wash soln. (<12—
15 mL soln should be used for rinsing and washing.) Continue
suction ca 5 min; then dry to const wt at 40° (ca 1 hr). Wt
obtained is crude rotenone-CCl 4 solvate.
Break up contents of crucible with spatula, mix thoroly, and
weigh 1.000 g into 50 mL erlenmeyer. Add 10 mL alcohol
previously satd with rotenone at room temp., swirl flask few
min, stopper tightly, and set aside ^4 hr, preferably overnight,
AOAC Official Methods of Analysis (1990)
Natural Products and Synergists 1 69
at same temp. Filter on weighed gooch fitted with filter paper
disk, Rinse crystals from flask and wash under suction with
alcohol satd with rotenone at temp, of recrystn (ca 10 mL usu-
ally required). Continue suction 3-5 min and then dry crucible
at 105° to const wt (ca 1 hr).
Multiply g residue by g total crude rotenone-CCl 4 solvate,
and add 0.07 g to product as correction for rotenone held in
soln in the 25 mL CC1 4 used in crystn. If any pure rotenone
was added, subtract its wt from value obtained. This gives wt
pure rotenone contained in aliquot of ext.
Note: Most important precaution in using this method is to
keep temp, of CCl 4 -rotenone wash soln and crucibles as near
0° as possible. Keep wash soln surrounded by crushed ice ex-
cept when actually being used. In warm weather keep cruci-
bles in refrigerator until ready to use.
Refs.: Ind. Eng. Chem. Anal. Ed. 10, 19(1938). JAOAC 21,
148(1938); 22, 408(1939); 24, 70(1941); 43, 376(1960).
CAS-83-79-4 (rotenone)
961.03 Rotenone in Derris and Cube Powder
Infrared Spectroscopic Method
First Action
(Not applicable to derris products)
A. Standardization
Prep, std solns of purified rotenone, 938.01 A(a), in CHC1 3
at concns of 5, 10, 15, and 20 mg/mL. Scan each std soln
from 7.0 to 8.0 fxm at speed of 6 min/|xm and scale of 10
cm/|xm, using 0.1 mm cell and accurately matching cell filled
with CHC1 3 as ref. Scan each in duplicate. Obtain av. A of
each concn, using 7.57 |mm as base point and 7.65 (Jim as peak.
Plot A against concn.
B. Determination
Weigh sample contg 250-300 mg rotenone into 25 x 200
mm culture tube. Add 1-2 g anhyd. Na 2 S0 4 , 2 g activated
charcoal, and 50 mL CHC1 3 by pipet. Close securely with Tef-
lon-lined screw cap and tumble end over end 1 hr at ca 35
rpm. Filter thru medium paper, avoiding evapn losses. Trans-
fer 20 mL aliquot to 50 mL erlenmeyer and evap. on steam
bath with current of air. Transfer residue to 10 mL g-s vol.
flask and dil. to vol. with CHC1 3 . Stopper, and mix thoroly.
Scan from 7.0 to 8.0 |xm, using 0.1 mm cell and matched
cell filled with CHC1 3 as ref. Det. A by baseline method from
7.57 to 7.75 )xm and peak at 7.65 |xm, using same scanning
speed and scale expansion as in stdzn.
Calc. % rotenone from std curve and wt sample in final diln.
Refs.: JAOAC 44, 580(1961); 46, 668(1963); 59, 380(1976).
CAS-83-79-4 (rotenone)
940.04
Ether Extract of Derris
and Cube Powder
Final Action
flam-
(Caution: See safety notes on monitoring equipment,
mable solvents, diethyl ether, and peroxides.)
Ext 5 g finely powd root with ether 48 hr in Soxhlet or other
efficient extn app. Cone, ext and filter off any insol. material
present. Receive filtrate in tared beaker, evap. ether on steam
bath, and dry in oven at 105° to const wt.
983.06 Rotenone in Pesticide Formulations
Liquid Chromatographic Method
First Action 1983
A. Principie
Sample is extd with dioxane, and rotenone is detd by reverse
phase LC with UV detection at 280 nm.
B. Apparatus and Reagents
(a) Liquid chromato graph. — M6000A pump, U6K injector,
Model 450 variable UV detector (all Waters Associates, Inc.),
and Omni- Scribe recorder (Houston Instrument, 8500 Cam-
eron Rd, Austin, TX 78753), or equiv. system. Operating con-
ditions: column ambient; flow rate 1.0 mL/min for Partisil
column, 1.5 mL/min for Zorbax column, 1.2 mL/min for
Bondapak column; injection vol. 5 pJL for Partisil column and
10 |xL for others; detector wavelength 280 nm; absorbance range
0.4 AUFS; chart speed 1 cm/min.
(b) Chromatographic columns. — Partisil 5 ODS-3, 5 fxm
particle size, stainless steel, 25 cm X 4.6 mm id (Whatman
Inc.). Zorbax C 8 , 10 \xm particle size, stainless steel, 25 cm
x 4.6 mm id (DuPont Co.). jxBondapak C 18 , 10 \xm particle
size, stainless steel, 30 cm X 3.9 mm id (Waters Associates,
Inc.).
(c) Mobile phases. — Use LC grade org. solvs (Fisher Sci-
entific Co.). Use glass-distd H 2 treated to remove org. compds
by passing thru C 18 column system (Millipore Corp.) or use
LC grade H 2 0. Use MeOH-H 2 (75 + 25), (68+32), and
(66+34) for Partisil, Zorbax, and Bondapak, resp. If neces-
sary, adjust mobile phase to give adequate sepn of tephrosin,
rotenone, and deguelin in test soln (Fig. 983.06).
(d) Test soln. — Accurately weigh portion of well mixed
sample of Noxfish Fish Toxicant or powd cube root ext (Rous-
sel BioCorp, 400 Sylvan Ave, PO Box 1077, Lyndhurst, NJ
07071) contg ca 20 mg rotenone into 125 mL g-s erlenmeyer.
Add 50 mL dioxane, and mix.
(e) Std soln. — Accurately weigh ca 20 mg 99% pure rote-
none (Penick Co.) into 50 mL vol. flask and dil. to vol. with
w
_JUU
10
12
14
TIME(min)
FIG. 983.06 — Liq. chromatogram of rotenone sample with
Whatman column: a, rotenolone; b, tephrosin and an un-
known; c, rotenone; d, deguelin
170
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
dioxane (reagent grade). Keep rotenone from light or store in
actinic glassware.
(f) Sample extraction solvent. — Reagent grade dioxane.
C. Preparation of Sample
(a) Solid formulations. — Accurately weigh portion of well
mixed sample contg ca 20 mg rotenone into 125 mL g-s er-
lenmeyer. Pipet in 50 mL dioxane, stopper, and shake 1 i / 2 hr
on rotary shaker. Let settle and filter aliquot thru 0.45 |xm
organic filter (Millipore Corp.), or equiv.
(b) Liquid formulations. — Use same procedure as above,
omitting rotary shaking and settling.
D. Determination and Calculation
Inject std soln followed by 2 injections of sample soln and
another injection of std soln. Measure peak hts, average, and
calc. as follows:
% Rotenone - (PH/PH') X (W /W) X % purity of std
where PH and PM' = av. peak hts of sample and std solns,
resp.; W = g rotenone std/50 mL; and W - g sample extd.
Ref.: J AOAC 66, 796(1983).
CAS-83-79-4 (rotenone)
960.11 Piperonyl Butoxide
in Pesticide Formulations
Colorimetric Method
First Action 1960
Final Action 1961
A. Apparatus and Reagents
(a) Photoelectric colorimeter. — Equipped with narrow band-
pass interference type filter with central wavelength 630 nm.
(Filter is available from PTR Optics Corp, 145 Newton St,
Waltham, MA 02154.) Spectrophtr set at wavelength in range
625-635 nm may also be used.
(b) Purified tannic acid. — Purify as follows: To 20 g tannic
acid (USP reagent grade) add 100 mL EtOAc (99%) and stir
mech. ca 1 hr. Filter by suction thru fritted glass funnel, and
wash residue with three 5 mL portions EtOAc. To combined
filtrate and washings add 2 g finely powd Darco G-60 (or equiv.
decolorizing C), and stir mech. ca 0.5 hr. Filter by gravity
thru double thickness Whatman No. 1, or equiv., paper into
graduated dropping funnel. Wash residue several times with
EtOAc until vol. filtrate and washings is ca 125 mL. Place
dropping funnel over 1 L, 3 -neck, r-b flask, equipped with
mech. stirrer, and with vigorous agitation in flask, add filtrate
dropwise to 5 times its vol. of toluene. Purified tannic acid is
pptd immediately.
Filter by suction thru fritted glass funnel, and wash product
thoroly with toluene, stirring solids with toluene to assure
complete removal of EtOAc. Continue suction until practically
all toluene is removed. Dry purified tannic acid in vac. oven
at ca 40°, and place in tightly stoppered bottle.
(c) Tannic acid reagent. — Completely dissolve exactly 0.025
g purified tannic acid in 20 mL HOAc by shaking at room
temp. Add 80 mL H 3 P0 4 and mix thoroly. Prep, fresh daily.
Store tightly stoppered, as it is hygroscopic.
(d) Purified piperonyl butoxide. — Purify by low pressure
fractional distn of tech. product. (Caution: See safety notes on
vacuum.) Also available from Fairfield American Corp., 3932
Salt Rd, Medina, NY 14103.
(e) Piperonyl butoxide std soln. — 50 juLg/0. 1 mL. Weigh
exactly 1.000 g purified piperonyl butoxide into 100 mL vol.
flask. (Hypodermic syringe and needle are convenient for add-
ing compd to flask.) Dil. to vol. with deodorized kerosene and
mix well. Pipet 10 mL of this soln into 200 mL vol. flask.
Dil. to vol. with deodorized kerosene and mix well. This soln
is stable for several months. If std is to be used with sample
contg pyrethrum, add enough pyrethrum ext to std before ini-
tial diln to give ratio piperonyl butoxide to pyrethrins similar
to sample.
B. Preparation of Sample
Accurately weigh sample contg 0.5-1.5 g piperonyl butox-
ide into tared 100 mL vol. flask, dil. to vol. with deodorized
kerosene, and mix well. Pipet 10 mL into 200 mL vol. flask,
dil. to vol. with deodorized kerosene, and mix well.
C. Determination
Pipet 0.1 mL (from 1 mL pipet graduated in 0.1 mL) sample
soln into 18 x 150 mm test tube. Add exactly 5 mL tannic
acid reagent and shake vigorously 1 min. Treat std and blank,
consisting of 0. 1 mL deodorized kerosene, simultaneously in
same manner.
Place test tubes in test-tube basket and place in vigorously
boiling H 2 bath 5 min. Remove basket and let tubes cool to
room temp. Transfer solns to colorimeter tubes and read, against
H 2 0, using 625-635 nm filter or setting. (After cooling to
room temp, there is no appreciable change in A for several
hr.)
Subtract A of deodorized kerosene from readings of both
sample, A, and std, A f .
mg Piperonyl butoxide = A x 0.05 /A'
Refs.: JAOAC 35, 771(1952); 43, 350(1960).
CAS-5 1-03-6 (piperonyl butoxide)
936.05 Pyrethrin in Pesticide Formulations
Mercury Reduction Method (1)
Final Action
(Caution: See safety notes on flammable solvents, diethyl ether,
peroxides, and petroleum ether.)
A. Reagents
(a) Deniges reagent. — Mix 5 g yellow HgO with 40 mL
H 2 0, and, while stirring, slowly add 20 mL H 2 S0 4 ; then add
addnl 40 mL H 2 and stir until all dissolves. Test for absence
of mercurous Hg by adding few drops of (b) to 10 mL and
titrg with (c) as in 936. 05C, par. 2, beginning "Add 50 mL
previously prepd and cooled dil. HO ..."
(b) Iodine monochloride soln. — Dissolve 10 g KI and 6.44
g KI0 3 in 75 mL H 2 in g-s bottle; add 75 mL HC1 and 5 mL
CHCI3, and adjust to faint I color (in CHC1 3 ) by adding dil.
KI or KIO3 soln. If much I is liberated, use stronger soln of
KIO3 than 0.01 M at first, making final adjustment with 0.0 1M
soln. Keep in dark and readjust when necessary. Do not store
in refrigerator.
(c) Potassium iodate std soln. — 0.01 A/. Dissolve 2.14 g pure
K10 3 , previously dried at 105°, in H 2 and dil. to 1 L. 1 mL
= 0.0057 g pyrethrin I and needs no further stdzn.
(d) Alcoholic sodium hydroxide soln. — (/) J.0N. — Dis-
solve 40 g NaOH in alcohol and dil. to 1 L with alcohol. (2)
0SN— Dil. LOW with alcohol (1 + 1).
(e) Petroleum ether. — Aromatic-free, bp range 30-60°.
(f) Ethyl ether. — Peroxide-free, reagent grade.
B. Preparation
(a) Pyrethrum powder. — Ext sample contg 40-150 mg to-
tal pyrethrins in Soxhlet or other efficient extn app. 7 hr with
AOAC Official Methods of Analysis (1990)
Natural Products and Synergists
171
pet ether. After extn is complete, evap. pet ether to c a 40 triL,
stopper flask, and place in refrigerator at ± 0.5° overnight.
Filter cold ext thru cotton plug satd with cold pet ether, in stem
of funnel, collecting filtrate in 250 mL erlenmeyer. Wash with
three 15 mL portions cold pet ether. Evap. filtrate and wash-
ings on H 2 bath, using air current, until <1 mL solv. re-
mains.
Add 15-20 mL 0.5/V ale. NaOH to evapd ext, connect to
reflux condenser, and boil gently 1-1.5 hr. Transfer to 600
mL beaker and add enough H 2 to bring vol. to 200 mL. Add
few glass beads, or preferably use boiling tube, and boil down
to 150 mL. Transfer to 250 mL vol. flask and add 1 g Filter-
Cel and 10 mL 10% BaCl 2 soln. Do not shake before dilg to
vol. Dil. to vol., mix thoroly, filter off 200 mL, neutze with
H 2 S0 4 (1 +4), using 1 drop phthJn, and add 1 mL excess.
(If necessary to hold soln overnight at this point, leave in alk.
condition.)
(b) Pyrethrum extracts in mineral oil. — Weigh or measure
sample contg 40-150 mg total pyrethrins, add 50 mL pet ether
and 1 g Filter-Cel, and place in refrigerator at ± 0.5° over-
night. Filter thru gooch into 300 mL erlenmeyer and wash with
three 15 mL portions cold pet ether. Evap. filtrate and wash-
ings on H 2 bath, using air current, until <1 mL solv. re-
mains.
Add 20 mL \N ale. NaOH, or more if necessary, to ext,
connect to reflux condenser, and boil gently 1-1.5 hr. Trans-
fer to 600 mL beaker and add enough H 2 to make aq. layer
200 mL. If >20 mL ale. NaOH soln was used, add enough
H 2 so that all alcohol is removed when vol. is reduced to
150 mL. Add few glass beads, or preferably use boiling tube,
and boil aq. layer down to 150 mL. Transfer to 500 mL sep-
arator and drain aq. layer into 250 mL vol. flask. Wash oil
layer once with H 2 and add wash H 2 to aq. portion. If slight
emulsion still persists after draining aq. layer and washings,
add 2-3 mL 10% BaCl 2 soln, but do not shake vigorously after
adding BaCI 2 because reversed emulsion difficult to sep. may
form. To aq. soln in 250 mL flask add 1 g Filter-Cel and >10
mL of the BaCl 2 soln. Swirl gently and let stand 30 min. Dil.
to vol., mix thoroly, and filter off 200 mL. Test filtrate with
BaCl 2 soln to see if enough has been added to obtain clear
soln. Neutze with H 2 S0 4 (1 + 4), using 1 drop phthln, and
add 1 mL excess. (If necessary to hold soln overnight at this
point, leave in alk. condition.)
C. Determination of Pyrethrin I
Filter acid soln from 936.05B(a) or (b) thru 7 cm paper,
coated lightly with suspension of Filter-Cel in H 2 0, on buch-
ner, and wash with three 15 mL portions H 2 0. Transfer to 500
mL g-s separator and ext with two 50 mL portions pet ether.
Shake each ext >1 min, releasing pressure if necessary by in-
verting separator and carefully venting thru stopcock. Let lay-
ers sep. >5 min or until aq. layer is clear before draining and
re-extn. Reserve aq. layer for pyrethrin II detn. Do not com-
bine pet ether exts but wash each in sequence with same three
10 mL portions H 2 0, and filter pet ether exts thru small cotton
plug into clean 250 mL separator. Wash separators and cotton
in sequence with 5 mL pet ether. Ext combined pet ether solns
with 5 mL 0. \N NaOH, shaking vigorously >1 min. Let lay-
ers sep. >:5 min before draining aq. layer into 100 mL beaker.
Wash pet ether with addnl 5 mL portion 0. 17V NaOH and with
5 mL H 2 0, adding washings to beaker. Add 10 mL Deniges
reagent and let stand in complete darkness 1 hr at 25 ± 2°.
Add 20 mL alcohol and ppt HgCI with 3 mL satd NaCl
so /n. Warm to ca 60° and let stand several min until ppt co-
agulates and settles. Filter thru small paper, transferring all ppt
to paper, and wash with >10 mL hot alcohol. Wash with 2
or more 10 mL portions hot CHC1 3 and place paper and con-
tents in 250 mL g-s erlenmeyer. Add 50 mL previously prepd
and cooled dil. HC1 (3 + 2). Add 5 mL CHC1 3 or CC1 4 and
1 mL freshly adjusted IC1 soln, and titr. with 0.01M KI0 3
soln, shaking vigorously >30 sec after each addn, until no I
color remains in CHCl 3 or CC1 4 layer. Take as end point when
red color disappears from solv. layer and does not return within
1-3 min. From mL std K10 3 soln used in titrn and blank on
Deniges reagent, calc. % pyrethrin I.
(Reactions:
2Hg 2 Cl 2 + 4IC1 = 4HgCl 2 + 2I 2
2I 2 + KIO3 + 6HC1 = KC1 + 5IC1 4- 3H 2
Addn of IC1 does not change vol. relationship between mer-
curous Hg and KI0 3 soln, and aids in detg end point in titrn
of small amts of Hg.)
Note: Chrysanthemum monocarboxylic acid reacts with De-
niges reagent to form series of colors beginning with phthln
red, which gradually changes to purple, then to blue, and fi-
nally to bluish green. Color reaction is very distinct with 5 mg
monocarboxylic acid, and amts as low as 1 mg can usually be
detected. Therefore no pyrethrin I should be reported if color
reaction is neg.
With samples contg much perfume or other saponifiable in-
gredients, it may be necessary to use as much as 50 mL IN
ale. NaOH. When lethanes are present, after washing HgCI
ppt with alcohol and CHC1 3 , wash once more with alcohol and
then several times with hot H 2 0.
D. Determination of Pyrethrin II (2)
If necessary, filter aq. residue from pet ether extn thru gooch.
Cone, filtrate to ca 50 mL and transfer to 500 mL g-s sepa-
rator. Wash beaker with three 15 mL portions H 2 0. Acidify
with 10 mL HC1 and sat. with NaCl. (Acidified aq. layer must
contain visible NaCl crystals thruout following extns.)
Ext with 50 mL ether, drain aq. layer into second separator,
and ext again with 50 mL ether. Continue extg and draining
aq. layer, using 35 mL for third and fourth extns. Shake each
ext >: 1 min, releasing pressure, if necessary, by inverting sep-
arator and carefully venting thru stopcock. Let layers sep. >5
min or until aq. layer is clear before subsequent draining and
extn. Combine ether exts, drain, and wash with three 10 mL
portions satd NaCl soln. Filter ether exts thru cotton plug into
500 mL erlenmeyer and wash separator and cotton with addnl
10 mL ether. Evap. ether on H 2 bath, and remove any fumes
of HC1 with air current and continued heating <5 min. Dry
10 min at 100°.
(a) For crude pyrethrum exts. — Treat residue with 75 mL
boiling H 2 and filter thru 9-11 cm Whatman No. 1 , or equiv.,
paper. Wash flask and paper with five 20 mL portions boiling
H 2 or until filtrate from final wash is neut. to litmus. Add
1-2 drops phthln and rapidly titr. with 0.027V NaOH (1 mL
= 0.00374 g pyrethrin IT). Check normality of 0.02/V NaOH
same day sample is titrd.
(b) For refined pyrethrum exts. — Add 2 mL neut. alcohol
and 20 mL H 2 0, and heat to dissolve acid. Cool, filter thru
gooch if necessary, add 1-2 drops phthln, and titr. with 0.02/V
NaOH (1 mL = 0.00374 g pyrethrin II). Check normality of
0.02jV NaOH same day as sample is titrd.
Refs.: (1) Contrib. Boyce Thompson Inst. 8, No. 3, 175(1936).
Ind. Eng. Chem. Anal. Ed. 10, 5(1938). JAOAC 43,
358(1960).
(2) Soap 10, No. 5, 89(1934). JAOAC 43, 354(1960);
46, 664(1963); 56, 915(1973).
CAS-12L21-1, 121-29-9 (pyrethrins)
172
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
982.02 Pyrethrins and Piperonyl Butoxide
in Pesticide Formulations
Gas Chromatographic Method
First Action 1982
Final Action 1983
A. Principle
Sample is dild with acetone contg dicyclohexyl phthalate in-
ternal std and detd by GC with flame ionization detection.
Method is applicable to tech. piperonyl butoxide [80% butyl-
carbityl 6-propylpiperonyl ether and 20% related compds] and
most formulations contg pyrethrins and piperonyl butoxide ex-
cept shampoo products. Occasionally, an oil diluent will in-
terfere with GC detn. Method may not be applicable to sam-
ples contg <0.1% pyrethrins. Variation in active constituents
of pyrethrin ext may cause minor deviations from expected
results.
B. Apparatus and Reagents
(a) Gas chromato graph. — Equipped with flame ionization
detector and 122 cm X 4 mm id glass column packed with 5%
OV-101 or 5% OV-I (Analabs, Inc.) on 80-100 mesh Chro-
mosorb W(HP). Operating conditions: column 210°, injection
port 250°, detector 250°; gas flows (mL/min) — N carrier gas
flow 50, air 350-400, and H 40-50; sensitivity 10" 10 AUFS.
Adjust attenuation to maintain 50-75% FSD for 1.0-1.5 fxg
piperonyl butoxide. Before use, condition column 2-3 h at
275° with N flow 50 mL/min. If necessary, vary column temp,
or gas flow to attain retention times of ca 13-15 min for in-
ternal std. Theoretical plates /ft must be >400, based on di-
cyclohexyl phthalate peak,
Calc. theoretical plates/ft (N) as follows: N = 16 x (L 2 /M 2
x F), where L = retention of GC peak (mm); M = peak base-
line (mm) produced by drawing tangents to points of inflection
of peak; and F = length of column (ft).
(b) Internal std soln. — 8.0 mg dicyclohexyl phthalate (Pfaltz
and Bauer, Inc., 172 Aurora St., Waterbury, CT 06708)/mL
acetone.
(c) Std soln. — (/) Std soln A. — 0.5 mg piperonyl butoxide/
mL. Accurately weigh ca 0.25 g piperonyl butoxide (available
from McLaughlin Gormley King Co., 8810 Tenth Ave N,
Minneapolis, MN 55427) into 50 mL vol. flask and dil. to vol.
with acetone. Pipet 10 mL this soln into 100 mL vol. flask,
add 5 mL internal std soln by pipet, and dil. to vol. with ace-
tone. Use this soln for detn of tech. piperonyl butoxide. (2)
Std soln B. — Accurately weigh ca 0.25 g piperonyl butoxide
into 50 mL vol. flask. Add weighed amt of pyrethrins such
that ratio of active ingredients closely resembles that which is
expected in sample. Dil. to vol. with acetone. Pipet 10 mL of
this soln into 100 mL vol. flask, add 5 mL internal std soln
by pipet, and dil. to vol. with acetone. Use this soln for detn
of pyrethrins and piperonyl butoxide in formulations.
C. Preparation of Sample
(a) Technical piperonyl butoxide . — Accurately weigh ca . 25
g sample into 50 mL vol. flask and dil. to vol. with acetone.
Pipet 10 mL this soln into 100 mL vol. flask, add 5 mL in-
ternal std soln by pipet, and dil. to vol. with acetone.
(b) Pyrethrins-piperonyl butoxide formulations . — (./) Liqs. —
Accurately weigh sample contg ca 0.05 g piperonyl butoxide
into 100 mL vol. flask, add 5 mL internal std soln by pipet,
and dil. to vol. with acetone. (2) Aerosol formulations . — Cau-
tion: Open aerosol behind safety shield and in hood. Weigh
aerosol can to nearest 0.1 g (G). Puncture as small a hole as
possible in top of can with sharp punch and hammer to allow
propellant to release very slowly, (Best results can be obtained
by allowing punctured can to stand overnight.) After hiss of
escaping propellant is no longer evident, cut open top of can
with hand can opener. Leave ca 1 cm attached to can and bend
top open. Carefully warm can in beaker of warm tap H 2 sev-
eral minutes to ensure complete removal of propellant. Trans-
fer aerosol nonvolatiles to vol. flask with aid of acetone. Rinse
can thoroly, adding rinses to vol. flask. If aerosol is 8 oz, use
2 L (V) vol. flask. This vol. is necessary to ensure complete
miscibility of oil phase of aerosol contents. Dil. to vol. with
acetone and mix thoroly. Dry empty can and weigh (T). Trans-
fer aliquot (A) (must be ^90 mL), equiv. to 50 mg piperonyl
butoxide, to 100 mL vol. flask, add 5 mL internal std soln by
pipet, and dil. to vol. with acetone.
Wt sample = (G - 7^ X (A/V)
D. Gas Chromatography
Inject 2-3 julL aliquots of std soln until internal std ratios
vary <2% for successive injections. Det. baseline by drawing
straight line to min. on either side of peak of interest. For
pyrethrins, use combined ht of cmerin I and pyrethrin I peaks
for internal std ratio. Repeat injection procedure with sample
soln, followed by injection of std soln. If std peak ratios differ
2: ±2.0%, repeat series of injections. Injection vol. should not
vary >±10%. Calc. peak ht ratios (sample peak ht/internal
std peak ht) of std injections before and after sample injections
and average std ratio preceding and following sample injec-
tions. Calc. av. peak ht ratios for sample injections. After elu-
tion of piperonyl butoxide, allow ca 7 min for elution of ex-
traneous peaks.
% Piperonyl butoxide or pyrethrins = (R/R') X (W /W) X P
where W = g std in final diln; W = g sample in final diln; P
~ % purity of std; R ! = ratio of std; and R ~ ratio of sample.
Ref.: JAOAC65, 249(1982).
CAS-51-03-6 (piperonyl butoxide)
CAS-121-21-1, 121-29-9 (pyrethrins)
980.04 W-Octyl Bicycloheptene Dicarboximide
in Pesticide Formulations
Gas Chromatographic Method
First Action 1980
Final Action 1981
(Caution: See safety notes on pesticides.)
A. Principle
Sample is dild with acetone contg dibutyl phthalate as in-
ternal std. GC peak ht or area ratios of MGK 264 (/V-octyl
bicycloheptene dicarboximide) to dibutyl phthalate peak of
sample and std are compared for quantitation. Method is ap-
plicable to technical MGK 264 and to several formulations.
Not applicable to formulations contg Dursban® and isopropyl
palmitate. Presence of large amts of MGK® Repellent 326 causes
slightly high results.
B. Apparatus and Reagents
(a) Gas chromato graph. — With flame ionization detector
and 120 cm x 4 mm (id) glass column packed with 5% OV-
1 (Analabs, Inc.) on 80-100 mesh Chromosorb VV(HP). Op-
erating conditions: temps (°) — column 170, injection port 250,
detector 250; gas flows (mL/min) — N carrier gas 60, air 350-
400, H 40-50; sensitivity — 10" 10 amp full scale, attenuation
16 x . Before use, condition column 2-3 hr at 275° with N flow
50 mL/min. If necessary, vary column temp, or gas flow to
attain retention times of ca 6 and 8 -min for internal std and
MGK 264, resp. Also vary detector sensitivity or injection vol.
AOAC Official Methods of Analysis (1990)
Natural Products and Synergists 1 73
to attain >100 mm peak ht for each compd (ca 9 fxg MGK
264). Theoretical plates/ft must be >300.
Calc. theoretical plates/ft (TV) as follows: TV - 16 L 2 /M 2 x
F), where L = retention GC peak in mm; M = peak baseline
produced by drawing tangents to points of inflection of peak;
and F = length of column (ft).
(b) Internal std soln. — 5.0 mg dibutyl phthalate (Monsanto
Co., 98%)/mL acetone.
(c) MGK 264 std soln. — Accurately weigh ca 0.15 g MGK
(available from McLaughlin Gormley King Co., 8810 Tenth
Ave N, Minneapolis, MN 55427) into 50 m'L vol. flask, add
10.0 mL internal std soln, and dil. to vol. with acetone.
C. Determination
Accurately weigh sample contg ca 0.15 g MGK 264 into 50
mL vol. flask, add 10.0 mL internal std, soln, and dil. to vol.
with acetone.
Inject aliquots (2-3 julL) std soln until ratio of MGK 264 to
dibutyl phthalate peak hts (larger peak) or area (use area of
both MGK 264 peaks) varies <1% for successive injections.
Repeat with sample soln, followed by duplicate injections of
std soln. If std peak ratios differ by more than ±1.5% repeat
series of injections. Injection vols should not vary more than
±10%. (After eiution of MGK 264, it is advantageous to in-
crease column temp, to reduce retention time of subsequent
peaks, such as pyrethrins and piperonyl butoxide.) Calc. peak
ht or area ratios for duplicate std injections before and after
sample injections and average the 4 values. Calc. and average
peak ht or area ratios for sample injections.
% MGK 264 - (/?//?') x (W'/W) x P
where W = g std; W - g sample; P = purity of std; R' =
ratio of std; and R = ratio of sample.
Ref.: JAOAC 63, 128(1980).
CAS- 113-48-4 (N-octyl bicycloheptene dicarboximide)
960.12 SabadilBa Alkaloids
in Pesticide Formulations
Gravimetric Method
First Action 1960
Final Action 1961
(In dust formulations)
A. Determination
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Weigh 10 g mixed 50% sabadilla dust (or corresponding amt
of lesser concn) into 500 mL g-s erlenmeyer. Add exactly 300
mL ether-CHCl 3 (3+1), and shake 5 min. Make alk. with 10
mL NH 4 OH and shake mecfi. 2 hr. Let stand overnight; then
shake 1 hr.
Filter, avoiding evapn. Place 200 mL aliquot in 500 mL
separator, acidify with H 2 S0 4 (3+97), and shake; withdraw
small amt aq. layer and test with litmus paper, returning soln
to separator. Add 50 mL of the dil. H 2 S0 4 and shake. Let sep.
and transfer acid ext to second 500 mL separator. Add 50 mL
pet ether to acid ext and shake. Let layers sep. and transfer
acid ext to third separator. Repeat extn of soln in first separator
with two 50 mL portions of the dil. H 2 S0 4 , using same 50 mL
pet ether in second separator for washing. Collect acid exts in
third separator.
Make acid exts alk. to phthln with NH 4 OH. Ext with three
50 mL portions CHC1 3 . Wash each CHCI 3 ext by shaking gently
with same 100 mL portion H 2 in fourth separator. (If emul-
sion forms, add small amt anhyd. Na 2 S0 4 .)
Filter each CHC1 3 ext thru cotton into weighed 250 mL flask.
Evap. CHCI3 on steam bath. Add few mL alcohol, and evap.
again. Dry 1 hr at 100° and weigh sabadilla alkaloids. Calc.
% total alkaloids.
B. Qualitative Test
Add 1-2 mL H 2 S0 4 to few mg of residue, 960. 12 A. Pres-
ence of sabadilla alkaloids is indicated by yellow that gradu-
ally becomes intensely red with greenish fluorescence.
Ref.: JAOAC 43, 374(1960).
CAS-8082-57-7 (sabadilla alkaloids)
920.35 Nicotine in Tobacco Products
Silicotungstic Acid Method
Final Action
(Note: Nicotine is very toxic. Avoid contact with skin.)
(Includes nornicotine)
A. Reagent
Silicotungstic acid soln. — Dissolve 120 g silicotungstic acid
(4H 2 O.Si0 2 .12W0 3 .22H 2 or Si0 2 .12W0 3 .26H 2 0) in H 2
and dil. to I L. (Acid should be white or pale yellow crystals,
free from green color; soln should be free from cloudiness and
green color. Of the several silicotungstic acids, 4H 2 O.Si<3 2 .
10WO 3 .3H 2 O and 4H 2 O.SiO 2 .12WO 3 .20H 2 O do not give cryst.
ppts with nicotine and should not be used.)
B. Determination
Weigh sample contg preferably 0. 1-1 .0 g nicotine. If sam-
ple contains very little nicotine (ca 0. 1%), do not increase amt
to point where it interferes with distn. Wash with H 2 into
500 mL Kjeldahl flask, and if necessary add little paraffin to
prevent frothing and few small pieces pumice to prevent
bumping. Add 10 g NaCl and 10 mL NaOH soln (30% by wt),
and close flask with rubber stopper thru which passes stem of
trap bulb and inlet tube for steam. Connect trap bulb to well-
cooled condenser, lower end of which dips below surface of
10 mL HC1 (1+4) in suitable receiving flask. Steam distil rap-
idly. When distn is well under way, heat flask to reduce vol.
of liq. as far as practicable without bumping or excessive sepn
of insol. matter. Distil until few mL distillate shows no cloud
or opalescence when treated with drop silicotungstic acid soln
and drop HO (1+4). Confirm alky of residue in distn flask
with phthln.
Adjust distillate, which may total 1.0-1.5 L, to convenient
exact vol. (soln may be coned on steam bath without loss of
nicotine), mix well, and pass thru dry filter if not clear. Test
distillate with Me orange to confirm acidity. Pipet aliquot contg
ca 0. 1 g nicotine into beaker. (If samples contain very small
amts of nicotine, aliquot contg as little as 0.01 g nicotine may
be used.) To each 100 mL liq., add 3 mL HCI (1+4) and 1
mL silicotungstic acid for each 0.01 g nicotine supposed to be
present. Stir thoroly and let stand overnight at room temp. Be-
fore filtering, stir ppt to see that it settles quickly and is in
cryst. form. Filter on either ashless paper or gooch and wash
with HCI (1 + 1000) at room temp. Continue washing for 2 or
3 fillings of filter after no more opalescence appears when few
mL fresh filtrate is trested with few drops nicotine distillate.
With paper, transfer paper and ppt to weighed Pt crucible, dry
carefully, and ignite until all C is destroyed. Finally heat over
Meker burner <10 min. Wt residue x 0.1141 = wt nicotine
174
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
in aliquot. With gooch, dry in oven 3 hr at 105° and weigh.
Wt residue X 0.1012 = wt nicotine in aliquot.
Ref.: USDA Bur. Animal Ind. Bull. 133.
CAS-54-11-5 (nicotine)
ORGANOHALOGEN PESTICIDES
985.04 AEachlor In Pesticide Formulations
Gas Chromatographic Method
First Action 1985
Final Action 1987
(Method is suitable for formulated products, including emul-
sifiable cones and granulated formulations.)
A. Principle
Sample is dissolved in acetone contg di~/7~pentyl phthalate
as internal std, analyzed by gas chromatgy with flame ioni-
zation detector, and quantd by comparison with internal std.
B, Apparatus
(a) Gas chromatograph.— With flame ionization detector
and on-column injection ports. Temps (°)— column oven 230,
injection port 250, detector 260; gas flows (mL/min) — He
carrier gas 35, H 30; air 250; sample size 1.0 |ulL; run time
15 min.
(b) Column. — 6 ft x 2 mm (id) glass column (on-column
configuration) packed with 10% SP-2250 on 100-120 mesh
SupeJcoport (Supelco, Inc., Cat. No. 1-2132), or equiv. SP-
2250 is methyl-phenyl silicone (50 + 50). Precondition over-
night at 250° before use. Retention times for alachlor and in-
ternal std are ea 5.5 and 11.2 min, resp.
C* Reagents
(a) Acetone. — Pesticide grade, Fisher, or equiv.
(b) Di-n-penty I phthalate internal std. — (CTC Organics, PO
Box 6933, Atlanta, GA 30315). Weigh 5.3 g di-rt-pentyl
phthalate into 1 L vol. flask. Dissolve in acetone and dil. to
vol. with acetone.
(c) Alachlor, — Recrystallized from MeOH (Monsanto Co.,
Muscatine, 1A 52761). Accurately weigh 0.2 g alachlor into
small flask. Add by pipet 30.0 mL internal std soln and shake
to dissolve,
D. Determination
Accurately weigh sample contg ca 0.2 g alachlor into small
flask. Add by pipet 30.0 mL internal std soln and shake well
to ext alachlor. For granular formulation, mix ^5 min on mech.
shaker.
Make replicate I |ulL injections of alachlor std soln and mea-
sure response ratios, R (area alachlor peak/area internal std
peak) for each injection. Repeat until consecutive response ra-
tios R agree within 0.5%.
Make duplicate injections of sample soln and det. av. R.
Follow with injection of alachlor std soln; det. av. R 1 for std
before and after sample injection.
% Alachlor = (R/R r ) x (W /W) x p
where R and R' = av. response ratios for sample and std, resp.;
W and W = wt (g) of sample and std, resp.; P = % purity
of std.
Ref.; JAOAC68, 573(1985).
CAS- 15972-60-8 (alachlor)
988.03 Alachlor in Microencapsulated
Pesticide Formulations
Gas Chromatographic Method
First Action 1988
AOAC-CIPAC Method
A. Principle
Sample is dissolved in acetone contg di-n-penty 1 phthalate
as internal std, analyzed by gas chromatgy with flame ioni-
zation detection, and quantitated by comparison of integrated
peak areas.
B. Safety
LD 50 of alachlor has been found to be 930 mg/kg in rat
acute oral studies (Monsanto Co., 1985, MSDS No.
015972608). Alachlor has been detd to produce tumors in lab-
oratory animals. Wear protective clothing to avoid excessive
exposure,
C. Apparatus
(a) Gas chromatography — With flame ionization detector
and on-column injection ports. Operating conditions: temps^
column oven 230°, injection port 250°, detector 260°; gas flows
(mL/min) — He carrier gas 35, H 30, air 250; sample size 1,0
jjlL; run time 15 min.
(b) Column. — Glass, 6 ft x 2 mm id (on-column config-
uration), packed with 10% SP-2250 on 100-120 mesh Supel-
coport (Supelco Inc.), or equiv. SP-2250 is methyl -phenyl sil-
icone (50 + 50).
D. Reagents
(a) Acetone, — Pesticide grade (Fisher or equiv.).
(b) Di-n-pentyl phthalate internal std soln. — Weigh 5,3 g
di-rt-pentyl phthalate (CTC Organics, PO Box 6933, Atlanta,
GA 30315) into 1 L vol. flask. Dissolve in acetone and dil.
to vol. with acetone,
(c) Alachlor std jro/re.— Recrystallize alachlor (Monsanto Co. ,
PO Box 473, Muscatine, IA 52761) from MeOH. Accurately
weigh 0.2 g recrystd alachlor into small flask. Add by pipet
30.0 mL internal std soln and shake mixt. to dissolve.
£ Instrument Setup and Calibration
Condition chromatgc column overnight at 250° with He flow
at 35 mL/min, Suggested conditions represent best compro-
mise for sepn and quantitation of empds of interest. Some
minor adjustments may be required in other instruments and
columns. Column, when working properly, should generate
4000-5000 plates calcd as follows: N - \6(x/y) 2 , where TV =
no. of theoretical plates, x = distance from point of injection
to peak max,, and y = distance along baseline between inter-
cept points of lines drawn tangent to slope of peak, with jc and
y measured in same units. Typical retention times for alachlor
and internal std are ca 6 and 11.5 min, resp. Impurity in in-
ternal std (peak C), which elutes at ca 9.9 min, should be
completely resolved from internal std peak at ca 1 1 .5 min (Fig.
988.03), Internal std contains another impurity that elutes slightly
after internal std causing slight tail on that peak. Careful con-
trol of integrator conditions is required to integrate internal std
peak.
After instrument equilibration, make ^3 injections of std
soln before calibration.
F. Determination
Accurately weigh, to nearest 0.1 mg, ca 0.45 g alachlor mi-
croencapsulated formulation into 2 oz sample bottle. Avoid
spilling sample on inside wall or neck of bottle; entire sample
should be on bottom of bottle.
AOAC Official Methods of Analysis (1990)
Organohalogens 1 75
4.41
4.33
£ 4.24
4.15
4.06
3.S7
1.87 3.75 5.G2 7.50 S.37 11.25 13.12 15,0
MINUTES
FIG. 988.03— GC chromatogram of alachlor standard (A), internal standard di-n-pentyl phthalate (B), and unknown from internal
standard (C)
Pipel 30.0 mL internal std soln (b) into sample bottle. To
reduce stirring time, use liq. stream from pipet to remove most
of alachlor sample from bottom of bottle. Add mag. stirring
bar (13 x 15 mm) and cap bottle with polyethylene-lined cap.
Mag. stir mixt. until sample is completely removed from
inside wall and bottom of bottle. During stirring, aggregated
sample turns fluffy and easily floats in acetone. For most sam-
ples, this requires ca 2-3 min moderately fast stirring. Then
place bottles on shaker and shake 10 min at high speed.
Let solids settle and pipet off clear acetone soln.
Make replicate 1 jx'L injections of alachlor std soln and mea-
sure response ratio, R (area alachlor peak/area internal std peak)
for each injection. Repeat until consecutive response ratios agree
within 0.5%.
Make duplicate injections of acetone sample soln and det.
av. R. Follow with injection of alachlor std soln. Det. av. R
for std before and after sample injection.
Alachlor, % - (R/R f ) X (W /W) x P
where R and R' - av. response for sample and std, resp.; W
and W — wt (g) of sample and std, resp.; P = % purity of
std.
Ref.: J AOAC 70, 1056 (1987).
CAS- 15972-60-8 (alachlor)
961.04* Chlorine (Total) in Organohalogen
Pesticide Formulations
Sodium Biphenyl Reduction Method
First Action 1961
Final Action 1962
Surplus 1978
(Applicable to aldrin, dieldrin, and endrin)
See 6.177-6.181, 13th ed.
961.05 Aldrin, Dieldrin, and Endrin
in Pesticide Formulations
Infrared Spectroscopic Method
First Action 1961
Final Action 1962
A. Principle
Dieldrin and endrin in dusts, granules, wettable powders,
emulsifiable concns, and so Ins are purified on adsorbent col-
umns. Hexachloro-epoxy-octahydro-endo,exo-dimethanona-
phthalene (HEOD) content of the purified dieldrin or of tech.
dieldrin is detd by IR, using baseline technic, and dieldrin is
calcd assuming 85% HEOD content. Endrin content of puri-
fied or tech. endrin is detd as hexachloro-epoxy-octa-hydro-
endo ,endo-dimethanonaphthalene similarly .
Aldrin is extd from dusts, wettable powders, and inorg. fer-
tilizers on adsorbent column. Hexachloro-hexahydro-endo,exo-
dimethanonaphthalene (HHDN) content of the ext or of tech.
aldrin is detd by IR, using baseline technic, and aldrin is calcd
assuming 95% HHDN content. Method is not applicable to
emulsifiable concns or granules contg petroleum hydrocarbon
solvs or to mixts contg other common pesticides or adjuvants
that absorb in same wavelength region as HHDN.
B. Reagents and Apparatus
(a) Chromatographic solvent A. — Mix 1 vol. CHC1 3 with
19 vols hexane.
(b) Chromatographic solvent B. — Mix 1.5 vols acetone with
98.5 vols chromtgc solvent A.
(c) Extraction solvent. — Mix 1 vol. acetone with 19 vols
CS 2 .
(d) Infrared spectrophotometer. — With sealed liq. cells with
NaCl windows, having optical path length of ca 0. 1 mm (diel-
drin and endrin) and 0.2 mm (aldrin).
C. Preparation of Standard Solutions
(a) HEOD std soln for dieldrin. — Accurately weigh ca 100,
200, 300, 400, 500, and 600 mg std hexachloro-epoxy-
octahydro-endo,exo-dimethanonaphthalene (HEOD) into 10 mL
176
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
vol. flasks, dissolve in CS 2 , and dil. to vol. Concns will be
1, 2, 3, 4, 5, and 6 g/lOcTmL, resp.
(b) Std solnfor endrin. — Accurately weigh ca 50, 100, 150,
200, 300, and 400 mg std hexachloro-epoxy-octahydro-
endo,endo-dimethanonaphthalene (endrin) into 10 mL vol.
flasks, dissolve in CS 2 , and dil. to vol. Concns will be 0.5,
1.0, 1.5, 2.0, 3.0, and 4.0 g/100 mL, resp.
(c) HHDN std solnfor aldrin. — Accurately weigh ca 100,
150, 200, 250, 300, and 350 mg std hexachloro-hexahydro-
endo,exo-dimethanonaphthalene (HHDN) into 10 mL vol.
flasks, dissolve in CS 2 , and dil. to vol. Concns will be 1.0,
1.5, 2.0, 2.5, 3.0, and 3.5 g/100 mL, resp.
D. Preparation of Standard Curve
Fill 0.1 mm cell (0.2 mm for aldrin) with most dil. of stds
solns, using hypodermic syringe. Adjust spectrophtr to opti-
mum settings for gain, slit width, response, speed, and drum
drive. Make duplicate scans of CS 2 soln over scanning range
indicated in Table 961.05 and repeat with each of other std
solns at same instrument settings.
For each of scans of the 6 std solns of each compd, draw
line between baseline points indicated in table. Draw perpen-
dicular from zero radiation line thru absorption peak to base-
line and measure distance from line to peak, P, and to base-
line P . Calc. A (— log P /P) and plot as ordinate against
concn in g/100 mL as abscissa.
Since std curve intersects abscissa at pos. concn value, method
is not applicable to concns below this value.
Peak wavelengths given in table are characteristic for low
concns and they shift at higher concns. P is always detd as
distance from line to point of max. absorption.
E. Preparation of Sample
(Caution: See safety notes on distillation, pesticides, acetone,
chloroform, and hexane.)
(a) Dusts and wettable powders. — Transfer 3-20 g sample,
depending on concn (75-0.5%), weighed to nearest 0.01 g,
to chromatgc tube contg 25-50 mm (ca 5.5 g) Hyflo Super-
Cel. (For finely divided dieldrin or endrin powder, use 3 g
activated C instead of Super-Cel.) Tamp or vibrate column
slightly to settle contents. Place 250 mL wide-mouth erlen-
meyer or 500 mL evapg dish under tip of column.
Working in well ventilated hood, add 50 mL portions extn
solv. to column (if S is present, ext with acetone instead of
extn solv.), letting solv. percolate thru column between addns,
until 150 mL ext collects. Rinse tip of column with addnl 10
mL extn solv.
Evap. solv. almost to dryness on steam bath under N. Dry
HEOD or HHDN residues 15 min at 75°; dry endrin in vac.
oven 15 min at 30° and 10 mm pressure. (Extd endrin may no
longer be associated with its inhibitors. Residue must not be
exposed to elevated temps and must be dissolved promptly to
avoid decomposition.)
Cool residue and dissolve in few mL CS 2 . Quant, transfer
to vol. flask of such size (5-100 mL) as to give optimum concn
of 3 g HEOD, 2 g endrin, or 2 g HHDN/100 mL, dil. to vol.
Table 961.05 Characteristic Wavelength Points for Infrared
Determination of Dieldrin, Endrin, and Aldrin,
Compound
Scanning
Range
Baseline
Points
Peak at
Low Concn
HEOD
Endrin
HHDN
11.59-12.18
11.43-12.04
11.79-12.24
1 1 .64,
11.50,
11.85,
12.18
11.97
12.24
11.80
11.76
12.01
with CS 2 , and mix thoroly. If soln is cloudy from H 2 0, add
little NaCl, shake, and let settle.
(b) Granules containing dieldrin or endrin. —Slurry 40 g
Florisil in 200 mL beaker with 100 mL hexane. Transfer to
chromatgc column with stream of hexane from wash bottle.
Eliminate any bubbles or voids by vibration or agitation. Let
hexane drain until only 2-3 mm layer remains above surface
of column. Add small layer of Na 2 S0 4 to top of column.
Transfer 2-10 g finely ground sample, depending on concn
(10-1%), to prepd column. Rinse down column walls with
three 10 mL portions chromatgc solv. A, letting each portion
enter column before adding next. Add 170 mL chromatgc solv.
A, let percolate thru column, and discard.
Gently flow 10 mL chromatgc solv. B down walls of tube,
avoiding disturbing surface of adsorbent. After solv. sinks into
column, repeat washing with 2 addnl 10 mL portions. Add 220
mL chromatgc solv. B and let flow at rate of 2-5 mL/min,
collecting eluate in 500 mL wide-mouth erlenmeyer or evapg
dish. Evap. solv. to dryness on steam bath, avoiding spatter-
ing, and proceed as in (a), using 5-10 mL vol. flask.
(c) Emulsifiable concentrates and solns. — Weigh 1.5 g
dieldrin cone. (1.5 lb/gal.), 1.0 g endrin cone. (1.6 lb /gal.),
or 30.0 g 0.5% dieldrin soln, and add 5 mL hexane. Transfer
to prepd column and proceed as in (b).
(d) Technical materials. — Transfer sample contg 1.75-4.00
g dieldrin, 1.50-3.00 g endrin, or 1.00-2.00 g aldrin, weighed
to 0.01 g, to 100 mL vol. flask. Dissolve in CS 2 and dil. to
vol. with CS 2 .
F. Determination
Fill same 0.1 mm cell (0.2 mm for aldrin) used for prepn
of std curve with sample soln. Make duplicate scans, and calc.
A and mean A as in prepn of std curve. From appropriate std
curve, obtain g HEOD, endrin, or HHDN/100 mL sample
soln, W.
% dieldrin- WxVx 1.175/5
% endrin = WXV/S
% aldrin = W XVX 1.053/5
where V = mL sample soln; 5 = g sample; 1.175 and 1.053
= conversion factors HEOD to dieldrin and HHDN to aldrin,
resp.
Ref.: JAOAC 44, 595(1961).
CAS-309-00-2 (aldrin)
CAS-60-57-1 (dieldrin)
CAS-72-20-8 (endrin)
949.05 gamma-BHC (Lindane)
in Pesticide Formulations (1)
Partition Chromatographic Method
Final Action
(Caution: See safety notes on monitoring equipment, distil-
lation, flammable solvents, toxic solvents, pesticides, and
diethyl ether.)
A. Apparatus
(a) Partition column. — Column and O type reduction valve
are shown in Fig, 949. 05 A. Construct column of heavy- wall
Pyrex tubing ca 3.5 mm thick, 90 cm long x 2.5 cm diam.
Seal coarse porosity fritted glass disk in place and attach No.
18/9 & joint 5 cm below disk. Supply pressure from laboratory
supply line. (Column available from Lurex Scientific, No, JC
1800-0104 constructed from heavy rather than std wall tub-
ing.)
AOAC Official Methods of Analysis (1990)
Organohalogens 1 77
FIG. 949.05A— Partition column and solvent evaporator
(b) Solvent evaporator. — Fig. 949. 05 A. Evap. fractions to
dryness under reduced pressure at 60°, with aid of H 2 pump.
Recover solv. in trap consisting of Kjeldahl flask immersed in
mixt. of NaCl and ice.
(c) Melting point apparatus. — Use Thiele mp app. equipped
with mech. stirrer. App. shown in Fig. 949. 05B, or Hershberg
modification (2) (available from Ace Glass, Inc., Cat No. 7686)
is suitable.
(d) Thermometer. — Precision grade, meeting NIST speci-
FIG. 949.05B— Melting point apparatus
fications: partial immersion; range 90-120° in 0.2° subdivi-
sions. Calibrated by NIST or against thermometer checked by
NBS.
(e) Melting point tubes. — 1-2 mm capillary tubes of uni-
form wall thickness and diam.
B. Reagents
(a) n-Hexane. — Com. grade, distd before use.
'(b) Nitromethane. — Reflux com. grade material 4 hr and
distil. No visible residue is left after evapn of 10 ml purified
material .
(c) Silicic acid. — Use Mallinckrodt reagent grade (for chro-
matgy) which meets following requirements: When column
prepd as in 949. 05D is used for detn on sample contg known
amt of 7-isomer, flow rate and packing characteristics should
be similar to those of an H 2 SI0 3 known to be satisfactory, and
recovery of 7-BHC should be within ±3% of the 7-BHC con-
tent.
(d) Dye soln. — Dissolve 25 mg D&C Violet No. 2 (1-
hydroxy-4-p-toluidino-anthraquinone) in 50 mL mobile solv.
and store in g-s bottle. (Available from Sigma Chemical Co.)
(e) Mobile solvent. — Satd soln nitromethane in n-hexane.
Vigorously shake 2 L n-hexane with excess nitromethane in
g-s bottle. Decant mobile solvent from nitromethane as needed.
C. Preparation of Sample
(a) Powders containing more than 10% y-BHC. — Crush and
thoroly mix sample with mortar and pestle. Weigh enough
sample into tared 125 mL erlenmeyer to provide ca 0.2 g 7-
isomer after extg and aliquoting. Add 25 mL mobile solv.,
heat just to bp on steam bath, and cool to room temp., shaking
occasionally. Decant ext thru buchner with ca 34 mm medium
porosity fritted disk into 100 mL Kohlrausch flask, with gentle
suction. Re-ext residue in flask, using 10 mL mobile solv.
Wash residue and flask with five 10 mL portions cold mobile
solv., decanting each wash thru buchner. Add 2 mL dye soln
and dil. to vol. with mobile solv.
(b) Dusts containing less than 10% y-BHC. — Weigh enough
sample to provide 1.75-2.00 g 7-isomer. Transfer to Soxhlet
extractor and ext overnight with ether. Evap. most of ether on
steam bath and evap. remainder at room temp, under vac. Ext
7-isomer from residue with mobile solv. as in (a).
178
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
D. Preparation of Column
(Caution: See safety notes on blenders and hexane.)
Transfer 100±0.5 g H 2 Si0 3 to high-speed blender, add 300
mL mobile solv., and with mixing, add 55 ml nitromethane.
Mix 15-30 sec; then pour into column thru glass funnel. Stir
slurry with long glass stirring rod to displace air bubbles. Wash
down sides of column with few mL mobile solv. and apply 5
lb pressure to pack column and force out excess solv.; tap col-
umn gently to aid packing. When boundary between solv. and
H 2 Si0 3 remains stationary, release pressure cautiously, pipet
out most of excess solv., and reapply pressure until ca 3 mm
solv. remains above adsorbent.
E. Determination
(Caution: See safety notes on vacuum and pipets.)
Pipet 10 mL aliquot of sample soln onto column by letting
it flow slowly down inside of column without disturbing H 2 Si0 3
surface. Wash down side of column with 2 mL mobile solv.
and force soln into column by applying 2-3 lb pressure, re-
leasing pressure when all solv. has entered column. Add 10
mL mobile solv. and force into column. Release pressure and
slowly add mobile solv. to within 7-12 cm from top of col-
umn. Apply enough pressure to force solv. thru column at 3-
4 mL/min. Just before last trace of dye leaves column, begin
to collect 10 mL fractions, alternately using two 10 mL grad-
uates. Transfer each fraction to 125 mL erlenmeyer and evap.
to dryness, using solv. evaporator. (Evap. fractions without
boiling; if boiling begins, raise flask momentarily from H 2
bath.)
Appearance of 7-isomer upon evapn is recognized by its ten-
dency to cover bottom of flask as white residua] film with typ-
ical crystal formation. When first residue of 7-isomer is rec-
ognized, begin to collect 10 mL fractions until all 7-isomer is
obtained (usually <8 fractions). Dissolve residue in each flask
with 5 mL /i-hexane and transfer to weighed flask, rinsing flasks
successively with 5 mL portions /i-hexane. Evap. solv., using
solv. evaporator. Evacuate flask ca 20 min at room temp, with
vac. pump. (There is little danger in evacuating 125 mL er-
lenmeyer; larger size erlenmeyer, however, is likely to col-
lapse under vac.) Release vac, wipe with clean, moist towel,
and let stand 5 min. Weigh, and calc. % 7-benzene hexachlo-
ride in original sample.
F. Melting Point Determination of the Gamma Fraction
Dissolve residue in min. amt acetone and transfer quant, to
10 mL beaker. Evap. acetone at 40° , using filtered air stream.
Scrape residue from beaker for mp detn. (Beaker may be set
on piece of solid C0 2 to ensure prepn of finely powd product.)
Place material in agate mortar and mix thoroly with pestle.
Select 2 clean, dry capillary tubes and fill with sample. Be
sure material is well packed into bottom of tube to ensure max.
contact between sample and wall of tube. Insert tubes and ther-
mometer bulb in Thiele tube so that samples and thermometer
bulb touch. Start stirrer and heater, and adjust heating rate to
r/min at 90°. Continue heating until sample melts or reaches
106°. Reduce heating rate to 0.5°/min and continue heating
until sample melts.
Sample mp is corrected temp, of bath when last solid dis-
appears into the clear melt. If mp is <108°, check result by
IR method, 947.01.
Refs.: (1) JAOAC 32, 684(1949); 39, 373(1956).
(2) Ind. Eng. Chem. 8, 312(1936).
CAS-58-89-9 (7-BHC)
953.06* gamma-BHC
in Pesticide Formulations
Radioactive Tracer Method
First Action
Surplus 1970
&?£ 6.257-6.260, 11th ed.
984.05 gamma-BHC in Technical BHC,
Pesticide Formulations, and
Lindane Shampoos and Lotions
Gas Chromatographic Method
First Action 1984
Final Action 1986
CIPAC-AOAC Method
(Applicable to tech., emulsifiable cone, and H 2 0-dispersible
powd. formulations and in lindane shampoo and lotion)
A. Principle
Samples of tech. BHC (benzene hexachloride) and formu-
lations are dissolved in EtOAc with dipropyl phthalate added
as internal std. Lindane (7-BHC) shampoo and lotion samples
are extd with EtOAc-isooctane contg dipropyl phthalate inter-
nal std. EtOAc-isooctane layer is then extd with CH 3 CN to
remove interferences. Std and sample solns are carried thru
same extn procedures. 7-BHC content is detd by GC using
flame ionization detector.
B. Apparatus and Reagents
(a) Gas chromato graph. — Suitable for on-column injec-
tion; equipped with flame ionization detector.
(b) Lindane lotion and shampoo. — Accurately weigh ca 100
mg pure 7-BHC into 50 mL screw-cap centrf. tube. Add by
pipet 10.0 mL internal std soln. Agitate to dissolve 7-BHC
and add 20 mL H 2 0. Accurately weigh 10 g .1 % lindane lotion
or shampoo into 50 mL screw-cap centrf. tube. Add 20 mL
H 2 0. Gently agitate contents by tapping tube with fingers to
mix H 2 and shampoo or lotion. Add by pipet 10.0 mL in-
ternal std soln. Vigorously shake std and samples by hand 1
min. Add 5 drops isoamyl alcohol to each tube and centrf.
Transfer by pipet 3.0 mL each ext (top layer) to sep. 15 mL
screw-cap centrf. tubes. Add by pipet 5.0 mL isooctane fol-
lowed by 3.0 mL CH 3 CN. Vigorously shake each tube by hand
30 s. After phases sep. , withdraw by disposable pipet a portion
of CH 3 CN phase (lower layer) for GC analysis.
D. Analysis of Solutions
(a) Technical BHC and formulations. — Inject 1 (xL por-
tions of std soln of 7-BHC until response ratios (area or peak
ht) for 7-BHC to internal std agree ±2%. Make duplicate in-
jections of std soln followed by duplicate injections of sample
solns. Recalibrate after not more than 4 injections of sample
solns.
(b) Lindane lotion and shampoo. — Inject I jmL portions of
ext of std soln of 7-BHC until response ratios (area or peak
ht) for 7-BHC to internal standard agree ±2%. Make duplicate
injections of std soln followed by duplicate injections of un-
known exts. Recalibrate after not more than 4 injections of
unknown exts.
E. Calculations
For each injection, calc. response ratio (R) = area (or peak
ht) of 7-BHC peak to area (or peak ht) of internal std peak.
7-BHC, wt% = (R/R')(W'/W) X P
AOAC Official Methods of Analysis (1990)
Organohalogens 1 79
where R r and R = av. response ratio for std and sample solns,
resp.; W and W = wt (mg) of 7-BHC in std and in sample,
resp.; and P = purity of 7-BHC std (%).
Ref.: JAOAC 67, 834(1984).
CAS-58-89-9 (7-BHC)
947.01 Benzene Hexachloride
in Pesticide Formulations
Infrared Spectrophotometric Method
Final Action
(Applicable to tech. BHC. Caution: See safety notes on pipets,
pesticides, and carbon disulfide.)
A. Apparatus
Infrared spectrometer.
tion cells, 0.5-
With matched pair of liq. absorp-
mm thick.
B. Calibration of Cells
Det., in spectrometer, difference between deflections of the
2 cells filled with CS 2 . Plainly mark one cell to be used as
sample cell for reading /. Correct values of / obtained with
other cell by adding or subtracting difference between cells
and refer to this as cell factor F. Check factor every 10-14
days.
C. Preparation of Standards and Working Curves
Obtain a, p, 7, and 8 isomers of BHC, either by fractional
crystn from tech. material or as sepd materials, and recrystal-
lize several times from solvs that have been redistd from all-
glass app. Recrystallize from following solvs until mps by
capillary tube method become const: a isomer from benzene
followed by MeOH (mp ca 158°); P isomer from toluene (mp
ca 210.5°, sealed capillary); 7 isomer from MeOH (mp ca 113°);
and 8 isomer from CC1 4 followed by CHC1 3 (mp ca 138.5°).
Confirm purity of each isomer as follows: Evap. to dryness
enough mother liquor from last crystn to yield ^1 g dissolved
solids, grind residue, and dry overnight in evacuated desic-
cator. Weigh and dissolve in enough CS 2 to make 4 g/100 mL
soln. Prep, corresponding soln of recrystd isomer as std. Com-
pare solns of residue and std in spectrometer at wavelength
points used for analysis of other isomers. Consider purity of
isomer satisfactory if A of residue soln is not significantly greater
than that of std at these points.
Prep, working curves of the isomers by detg T of their solns
in CS 2 at various concns as in 947. 01D. Calc. A and plot against
concn in g/L.
D. Determination
Reduce sample of tech. BHC to ca 2 g by grinding and quar-
tering, and dry 24 hr in vacuo at room temp. Weigh 1.5000
g dried material into 50 mL vol. flask and dil. to vol. with
CS 2 (equiv. to 30 g/L). Shake vigorously to dissolve (p iso-
mer is not completely sol. and will settle out). Pipet 25 mL
of this sample soln into another 50 mL vol. flask and again
dil. to vol. with CS 2 (equiv. to 15 g/L). Fill sample cell with
the coned soln for reading /, and fill blank cell with CS 2 , place
in spectrometer, and read T in duplicate at following wave-
lengths:
Isomer
Wavelength, ^m
Alpha
Beta
Gamma
Delta
Epsilon
12.58
13.46
14.53
13.22
13.96
Average duplicates for calcns. Repeat readings with dil. soln
(15 g/L) at a and 7 wavelengths. Calc. A of each of isomers
at the various wavelengths from T measurements by equation:
(F x /„) - (Fxl b x% Set)
Log = A
I s - (F x 4 x % Set)
where F = cell factor, l b = reading of blank cell, % Set = %
scatter, / s = reading of sample cell, and A = absorbance.
Obtain approx. concns from working curves, 947.01C. Cor-
rect A at each wavelength for absorption of interfering com-
ponents. (Altho p isomer has low solubility in CS 2 , this isomer
interferes with 8 analytical point; therefore det. A of p isomer
in CS 2 at this point and apply as correction.) Since these new
values are overcorrected, make repeated evaluations until suc-
cessive values are const, within desired precision.
Refs.: Anal. Chem. 19, 779(1947); Report No. 4760; May
15, 1949, Phys. Chem. Lab., Hooker Electrochemical
Co., Niagara Falls, NY.
CAS-608-73-1 (benzene hexachloride)
973.13 Benfluralin or Trifluralin
in Pesticide Formulations
Ultraviolet Spectroscopic Method
First Action 1973
Final Action 1975
A. Principle
Trifluralin or benfluralin is extd from solid carrier or dis-
solved in n-hexane if liq., purified by chromatgy on Florisil,
and detd by UV spectrometry at 376 nm.
B. Reagents
(a) Florisil. — 100-200 mesh. Test elution characteristics
of Florisil by adding 5 mL std soln to prepd column. Proceed
as in 973. 13E. Elution vol. should be >80 mL but <100 mL.
If elution vol. does not fall within this range, adjust H 2 con-
tent of Florisil by trial and error to obtain proper elution (add
H 2 to decrease elution time; dry at 130° to increase it).
(b) Std soln.—l. 25 mg/mL. Weigh 0.125 g trifluralin or
benfluralin Ref. Std (Blanco Products Co.), into 100 mL vol.
flask, dil. to vol. with n-hexane, and mix.
C. Preparation of Column
Insert glass wool plug in bottom of 25 x 400 mm glass tube
with Teflon stopcock. Add, with const tapping of column, 5
g anhyd. Na 2 S0 4 , stdzd Florisil, (a), to ht of 50 mm, and 5
g anhyd. Na 2 S0 4 . With stopcock open, add 50 mL n-hexane
and let drain to top of column. Close stopcock.
D. Preparation of Sample
(a) Dry formulations (containing more than J % trifluralin
or benfluralin). — Weigh sample contg 0.25 g trifluralin or
benfluralin into Soxhlet extn thimble (33 x 80 mm), cover
with glass wool, and ext with CHC1 3 1 hr beyond time when
no further color is extd. Quant, transfer ext to 200 mL vol.
flask with CHC1 3 , dil. to vol. with CHC1 3 , and mix. Transfer
5 mL to r-b flask and evap. just to dryness on rotary evapo-
rator.
(b) Dry formulations (containing 1% or less trifluralin or
benfluralin). — Weigh sample contg 0.05 g trifluralin or ben-
fluralin, ext, transfer to 200 mL vol. flask, and dil. as in (a).
Transfer 25 mL to r-b flask and evap. just to dryness on rotary
evaporator.
(c) Liquid formulations . — Weigh sample contg 0.12 g tri-
fluralin or benfluralin into 100 mL vol. flask. Dil. to vol. with
n-hexane and mix vigorously. Proceed as in 973. 13E.
180
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
E. Determination
Transfer 5 mL soln from (c) or residue from (a) or (b), with
aid of /i-hexane, to Florisil column. Transfer 5 mL std soln to
second Florisil column. Wash sample into column with small
portions n-hexane. Let each portion drain to top of column
before adding next. Fill column with w-hexane, discarding eluate
until band has moved ca 3 / 4 length of column. Collect eluate
contg trifluralin or benfluralin band (first yellow-orange band
to elute) in 100 mL vol. flask. (If band requires >100 mL vol.
to elute, replace vol. flask with r-b flask, evap., and transfer
quant, to 100 mL vol. flask.) Dil. to vol. with n-hexane and
mix. Det. A of sample and std solns in 1 cm cells at 376 nm
against n-hexane as ref.
F. Calculations
% Trifluralin or benfluralin
= (A x g std x F X P)/(A' x g sample)
where A and A' refer to sample and std solns, resp.; P — %
purity of std; and F = 2, 0.4, or 1 for sample prepns (a), (b),
or (c), resp.
Ref.: JAOAC 56, 567(1973).
CAS- 1861 -40-1 (benfluralin)
CAS-1582-09-8 (trifluralin)
973.14 Benfluralin or Trifluralin
in Pesticide Formulations
Gas Chromatographic Method
Final Action
A. Principle
Trifluralin or benfluralin is extd from solid carrier, or dis-
solved in acetone if liq., and detd by GC.
B. Reagents
(a) Diisobutyl phthalate internal std soln. — Weigh 0.625 g
diisobutyl phthalate (Eastman Kodak Co.) into 250 mL vol.
flask, dil. to vol. with acetone, and mix,
(b) Std soln. — 1 .6 mg/mL. Weigh 0.16 g trifluralin or ben-
fluralin Ref. Std into 100 mL vol. flask, dil. to vol. with ace-
tone, and mix.
C. Apparatus
(a) Gas chromatography — Equipped with flame ionization
detector; capable of programmed column temp, from 135 to
190° at 8°/min. Approx. instrumental conditions: inlet 205°,
detector 275°, N carrier gas 60 mL/min.
(b) Column. — 1.5 m (5') x x /s or V/ od, stainless steel or
Pyrex glass tube packed with 5% DC 200, 12,500 cstokes (An-
alabs, Inc.) on 80-100 mesh Chromosorb W (HP). Condition
newly prepd column at 230° overnight with N carrier gas.
D. Preparation of Sample
(a) Dry formulations (containing more than 1% trifluralin
or benfluralin). — Weigh sample contg 0.16 g trifluralin or
benfluralin into Soxhlet extn thimble (33 x 80 mm), cover
with glass wool, and ext with acetone 1 hr beyond time when
no further color is extd. (Caution: See safety notes on distil-
lation and acetone.) Evap. to ca 60 mL on steam bath with
stream of air directed into flask. Transfer quant, to 100 mL
vol. flask with acetone. Dil. to vol. with acetone and mix.
(b) Dry formulations (containing 1% or less trifluralin or
benfluralin).— Weigh sample contg 0.04 g trifluralin or ben-
fluralin, ext, and evap. as in (a). Transfer quant, to 100 mL
vol. flask with acetone and proceed as in 975. 14E without
dilg, beginning, ". . . add 10 mL internal std soln, ..."
(c) Liquid formulations. — Weigh sample contg 0.16 g tri-
fluralin or benfluralin into 100 mL vol. flask, dil. to vol. with
acetone, and mix.
E. Determination
Pipet 25 mL acetone soln, 973.140(a) or (c), and 25 mL
std soln, (b), into sep. 100 mL vol. flasks, add 10 mL internal
std soln, dil. to vol. with acetone, and mix.
Inject 2.5 (xL trifluralin or benfluralin std soln and start temp.
program to give symmetrical peak ca 70% scale deflection and
retention time 5,5 min. Diisobutyl phthalate internal std peak
appears ca 2 min after std peak. Repeat injection of std soln
until ratio of trifluralin or benfluralin peak area to internal std
peak area is reproducible.
Without changing conditions inject 2.5 jutL sample soln.
Calc. areas of trifluralin or benfluralin and diisobutyl phthal-
ate peaks. Divide area of trifluralin or benfluralin peak by area
of diisobutyl phthalate internal std peak to det. ratio, R.
% Trifluralin or Benfluralin - (R/R f ) X (W /W) X (P/F)
where R and R' = ratio for sample and std solns, resp.; W and
W — g sample and std, resp.; P - % purity of std; and F =
1, 1, or 4 for sample prepns (a), (c), or (b), resp.
Ref.: JAOAC 56, 567(1973).
CAS-1861-40-1 (benfluralin)
CAS-1582-09-8 (trifluralin)
980.05 Bromoxynil Octanoate
in Pesticide Formulations
Gas Chromatographic Method
First Action 1980
Final Action 1981
A. Principle
Bromoxynil octanoate formulations are dild with CHCl 3 , with
n-docosane as internal std, and ester is detd by GC with flame
ionization detection.
(Caution: See safety notes on pesticides and chloroform.)
B. Apparatus and Reagents
(a) Gas chromato graph. — Hewlett-Packard Model 5830,
or equiv., with flame ionization detector and 1.8 m (6') X 2
mm (id) glass column with 10% SP-2100 on 100-120 mesh
Supelcoport (Supelco, Inc.). Operating conditions: temps (°):
injection port 240, column 220, detector 300; carrier gas flow
40 mL He /min; sensitivity 10 X 64; injection vol. 2 |xL with
heated on-column injector. Retention times for n-docosane and
bromoxynil octanoate are 10 and 14 min, resp. Theoretical
plates of column for bromoxynil octanoate must be >3,000.
(b) Bromoxynil octanoate. — Anal, std purity (Rhone-
Poulenc Ag Co., 2 T.W. Alexander Dr, PO Box 12014, Re-
search Triangle Park, NC 27709).
(c) n-Docosane. — Aldrich Chemical Co., Inc.; Cat. No.
13445-7, or equiv.
(d) Internal std soln. — 8.0 g n-Docosane dild to 1 L with
CHC1 3 .
C. Preparation of Standard and Sample
(a) Std soln. — Weigh 0.500 g anal, std bromoxynil octan-
oate into 100 mL vol. flask. Pipet 25 mL internal std soln into
flask, dil. to vol. with CHC1 3 , and shake well to dissolve ester.
(b) Sample soln. — Weigh amt of formulation contg 0.500
g bromoxynil octanoate into 100 mL vol. flask, pipet in 25
mL internal std soln, dil. to 100 mL with CHCI3, and shake
well.
AOAC Official Methods of Analysis (1990)
Organohalogens
181
D. Determination
Inject std and sample soJns in duplicate. Response ratios for
each set of duplicate injections must not differ by >1%.
% Bromoxynil octanoate = (/?//?') X (W /W) X P
where R and R' = response ratio (area of bromoxynil octanoate
peak to internal std peak) for sample and std, resp. ; Wand W - g
sample and std, resp.; and P = % purity of std.
Ref.: JAOAC 62, 1215(1979).
CAS- 1689-99-2 (bromoxynil octanoate)
986.04 Butachlor in Pesticide Formulations
Gas Chromatographic Method
First Action 1986
Final Action 1988
AOAC-CIPAC Method
A. Principle
Sample is dissolved in acetone contg triphenyl phosphate as
internal std, analyzed by gas chromatgy with flame ionization
detection, and measured by comparison with internal std on
the basis of integrated relative peak areas.
B. Safety
LD 50 of butachlor has been found to be 4600 mg/kg in rat
acute oral studies (Monsanto). Avoid excessive exposure by
wearing protective clothing.
C. Apparatus
(a) Gas chromatography — With flame ionization detector and
on-column injection ports. Temps — column over 250°, injec-
tion port 280°, detector 300°; gas flows (mL/min) — He carrier
gas 30, H 34, air 430; sample size 1 .0 (xL; run time 25 min.
(b) Column. — 6 ft X 2 mm (id) glass column (on-column
configuration) packed with 10% SP-2250 on 100-120 mesh
Supelcoport (Supelco, Cat. No. 1-2132), or equiv. SP-2250 is
methyl-phenyl silicone (50 + 50). Precondition overnight at
250° before use. Retention times of butachlor and internal std
are ca 5.9 and 18.5 min, resp.
D. Reagents
(a) Acetone. — Pesticide grade (Fisher Scientific Co., or
equiv.).
(b) Triphenyl phosphate internal std soln. — Gold Label
(Aldrich Chemical Co., Inc.). Weigh 6.4 g into 1 L vol. flask.
Dissolve in and dil. to vol. with acetone.
(c) Butachlor std soln. — 99.7% (recrystd from hexane at
-40°; Monsanto Chemical Co.). Accurately weigh 0.2 g into
small flask. Add by pipet 25 mL internal std soln and shake
to dissolve.
E. Determination
Accurately weigh sample contg ca 0.2 g butachlor into small
flask. Add by pipet 25.0 mL internal std soln and shake >5
min to ext butachlor. For emulsifiable cones, use ca 0.3000 g.
Make replicate 1 uX injections of butachlor std soln and
measure response ratio, R (area butachlor peak/area internal
std peak) for each injection. Repeat until consecutive response
ratios agree ±0.5%.
Make duplicate injections of sample soln and det. av. R.
Follow with injection of butachlor std soln; average R' for std
before and after sample injection.
Butachlor, % = (R/R r ) x (W /W) x P
where R and R' — av. response ratios for sample and std, resp.;
W and W
of std.
wt (g) of sample and std, resp.; P = % purity
Ref.: JAOAC 69, 721(1986).
CAS-23 184-66-9 (butachlor)
971.05* Captan in Pesticide Formulations
Gas Chromatographic Method
First Action 1971
Final Action 1982
Surplus 1984
AOAC-CIPAC Method
See 6.247-6.251, 14th ed.
980.06 Captan in Pesticide Formulations
Liquid Chromatographic Method
First Action 1980
Final Action 1982
AOAC-CIPAC Method
(Method is suitable for tech. captan and formulations with cap-
tan as only active ingredient.)
A. Principle
Captan is extd from inerts with soln of diethyl phthalate in
CH 2 C1 2 . Soln is chromatgd on microparticulate silica gel col-
umn, using CH 2 C1 2 as mobile phase. Ratio of captan peak ht
to diethyl phthalate peak ht is calcd from UV response and
compared to std material for quantitation.
(Caution: See safety notes on pipets and pesticides.)
B. Apparatus and Reagents
(a) Liquid chromato graph. — Able to generate over 1000 psi
and measure A at 254 nm.
(b) Chromatographic column. — Large bore column contg
narrow-range (<10 p,m) porous silica gel particles. Partisil-10,
4.65 mm id x 25 cm is suitable. Available from Whatman,
Inc.
(c) Strip chart recorder. — Range to match output of LC de-
tector.
(d) Diethyl phthalate. — EM Science No. 1295.
(e) Reference std captan. — Chevron Chemical Co., PO Box
4010, Richmond, CA 94804.
(f) Methylene chloride. — Spectroscopic grade or distd in
glass. (Burdick and Jackson Laboratories, Inc.).
(g) Glass fiber filter paper. — Whatman GF/A, or equiv.
C. Preparation of Standard
(a) Internal std soln. — 0.312 mg diethyl phthalate /mL.
Weigh ca 156 mg diethyl phthalate and transfer to 500 mL
vol. flask. DiL to vol. with same CH 2 C1 2 to be used for mobile
phase. Concn may be varied to accommodate column and in-
strument differences. If necessary, adjust concn so that peak
ht of diethyl phthalate matches peak ht of captan within 20%.
(b) Std soln. — 0.8 mg captan /mL, which is in the optimum
linearity range. Accurately weigh ca 40 mg std captan into
glass bottle. Pipet in 50 mL internal std soln, shake mech. 15
min, and filter thru glass fiber paper. Prep, fresh std daily.
D. Preparation of Sample
Accurately weigh sample expected to contain 40 mg captan
into glass bottle. Pipet in 50 mL internal std soln. Place on
mech. shaker 15 min. Centrf. and filter supernate thru glass
fiber paper. Prep, fresh sample daily.
182
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
E Determination
Adjust operating parameters to cause captan to elute in 4-
6 min. Maintain all parameters const thruout analysis. Typical
values are: flow rate, 2.5 mL CH 2 Cl 2 /min, max.; pressure, ca
800 psi; chart speed 0.2"/min; mobile phase, degassed CH 2 C1 2
equilibrated with room air; A range, 0.04 AUFS; temp., am-
bient; injection vol., 20 \xL.
Changes in H 2 content of mobile phase affect retention
time and peak hts of captan and internal std, so use same source
of CH 2 C1 2 thruout analysis.
Adjust injection size and attenuation to give largest possible
on-scale peaks. Make repetitive injections of std until response
is stable and ratio of captan peak ht to diethyl phthalate peak
for successive injections agree to within ±2% of their mean.
Inject sample. Peak ht ratio must be within 10% of peak ht
ratio for std. If not, reweigh samples to match std. If within
10%, reinject sample. Peak ht ratios for 2 sample injections
must agree to within ±2% of their mean. If not, repeat detn
starting with std injections.
Reinject std twice. Av. peak ht ratios of 2 stds immediately
preceding and following sample injections must agree to within
±2% of their mean. If not, repeat detn.
F. Calculation
Measure peak hts to 3 significant figures, and calc. ratio for
each injection. Average 4 std ratios, and the 2 sample ratios.
% Captan = (R/R f ) x (W'/W) x P
where R = av. sample ratio (captan peak ht/diethyl phthalate
peak ht); R' = av. std ratio (captan peak ht/diethyl phthalate
peak ht); W = rag sample; W = mg std, and P = % purity
of std.
Ref.: JAOAC63, 1231(1980).
CAS- 133-06-2 (captan)
971.06 Chloramben in Pesticide Formulations
Spectrophotometric Method
First Action 1971
Final Action 1973
AOAC-CIPAC Method
A. Principle
Chloramben contains conjugated tt electron system of ben-
zene which absorbs strongly in UV. Absorption is measured
quant, at 297 mm. (Caution: See safety notes on pipets and
pesticides.)
B. Apparatus and Reagents
(a) Spectrophotometer. — For use in UV, with 1 cm cells.
(b) Shake-out flask. — 250 mL erlenmeyer, with screw cap.
(c) Chloramben std solns. — (7) Stock soln. — 0.38 mg/mL.
Accurately weigh 19 ±2 mg chloramben (Rhone-Poulenc Ag
Co., 2 T.W. Alexander Dr, PO Box 12014, Research Triangle
Park, NC 27709) into 50 mL vol. flask, add 25 mL 1% NaOH,
agitate until dissolved, dil. to vol., and mix. (2) Working soln. —
0.038 mg/mL. Pipet 5 mL stock soln into 50 mL vol. flask,
dil. to vol. with 1% NaOH soln, and mix.
C. Preparation of Sample
Mix 10 g granular sample on 12 x 12" paper by lifting al-
ternate corners.
D. Determination
(a) Dry granular formulations. — Add amt solid material and
1% NaOH soln specified in Table 971.06 to 250 mL shake-
out flask and shake 30 min. Filter, and transfer stated aliquot
to vol. flask. Dil. to vol. with 1% NaOH soln and mix. Det.
A at 360 and 297 nm against 1% NaOH. Calc. A A - A 291 -
A 360 . Det. A A' of working std soln similarly.
% Chloramben = (A A x (mg std/mL) x F)/(AA'
x g sample x 10)
where F = factor in Table 971.06.
(b) Liquid formulations. — Weigh amt liq. indicated in
Table 971.06 into 100 mL vol. flask, dil. to vol. with 1%
NaOH soln, and mix. Transfer 1 mL aliquot to 100 mL vol.
flask, dil. to vol. with 1% NaOH, and mix. Proceed as in (a).
Ref.: JAOAC53, 1155(1970).
CAS- 133-90-4 (chloramben)
962.05 Chlordane (Technical)
and Pesticide Formulations
Total Chlorine Method
First Action 1962
Final Action 1965
{Caution: See safety notes on distillation, pipets, sodium bi-
phenyl, flammable solvents, toxic solvents, pesticides, and
benzene.)
A. Preparation of Standard Solutions
(a) Sodium chloride std soln. — 0. IN. Dissolve 5.845 g NaCI,
previously dried 2 hr at 105°, in H 2 0, and dil. to 1 L in vol.
flask.
(b) Silver nitrate std soln. — 0.LV, Prep, as in 941. 18A. To
250 mL g-s erlenmeyer add 15.00 mL 0. 1W NaCI, (a), 50 mL
H 2 0, 10 mL HN0 3 (1 + 1), boiled to expel oxides of N, and
25.00 mL of the AgN0 3 soln. Add 3 mL nitrobenzene, stop-
per, and shake vigorously 15 sec, Add 5 mL ferric indicator,
929.04A(e), and back-titr. with O.IjV KSCN, (c), to reddish-
brown end point. (Potentiometric titrn using Ag indicator elec-
trode and Ag-AgCl or glass ref. electrode may be substituted
for indicator method, but must be used in both stdzn and detn.)
(c) Potassium thiocyanate std soln. — 0. IN. Prep, and titr.
against AgN0 3 soln, (b), as in 941.18D(b). Calc. F = mL
AgN0 3 soln/mL KSCN soln.
Normality AgN0 3 soln = mL NaCI soln X 0.1000/
(mL AgN0 3 soln - mL KSCN soln x F)
(d) Sodium biphenyl reagent. — 30% w/w. (Caution: See
safety notes on sodium metals.) Place 300 mL dry toluene and
58 g Na in dry 2 L 3-neck flask equipped with adjustable speed
sealed stirrer, inlet for N, and reflux condenser. With stirrer
off, and with slow stream of N passing thru flask, warm until
refluxing begins and Na is entirely melted. Agitate vigorously
until Na is finely dispersed; then cool to < 10° Remove reflux
condenser and add 1.25 L anhyd. ethylene glycol dimethyl
ether. Add 390 g biphenyl with moderate stirring and with
slow stream of N passing thru flask. Reaction should begin
within few min, indicated by blue or green color which grad-
ually darkens to black. Maintain temp, at <30° with oil bath
or other cooling medium not involving hazard should flask contg
Na break. Reaction should be complete in 1 hr. Reagent pro-
tected from moisture and air has useful life of 1-2 months at
25°.
(Premixed reagent, packed in 15 mL vials, each enough for
1 detn, is available from Southwestern Analytical Chemicals,
Inc., PO Box 485, Austin, TX 78767.)
B. Preparation of Sample
(a) Emulsifiable concentrate formulations. — Accurately
weigh sample contg 0.5 ±0.05 g tech. chlordane into 50 mL
AOAC Official Methods of Analysis (1990)
Organohalogens 183
Table 971 .06 Parameters for Sample Analysis
Sample
Chloramben,
Wt,
1% NaOH,
Aliquot,
Final
Factor
Sample
%
g±o.i
mL
mL
Diln
(F)
Dry granular
1.2
3.0
50
2
50
1,250
Dry granular
4
7.5
100
1
100
10,000
Dry granular
10
3.0
100
1
100
10,000
Liquid
21.6
1.8
100
1
100
10,000
vol. flask, dissolve, and dil. to vol. with toluene. Transfer 5
mL aliquot to 125 mL separator, add 15 mL or g Na biphenyl
reagent, 962.05A(d) above and then swirl. If soln is not dark
green, add more reagent. Let stand 3 min and add 3-5 mL
H 2 dropwise. With stopper removed, swirl soln gently to de-
compose excess reagent. Add 25 mL H 2 0, stopper, and mix
with gentle rocking motion. (Do not shake vigorously.) Let
layers sep. and drain lower aq. layer into 250 mL erlenmeyer.
Re-ext solv. layer with two 25 mL portions 3N HN0 3 and
combine aq. solns in erlenmeyer.
(b) Dusts, granular impregnates, and wettable powders. —
Accurately weigh sample contg 0.5 ±0.05 g tech. chlordane
into Soxhlet extn thimble. Ext with 80 mL benzene in Soxhlet
app. 1 hr. Transfer to 100 mL vol. flask, washing with several
3 mL portions benzene. Dil. to vol. with benzene and transfer
10 mL aliquot to 125 mL separator. Proceed as in (a).
C. Determination
Add 15.00 mL 0. \N AgN0 3 and 3 mL nitrobenzene to er-
lenmeyer, stopper, and shake vigorously 15 sec. Rinse stop-
per, add 5 mL ferric indicator, 929.04A(e), and back-titr. with
0.17V KSCN to reddish-brown end point. (Designate mL KSCN
asD.)
Det. blank on reagents by pipetting 5 mL toluene into 125
mL separator, add 15 mL or g Na biphenyl reagent, and pro-
ceed as in 962.056(a), thru combining aq. solns in erlen-
meyer. Add 15.00 mL 0.17V NaCl, 25.00 mL 0.17V AgN0 3 ,
and 3 mL nitrobenzene, and proceed as above. Calc. blank
correction factor, C = mL KSCN used in stdzn of AgN0 3 —
mL KSCN used in blank detn.
% Chlorine - [15 - (C + D) x F]
x normality AgN0 3 x 3.545/g sample
% Tech. chlordane - % CI X 1.56
Ref.: J AOAC 45, 513(1962).
CAS-57-74-9 (tech. chlordane)
965.1 4* Chlordane (Technical)
and Pesticide Formulations
Colorimetric Method
First Action 1965
Final Action 1967
Surplus 1982
A. Reagent
Hexachlorocyclopentadiene (HEX) std solns. — Stock soln. —
0.1 g/100 mL. Weigh 0.1000 g hexachlorocyclopentadiene
Ref. Std (available from Velsicol Chemical Corp., 5600 N.
River Rd, Rosemont, IL 60018-5119) in 100 mL vol. flask,
dil to vol. with MeOH, and shake to dissolve. Std soln 1 . —
0.005 g/100 mL. Dil. 5 mL stock soln to 100 mL with MeOH.
Std soln 2. — 0.002 g/100 mL. Dil. 2 mL stock soln to 100
mL with MeOH.
Method I
B. Calibration
With MeOH in both ref. and sample cells (matched 1 cm
silica), adjust and 100% settings on UV spectrophtr at 324
nm. Empty sample cell, rinse several times with, and then fill
with std soln /, and read A. Empty sample cell, rinse with
MeOH, then rinse and fill with std soln 2, and read A. Calc.
A factor, K, for each std soln - (g std HEX/100 mL)/A. Av-
erage the two K values.
C. Determination
Weigh 0.5 g sample in 100 mL vol. flask, dil. to vol. with
MeOH, and shake to dissolve. Proceed as in 966. 06B, treating
sample soln in same manner as stds.
% HEX in sample
= (A of sample soln
D. Method II
100 X K)/{g sample/100 mL)
(Includes corrections for other components of chlordane which
absorb at 324 nm)
Proceed as in 966. 06B and C, except det. A of all solns at
300, 324, and 350 nm. Settings of and 100% must be re-
peated at 300, 324, and 350 nm for A readings at those points.
Calc. K = (g std HEX/100 mL)/[A 324 - 0.5(A 30 o + ^350)] ■
% HEX in sample
- [A 324 - 0.5(A 3O o + ^350)] x 100 x K/(g sample/100 mL)
Ref.: J AOAC 49, 254(1966).
CAS-77-47-4 (hexachlorocyclopentadiene)
See 6.266-6.270, 14th ed.
966.06 Hexachlorocyclopentadiene
in Technical Chlordane
Spectrophotometric Methods
First Action 1966
Final Action 1967
(Applicable to tech. chlordane, but not to formulations)
973.15* Alpha and Gamma Isomers
in AG Chlordane Technical
Infrared Spectroscopic Method
Final Action 1974
Surplus 1982
(Not applicable to tech. chlordane or its formulations)
See 6.275-6.278, 14th ed.
184
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
973.16* AG Chlordane in Granular
Pesticide Formulations
Infrared Spectroscopic Method
First Action 1973
Final Action 1974
Surplus 1982
(Not applicable to tech. chlordane or its formulations)
See 6.279-6.283, 14th ed.
973.17* Heptachlor in AG Chlordane
Gas Chromatographic Method
Final Action 1974
Surplus 1982
(Not applicable to tech. chlordane or its formulations)
See 6.284-6.287, 14th ed.
985.05 Chlordimeform
in Pesticide Formulations
Gas Chromatographic Method
First Action 1985
Final Action 1987
AOAC-CIPAC Method
A. Principle
Chlordimeform is extd with CH 2 C1 2 and detd by flame ion-
ization gas chromatgy, using diethyl terephthalate as internal
std. Identity is verified simultaneously by comparing retention
times with std.
B. Apparatus
(a) Gas chromato graph. — Capable of temp, program, pref-
erably equipped with auto-injector, flame ionization detector,
and integration capabilities.
(b) Chromatographic column. — 2 mm id X 1.83 m (6 ft)
glass column packed with 3% CBWX-20M on 80-100 mesh
Gas-Chrom Q. Condition column >24 h at 225°, using carrier
gas at ca 20 mL/min. Operating conditions: injector 250°; de-
tector 250°; column 170° for 22 min, then to 225° at 20°/min,
and hold 15 min; He carrier gas flow ca 25 mL/min. Retention
times for internal std and chlordimeform are ca 11 and 14.8
min, resp.
C. Reagents
(a) Internal std soln. — 4 mg/mL. Dissolve 4.0 g diethyl
terephthalate in CH 2 C1 2 and dil. to 1 L with CH 2 C1 2 . Check
internal std soln for interfering components by injecting an ali-
quot into chromatograph.
(b) Chlordimeform std soln. — Accurately weigh 100 mg
chlordimeform std of known purity (Ciba-Geigy Corp., Pro-
duction Technical Dept, PO Box 18300, Greensboro, NC 27419)
into 4 oz bottle. Pipet 50.0 mL internal std soln into bottle,
cap, and shake 30 min.
D. Preparation of Sample
Accurately weigh amt of sample contg ca 100 mg chlordi-
meform into 4 oz bottle. Pipet 50.0 mL internal std soln into
bottle, cap, shake 30 min to ext.
E Determination
Set integration parameters, and stabilize instrument by in-
jecting 1-3 uX aliquots of std soln until area ratios of chlor-
dimeform to internal std vary less than 2% for successive in-
jections. Using same established injection vol. as for std, inject
sample. Sample area ratio should be ±10% of std area ratio.
Inject 2 aliquots of std and 2 aliquots of sample followed by
2 aliquots of 2nd sample and 2 aliquots of std. Repeat se-
quence until all samples are analyzed. Calc. response factor,
R, for each injection:
R - peak area (or ht) chlordimeform/
peak area (or ht) internal std
Chlordimeform, % - (R/R f ) x (W /W) x P
where R and R r = av. response factor for sample and std solns,
resp.; W and W - mg sample and std, resp.; and P = purity
(%) of std.
Ref.: JAOAC 68, 589(1985).
CAS-6 164-98-3 (chlordimeform)
978.05 2,4-D in Pesticide Formulations
Automated Liquid Chromatographic Method
First Action 1978
A. Principle
Esters of 2,4-D are saponified in situ; amine salts are con-
verted to H 2 0-sol. K salt of 2,4-D. Ionic 2,4-D is protonated
by pH 2.95 CH 3 CN-H 2 (1 +4) eluant, and sepd from all known
impurities and /?-bromophenol internal std on reversed phase
bonded microparticulate column. 2,4-D elutes between im-
purities 2,4- and 2,6-dichlorophenol.
B. Apparatus
(a) Liquid chromatograph. — Fitted with 5000 psi pressure
gage, 280 nm UV detector, line filter in mobile phase reser-
voir, and 10 mv full scale deflection strip chart recorder. Au-
tomated sampling system and computing integrator are op-
tional. Typical operating conditions: chart speed, 0.2 cm /min;
mobile phase flow rate, 0.9-3.0 mL/min depending upon psi
range of pump; detector sensitivity, 0.64 A unit full scale; temp.,
ambient; injection valve vol., 10 jiL.
(b) Liquid chromatographic column. — No. 316 stainless
steel, 250 X 4.6 (id) mm, Partisil® 10 fxm ODS column with
7 cm X 2.1 mm id CSKl guard column, Pellicular ODS (Nos.
4223-001 and 4390-413, resp., Whatman Inc.) Regenerate, if
necessary, by pumping CH 3 CN thru column until baseline is
stable. Repack first 5 mm of guard and main columns with
Co:Pell ODS if peaks begin to "tail."
C. Reagents
(a) Mobile phase. — pH 2.95. CH 3 CN (distd-in-glass)-H 2
(deionized, 0.4 jxm filtered) (1+4) contg NaOH added from
(1 + 1) aq. soln of known normality, 936.168(b), at final vol.
concn of 03M. Add H 3 P0 4 to adjust pH to 2.95.
(b) Saponification-internal std soln. — 4 gp-Bromophenol/
L 0.2W KOH in isopropanol-H 2 (2+1). Add KOH from (1 + 1 )
aq. soln of known normality.
(c) 2,4-D std soln. — 300 mg/25 mL. Accurately weigh ca
300 mg 2,4-D anal. ref. std (99+% isomer pure; available from
Dow Chemical Co., Sample Coordinator), previously dried 15
min at 100°, into 1 or 2 oz glass vial with polyethylene-lined
screw cap. Pipet in 25 mL saponification-internal std soln, and
shake to dissolve. Prep. 2,4-D std soln and sample soln, O,
at same time, using same pipet.
D. Preparation of Sample
Accurately weigh sample contg ca 300 mg 2,4-D acid equiv.
into 1 or 2 oz glass vial with polyethylene-lined screw cap.
Pipet in 25 mL saponification-internal std soln, and shake 15
AOAC Official Methods of Analysis (1990)
Organohalogens
185
min, warming ester formulations to 50° several min before
shaking. Filter prepd sample thru 9 cm Whatman glass micro-
fiber filter GF/A, or equiv., collecting major portion of aq.
phase for chromatography. Adjust isopropanol-H 2 ratio, if
necessary, to obtain complete dissoln of sample; e.g., amine
formulations are best prepd with isopropanol-H 2 (1 + 1).
E. Determination
Transfer ca 1 mL portions of samples and stds to automated
sampler vials, and cap. Place samples and stds in position, and
start automatic sampler. With programmed integrator use fol-
lowing calcn program automatically:
% 2,4-D - (R/R f ) x (W'/W) x P
where R and R' = peak ht or area ratios of 2,4-D to internal
std for sample and std, resp.; W = mg 2,4-D in std; W = mg
sample; and P = % purity of std. If automated sampler and
computing integrator are unavailable, inject 10 |xL samples and
stds and perform calcns manually. As check on calibration,
place stds in sample sequence at beginning, middle, and end.
Periodically confirm linearity by analyzing stds contg 200, 300,
and 400 mg 99 + % 2,4-D/25 mL saponification-internal std
soln. Continuously recycle and mag. stir mobile phase. Re-
place mobile phase after ca 200 injections/L.
Refs.: JAOAC 61, 1163(1978); 62, 334(1979).
CAS-94-75-7 (2,4-D)
962.06 Dalapon (Sodium Salt)
in Pesticide Formulations
Titrimetric Method
First Action 1962
Final Action 1966
(Caution: See safety notes on pesticides.)
A. Apparatus
(a) Reflux apparatus. — 250 mL erlenmeyer connected thru
dfc 35/25 ball joint to reflux condenser.
(b) Filtering apparatus. — 60 mL, medium porosity fritted
glass funnel attached to glass filter bell, 1 1 cm od, 18 cm high,
with bottom gasket and slide valve.
B. Reagents
(a) Mercuric -cupric nitrate soln. — (Caution: See safety notes
on mercury.) Dissolve 100.0 g yellow HgO and 60 g
Cu(N0 3 ) 2 .3H 2 in 500 mL 3.100±0.0037V HN0 3 , measured
from vol. flask, in 1 L vol. flask, dil. to vol. with H 2 0, and
filter.
(b) Potassium iodide soln. — Dissolve 150 g KI in H 2 0, dil.
to 1 L, and neutze to phthln.
C. Determination
Accurately weigh sample contg 0. 1 1-0.22 g Na salt of 2,2-
dichloropropionic acid, transfer to erlenmeyer of reflux app.,
and add 100 mL Hg-Cu nitrate soln. Add some boiling chips,
attach condenser, and reflux 15 min. Cool in H 2 bath. Filter
thru filtering app., washing flask and ppt acid-free with H 2
from wash bottle. Discard filtrate and washings, and place 250
mL narrow-mouth erlenmeyer in filtering bell.
Add 50 mL Kl soln to erlenmeyer to dissolve any remaining
ppt, transfer to funnel, and stir until ppt dissolves. Draw soln
into narrow-mouth erlenmeyer with vac. Wash flask and fun-
nel with <50 mL KI soln from wash bottle, adding washings
to filtrate. Add few boiling chips to filtrate and boil 1 min.
Cool in H 2 bath. Titr. immediately with 0.17V HC1, using
phthln.
% Na salt 2,2-dichloropropionic acid
- mLO.UVHCl x 0.004499 x 100/g sample
Refs.: Anal. Chem. 31, 418(1959). JAOAC 43, 382(1960);
45, 522(1962).
CAS- 127-20-8 (dalapon sodium salt)
984.06 Dalapon (Magnesium and/or
Sodium Salt) in Pesticide Formulations
Liquid Chromatographic Method
First Action 1984
Final Action 1987
A. Principle
Dalapon is sepd from related compds on reverse phase LC
column, using paired-ion eluant, and detected by UV spectro-
phtry.
&. Apparatus
(a) Liquid chromatograph. — Fitted with 5000 psig pressure
gage, 214 nm UV detector, line filter in eluant reservoir, 20
jxL loop-type injection valve, and strip chart recorder. Com-
puting integrator optional. Operating conditions: temp., 20-
30°; eluant flow rate, 160 mL/h; detector sensitivity, 0.25
AUFS.
(b) Liquid chromatographic columns. — No. 316 stainless
steel, 50 x 4.6 (id) mm Co: Pell ODS pellicular guard column
(No. 6561-404, Whatman Inc.); 100 x 8 (id) mm C-18, 10
fxm radial compression main column with RCM-100 column
holder (Waters Associates, or equivalent (such as Whatman
Partisil 10-25-ODS-3)). Columns slowly degrade in use. Re-
place when 30% loss of retention occurs and repack first 5 mm
of guard column. Radial compression column recommended
gives lower back-pressure and longer column life than std 316
stainless steel columns.
C. Reagents
(a) Eluant.— Dilute mixt. of 200 mL CH 3 CN (UV grade),
1 .6 mL n-octyl amine (Eastman P7588 or equivalent), and 2.4
g (NH 4 ) 2 HP04 (J.T. Baker, Inc., No. 0784, or equivalent) to
1 L with H 2 (0.4 \xm filtered). Adjust pH of eluant to 7.0
with H3PO4. Eluant may be recycled if reservoir is mag. stirred
and no more than 200 injections are made per L eluant.
(b) Dalapon std soln. — 130 mg/50 mL. Accurately weigh
ca 130 mg dalapon acid ref. std (99 + % isomer pure; available
from Dow Chemical Co.) into 50 mL vol. flask, add H 2 to
mark, and shake. Do not use std soln after 24 h. Det. % H 2
in dalapon acid ref. std by Karl Fisher titrn. Labeled purity of
ref. std is on anhyd. basis and must be appropriately reduced
according to H 2 content.
D. Preparation of Sample
Accurately weigh sample contg ca 130 mg dalapon acid equiv.
into 50 mL beaker. Quant, transfer to 50 mL vol. flask with
H 2 0. Fill to mark with H 2 and shake. Do not use sample
soln after 24 h. Dalapon acid and salts are hygroscopic; protect
from moisture in air by storing in well sealed bottle. Duplicate
sample and std prepn is recommended.
Note: Do not add mineral acids in sample prepn.
E. Determination
Inject std soln, sample soln, and then std soln. Calc. results
as
% Dalapon acid equiv. - (R/R r ) x (W'/W) x P
where R and R' — average peak ht or peak area of dalapon
peak for sample and std solns, resp.; W and W — mg sample
186
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
and std, resp.; and P = % purity of std. Note: Initial system
stability may be poor. Before injecting sample soln, repeat in-
jection of std soln to confirm system stability. Periodically
confirm linearity by analyzing stds contg 100, 130, and 160
mg 99 + % dalapon acid/50 mL H 2 0. Flush injection loop with
>1 mL sample or std soln before operating injection valve.
Refs.: JAOAC66, 1390(1983); 70, 265(1987).
CAS-75-99-0 (dalapon)
970.05 DCPA in Pesticide Formulations
Gas Chromatographic Method
First Action 1970
Final Action 1972
{Caution: See safety notes on distillation, pipets, flammable
solvents, toxic solvents, pesticides, benzene, acetone, and car-
bon disulfide.)
(Under conditions specified, other pesticides or ingredients may
interfere with GC analysis, e.g., aldrin has same retention time
as DCPA. Aldrin and DCPA may be sepd at 170° column temp.)
A. Apparatus
Gas chromato graph. — 1.8 m (6') X ] /s" id stainless steel
column contg 10% silicone UC-98 (Applied Science) on 80-
100 mesh silanized Diatoport S (Hewlett-Packard Co., Avon-
dale, Div.). Conditions (applicable to Hewlett-Packard F&M
Model 5750) — temps(°): column 200, injection port 240, flame
ionization detector 260; H, air, and He carrier flows, 1 15, 600,
and 25 mL/min, resp.; chart speed 0.25'Vmin; attenuation 4X;
range setting 10 2 (10~ 10 amp full scale).
B. Preparation of Standard Curve
(a) DCPA std solns. —Weigh 0.5 g DCPA (available from
Diamond Shamrock Corp., PO Box 348, Painesville, OH 44077)
into 100 mL vol. flask, add ca 90 mL acetone (soln is rapid),
and dil. to vol. Pipet 5, 10, and 15 mL into sep. 25 mL vol.
flasks and dil. to vol. with acetone.
(b) Hexochlorobenzene (HCB) std solns. — Weigh 0.5 g ref.
grade HCB into 100 mL vol. flask, add 90 mL benzene, and
dil. to vol. with benzene. Pipet 1, 2, and 3 mL into sep. 25
mL vol. flasks and evap. to dryness with current of dry air.
Add 20 mL acetone to each flask and dil. to vol. with acetone.
Inject 5 (ulL each dild HCB and DCPA std at least twice.
Prep, curve of peak area or ht against concn for DCPA and
peak ht against concn for HCB.
C. Determination
(a) Benzene extraction. — Grind granular product. Weigh
portion contg ca 300-400 mg DCPA into Whatman extn thim-
ble (33 x 88 mm). Cover with glass wool. Place thimble in
medium Soxhlet extractor; add 150-175 mL benzene and 3
glass beads. Ext 6 hr. Quant, transfer ext to 400 mL beaker
and evap. to ca 5 mL on steam bath with dry air current; re-
move and evap. to dryness with air current. Add ca 150 mL
acetone and let stand until soln is complete (white, flaky crys-
tals may indicate incomplete soln; soln may be hastened by
placing flask in ultrasonic cleaner). Filter soln thru glass wool
into 200 mL vol. flask. Wash beaker with acetone, transfer
washings to vol. flask, and dil. to vol.
(b) Alternative acetone extraction. — Substitute acetone for
benzene in extn. Proceed as in (a) thru "Ext 6 hr." Continue
with "Filter soln thru glass wool ..."
Inject duplicate 5 |ulL sample soln into gas chromatograph.
Compare peak ht or peak area to std curve to det. % hexa-
chlorobenzene (HCB) and DCPA.
Ref.: JAOAC 52, 1284(1969).
CAS-1861-32-1 (dimethyl tetrachloroterephthalate)
970.06 DCPA in Pesticide Formulations
Infrared Spectroscopic Method
First Action 1970
Final Action 1972
A. Preparation of Sample
Grind granular product. Weigh sample contg 200-500 mg
DCPA into Whatman extn thimble. Proceed as in 970.05C(a)
thru "... evap. to dryness with air current." Add 25 mL CS 2 ,
allow ca 30 min for complete soln, and transfer quant, to 50
mL vol. flask with CS 2 , filtering sample thru glass wool. Dil.
to vol.
B. Preparation of Standard Solution
Weigh 1.25 g DCPA into 100 mL vol. flask. Add ca 90 mL
CS 2 (soln may be hastened by placing flask in ultrasonic cleaner)
and dil. to vol. Pipet 10, 15, and 20 mL into sep. 25 mL vol.
flasks and dil. to vol.
C. Determination
Set spectrophtr at optimum operating condition. Use 0.5 mm
KBr (or NaCl) matched cells. Fill ref. cell with CS 2 . Transfer
dild stds to other cell and scan slowly from 1 100 to 900 cnrT 1 .
Repeat with samples. Construct baseline from 1030 to 925 cm" 1
and draw line from midpoint of max. A at ca 964 cm™ 1 to
intersect baseline. Compute A A at 964 cm™ 1 at point of in-
tersection of stds and sample.
Prep. A A -concn curve for std; Beer's law is obeyed over
concn range 2-15 mg DCPA/mL. Calc. % DCPA from std
curve.
Ref.: JAOAC 52, 1284(1969).
CAS-1861-32-1 (dimethyl tetrachloroterephthalate)
947.02* DDT in Pesticide Formulations
Total Benzene-Soluble Chlorine Method
Final Action
Surplus 1978
(Applicable in absence of other org. CI compds.)
See 6.254-6.256, 13th ed.
960.13 DDT in Pesticide Formulations
Infrared Spectroscopic Method
First Action 1960
Final Action 1961
(Caution; See safety notes on pesticides.)
A. Reagent
DDT std soln.— Weigh 0.250 g tech. DDT into 50 mL vol.
flask or g-s container and add exactly 25 mL CS 2 . If sample
to be analyzed contains S, add wt of S expected in portion of
sample to be taken for analysis. Shake to dissolve and add
small amt anhyd. Na 2 S0 4 . Centrf. portion of soln if it is not
clear.
B. Determination
Weigh sample contg ca 0.25 g DDT into 50 mL vol. flask
and add exactly 25 mL CS 2 and small amt anhyd. Na 2 S0 4 . Let
stand >30 min with occasional shaking. Transfer portion to
AOAC Official Methods of Analysis (1990)
Organohalogens
187
g-s test tube and centrf. short time. Transfer to NaCl cell and
scan with infrared spectrophtr, using 0.5 mm cell in region,
8.5-10.5 fim.
Scan std soln in same manner.
Measure A of DDT peak at 9.83 jxm with baseline from 9.4
to 10.2 |xm, and calc. % DDT.
Refs.: JAOAC 40, 286(1957); 43, 342(1960).
CAS-50-29-3 (p,p'-DDT)
969.07 Dicamba in Pesticide Formulations
Infrared Spectrophotometric Method
First Action 1969
Final Action 1972
A. Reagents and Apparatus
(a) Acetone. — Spectral grade.
(b) Dimethy lamine (DMA) soln. — 60% (w/w).
(c) Dicamba std. — Ref. grade (Sandoz, Inc., 480 Camino
del Rio South, Suite 204, San Diego, CA 92108).
(d) Infrared spectrophotometer, — With BaF 2 cells, 0.025
mm, and matched NaCl cells, 0.2 mm.
B. Preparation of Sample
(Sample wts are for cell thicknesses specified. For other cells,
adjust wts to yield peak between 30 and 60% 7".)
(a) Aqueous solns of DMA salt (4 lb/ gal.). — Pipet, using
same pipet as for std, 5.00 mL sample into tared 25 mL vol.
flask and weigh. Dil. to vol. with acetone. (Use this soln di-
rectly in 0.025 mm BaF 2 cell.)
(b) Solns of DMA salt (other concentrations). — Prep, as
in (a), adjusting sample size to yield 2.4 g dicamba/25 mL.
(c) Technical dicamba. — Weigh 0.2 ±0.005 g sample into
tared 25 mL vol. flask and dil. to vol. with CS 2 .
C. Preparation of Standard
(a) Liquid formulations. — (J) Aqueous solns of DMA salt
(4 lb/gal.): Weigh 11.98±0.02 g dicamba std into tared 50
mL beaker. Add 5 mL H 2 and 4 mL 60% DMA. Adjust pH
to 7.0 by titrg with 60% DMA soln, using mag. stirrer and
pH meter. (All solids should be dissolved at this time.) Rinse
each pH electrode with two 1 mL H 2 rinses (4 mL total),
collecting rinses in the 50 mL beaker. Cool soln to room temp,
and transfer to tared 25 mL vol. flask. Rinse beaker twice with
H 2 0, collecting rinses in flask. Dil. to vol. with H 2 and mix
thoroly. Weigh flask and contents to det. total wt of soln. Pipet
5.0 mL std formulation into tared 25 mL vol. flask, weigh,
and dil. to vol. with acetone.
(2) Aqueous solns of DMA salt (other concentrations): Prep,
as in (a)(7), adjusting dicamba content to required concn.
(b) Technical dicamba. — Weigh 0.2 ±0.005 g dicamba std
into tared 25 mL vol. flask and dil. to vol. with CS 2 .
D. Determination
(a) Liquid formulations . — Record spectra of std and sample
between 1070 and 930 cm" 1 (9.3-10.7 pun), using BaF 2 cell.
Use air in ref. beam. Obtain A A and A A' for sample and std,
resp., at 1012 cm -1 (9.89 |xm) from horizontal baseline tan-
gent to min. between 1020 and 1070 cm"" 1 (9.4-9.7 (xm).
(b) Technical dicamba. — Record spectra of std and sample
from 1 100 to 930 cm"' (9.1-10.7 p,m), using NaCl cells. Use
CS 2 in ref. cell. Obtain A A and A A' for sample and std, resp.,
at 1012 cm" 1 (9.89 \xm) from horizontal baseline tangent to
min. between 1075 and 1035 cm" 1 (9.3-9.66 umi).
B. Calculations
(a) Liquid formulations. -^Dicamba, ib/gal. = A A x
C/A A', where C = lb std/gal. = (g std x % purity of std x
8.35)/25.
% Dicamba by wt = (A A x F)/(g sample/25 mL), where
F = [(g std/25 mL) X % purity of std]/AA\
(b) Technical dicamba. — % Dicamba by wt = A A x F/g
sample, where F — (g std x % purity of std)/AA\
Ref.: JAOAC 51, 1301(1968).
CAS- 191 8-00-9 (dicamba)
971.07 Dicamba-MCPA
and Dicamba-2,4-D in Pesticide Formulations
Infrared Spectrophotometric Method
First Action 1971
Final Action 1973
AOAC-CIPAC Method
(Caution: See safety notes on pesticides.)
A. Principle
Method is applicable to aq. dimethylamine (DMA) salt for-
mulations of dicamba and 2-methyl- 4-chlorophenoxy acetic acid
(MCPA) or 2,4-D (2,4-dichlorophenoxyacetic acid). Active
ingredients are pptd by HO and extd with CHC1 3 . Sol v. is
evapd, residue dissolved in acetone, and A measured at char-
acteristic 1R wavelengths.
B. Preparation of Standard Solutions
(a) Dicamba-MCPA. — Accurately weigh 0.20±0.02 g di-
camba and 0.60±0.02 g MCPA into tared weighing bottle.
Pipet in 25 mL acetone and swirl until completely dissolved.
If cells other than 0.2 mm are used, adjust wts to give A of
0.2-0.5 (30-65% T) for both std and sample solns.
(b) Dicamba-2,4-D.— Prep, as in (a), using 0.20±0.02 g
dicamba and 0.40±0.02 g 2,4-D.
C. Preparation of Sample
(a) Dicamba-MCPA. — Accurately weigh sample contg
0.20±0.02 g dicamba and 0.60±0.02 g MCPA into tared
weighing bottle. Add 5 mL H 2 and transfer quant, to 125
mL separator with 5-10 mL H 2 0.
(b) Dicamba-2A-D. — Prep, as in (a), using 0.20±0.02 g
dicamba and 0.40±0.02 g 2,4-D.
D. Determination
To soln add HO drop wise with const swirling to pH 1 ; then
add 5 drops excess. Pipet in 25 mL CHC1 3 and shake to dis-
solve ppt. Drain CHC1 3 ext into 125 mL erlenmeyer and re-
ext with two 15 mL portions CHC1 3 . Add boiling chips to com-
bined ext and evap. on steam bath to dryness. Let dry in hood
overnight at room temp. (Do not dry in air or vac. oven.) Pipet
in 25 mL acetone and swirl to completely dissolve residue.
Add few g granular anhyd. Na 2 S0 4 if any H 2 is present.
Record IR spectrum and measure A A in matched 0.2 mm
NaCl cells with acetone in ref. cell at following wavelengths:
(a) Dicamba-MCPA.— Range, 1135-930 cm" 1 (8.8-10.75
jxm); dicamba peak, 1012 cm" 1 (9.89 umi); MCPA peak, 1070
cm" 1 (9.35 |xm); baseline, horizontal tangent to min. at 970-
965 cm™ 1 (10.3-10.4 jutm) for both constituents.
(b) Dicamba-2A-D.— Range, 1130-945 cm" 1 (8.85-10.6
fxm); dicamba peak, 1012 cm" 1 (9.89 fjtm); 2,4-D peak, 1080
cm" 1 (9.26 |xm); baseline, horizontal tangent to min. at 970-
960 cm" 1 (10.3-10.4 |xm) for both constituents.
188
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
E. Calculations
% by wt of constituent = (AA/W)(W x P/AA'), where
A A and A A' = absorbance of constituent in sample and std
solns, resp.; W and W - g constituent in sample and std solns,
resp.; and P = % purity of constituent in ref. std.
lb/gal. = % by wt x sp gr x 8.345
Ref.: J AOAC 54, 706(1971).
CAS-94-75-7 (2,4-D)
CAS-1918-00-9 (dicamba)
CAS-94-76-6 (MCPA)
984.07 Dicamba, 2,4-D, and WICPP
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1984
A. Principle
Combinations of dicamba, 2,4-D, and MCPP in liq. for-
mulations as their salts are detd in any combination, using bi-
nary mobile phase 22% and 33% CH 3 CN-H 2 0, phosphate
buffered, pH 2.68-2.70, on reverse phase bonded micropar-
ticulate column. Specified column seps known impurities,
analytes, and internal stds salicylic acid and butyrophenone.
B. Apparatus
(a) Liquid chromato graph. — Fitted with 5000 psi pressure
gage, 280 nm detector, 10 |xL injector, eluant reservoir line
filter, and 10 mV FS strip chart recorder; flow rate 2.0 mL/
min.
(b) Liquid chromatographic column. — 250 X 4.6 (id) mm
Partisil 10-25 ODS-3 with Co:Pell ODS pellicular guard col-
umn (Whatman Inc.).
(c) Operating conditions. — Flow rate 2.0 mL/min; detec-
tor 280 nm; injection vol. 10 |ulL; temp, ambient (<76°F); set-
ting 0.2 AUFS for dicamba and 1 .0 AUFS for 2,4-D and MCPP;
chart speed 0.5 cm /min.
(d) Performance characteristics. — 10 fxL injection of 0.95
g salicylic acid/L causes ca 50% recorder deflection at 1 .0
AUFS. Optimum conditions for column sepn are obtained when
ochlorophenoxyacetic acid impurity, from technical 2,4-D, is
sepd between salicylic acid and dicamba in 33% eluant and
immediately after dicamba in 22% eluant.
C. Reagents
Prep, each eluant in 1 L erlenmeyer and re-circulate to con-
serve mobile phase.
(a) Eluant I. — 22% mobile phase: 725 mL H 2 (0.4 \xm-
filtered), 220 mL CH 3 CN (distd in glass), 16.9 mL NaOH (ca
17.7N, 1 + 1, 936.16B(b)); add H 3 P0 4 to pH 2.69 ± 0.01
(note vol.) and dil. to 1 L with H 2 0.
(b) Eluant II.— 33% mobile phase: 610 mL H 2 0, 330 mL
CH 3 CN, 16.9 mL NaOH (ca 17. 7N, 1 + 1, 936.16B(b)); add
H,P0 4 to pH 2.69 ± 0.01 (note vol.) and dil. to 1 L with
H 2 0.
(c) Diluting solvent. — Isopropanol-H 2 (2 + 1).
(d) Internal std soln. — 0.95 g salicylic acid (Aldrich Chem-
ical Co.) + 9.00 mL butyrophenone (Aldrich Chemical Co.);
dil. to 1 L with dilg sol v.
(e) Std soln. — Governing factor is highest peak. For ex-
pected 2,4-D in highest concn, weigh 0.150 g; for expected
MCPP A in highest concn, weigh 0.225 g. Weigh stds accord-
ing to expected sample composition and ratio of herbicides to
each other. Weigh into graduated, conical centrf. tube (Corn-
ing, Cat. No. 25330-50) or other stoppered, leak-proof con-
tainer marked at 25 mL. Pipet 20.0 mL internal std soln; dil.
to 25.0 mL with dilg solv.
D. Preparation of Sample Solution
Use same container as std soln and accurately weigh sample
equiv. to 0. 150 g 2,4-D or 0.225 g MCPP according to which
is expected to be in highest concn in formulation. For sample
having vol. >5 mL, dil. to 50 mL after addn of 20 mL internal
std soln. If sample is dild to 50 mL, also dil. internal std soln
to approx. same concn. Increase sensitivity to obtain approx.
same peak ht. Filter samples and stds thru Whatman 9.0 cm
glass fiber GF/C, or equiv. Use same pipet for adding internal
std soln to stds and samples.
E. Determination
Flush LC column of previous solv. with H 2 for several
minutes to stable baseline.
Dicamba: With eluant 1 flowing at 2.0 mL/min, inject 10
(xL salicylic acid (0.95 g/L in dilg solv.) to condition column.
Set detector to maximize peak (ca 0.2 AUFS), inject 10 (jlL
std soln, and, when pen returns to baseline after elution of
dicamba, inject 10 jxL sample soln.
2,4-D and /or MCPP must be flushed from column after
sample soln injection by the sequence: H 2 2 min, 50% CH 3 CN
until pen returns to stable baseline. Elution order is salicylic
acid first and dicamba second.
2,4-D and MCPP: With eluant II flowing at 2.0 mL/min,
and attenuation at 1.0 AUFS, inject 10.0 fxL std soln. When
pen returns to baseline after elution of butyrophenone, inject
10.0 (xL sample soln. Elution order is salicylic acid, dicamba,
2,4-D, MCPP, and butyrophenone.
F. Calculation
Use salicylic acid as internal std for calc. of dicamba; use
butyrophenone for calcg 2,4-D, MCPP. Either peak ht or com-
puter-integrated areas may be used.
% Compd = (W'/W) x (R/R') x P
where W = mg std; W = mg sample; P ~ % purity of std;
R and R' — peak ht or area ratios of compd to internal std for
sample and std, resp.
Ref.: JAOAC 67, 837(1984).
CAS-94-75-7 (2,4-D)
CAS-1918-00-9 (dicamba)
979.03 Dichlobenil
in Pesticide Formulations
Gas Chromatographic Method
Final Action
CIPAC-AOAC Method
(Caution: See safety notes on pesticides.)
A. Reagents and Apparatus
(a) Dichlobenil. — >99.5% purity (Duphar B.V., Box 2,
1380 A A Weesp, The Netherlands), or equiv.
(b) Methyl myristate.—Fluka AG Cat. No. 70129, >99.5%
purity (Fluka Chemie, AG, Industriestrasse 25, CH-9470,
Switzerland), or equiv.
(c) Mixed solvent soln. — 1 ,2-Dichloroethane-ether (1 + 1).
(d) Internal std soln. — Dissolve 0.80 g Me myristate, (b),
in 100 mL mixed solv. soln, (c).
(e) Calibration soln. — Accurately weigh ca 0.10 g dichlo-
benil, (a), into conical flask, pipet in 5 mL internal std soln,
(d), and add 45 mL solv. soln, (c).
(f ) Gas chromatograph. — With on-column injection, flame
ionization detector, injection port heating, and, preferably, de-
tector heating. Pyrex column 1.8 m x 3 mm id, packed with
AOAC Official Methods of Analysis (1 990)
Organohalogens
189
10% Carbowax 20M on 100-120 mesh Chromosorb P, acid
washed, dimethyldichlorosilane treated (available from Anal-
abs, Inc., Cat. No. GCP-009D). Operating temps (°): oven
200, injection port 210, detector 210. Carrier gas (N) flow rate
25 inL/min. Approx. retention times 7 and 12 min for Me
myristate and dichlobenil, resp.
B. Preparation of Sample
(a) Technical dichlobenil. — Accurately weigh ca 2.0 g
dichlobenil into 100 mL vol. flask. Dissolve in mixed solv.
soln, (c), and dil. to vol. Pipet 5 mL aliquot into 100 mL
conical flask, add 5.00 mL internal std soln, (d), and dil. to
50 mL with mixed solv., (c).
(b) W enable powders. — Accurately weigh sample contg ca
1.0 g dichlobenil into 100 mL vol. flask, add few mL mixed
solv., (c), swirl, and dil. to vol. with mixed solv. Let settle,
pipet 10 mL clear supernate into 100 mL conical flask, and
continue as in (a).
(c) Granules. — Accurately weigh ca 6.0 g sample into 100
mL conical flask. Add 20 mL dichloroethane and stir 10 min
on mag. stirrer. Filter with vac. thru glass filter paper (No.
G8, Fisher Scientific Co., or equiv.), supported on fritted glass
filter. Wash granules 5 times with 5 mL dichloroethane, col-
lect filtrate in 100 mL vol. flask, and dil. to vol. with ether.
Pipet aliquot of this soln, contg ca 0. 1 g dichlobenil (10 mL
for 20% granules, 25 mL for 7% granules), into 100 mL con-
ical flask, add 5.00 mL internal std soln, (d), and dil. to 50
mL with mixed solv., (c).
C. Determination
Inject 2 u,L portions of calibration soln, (e), until response
factor varies <1% for successive injections. Inject duplicate 2
|ulL portions of sample soln, followed by 2 [xL portions of cal-
ibration soln, (e). Measure peak areas of dichlobenil and Me
myristate, either by multiplying peak ht by retention time, or
by digital integration. Use av. of duplicate values.
D. Calculation
p = (j q x r X 20)/(I r X q)
where p — response factor, I q and I r — peak areas of internal
std and dichlobenil, resp., q — g internal std, r = g dichlobenil
in calibration soln. (Response factor is ca 1.5.)
% Dichlobenil = (I d x q x p x F x 100)/{I m x W x 20)
where l d and l m = peak areas of dichlobenil and internal std,
resp., p = response factor, F = diln factor for sample (100/
x, where x = mL taken to obtain final soln), and W — g sam-
ple.
Ref.: JAOAC 62, 8(1979).
CAS- 11 94-65-6 (dichlobenil)
976.02 Dicofol in Pesticide Formulations
Potentiometric Method
First Action 1976
A. Principle
Dicofol is hydrolyzed in ale. KOH under reflux, and hy-
drolyzable org. CI is converted to ionizable CI which is titrd
potentiometrically with std AgN0 3 .
B. Apparatus
(a) Condenser. — ¥ 24/40 Pyrex condenser, water cooled,
400 mm long with drip tip.
(b) Potentiometer. — Fisher Accumet Model 320 (new model
325) expanded scale pH meter, or equiv., with 50 mL buret
graduated in 0.1 mL, Ag billet indicating electrode (Fisher No.
13-639-122), and Ag-AgCl ref. electrode (Fisher No. 13-639-
53). Keep Ag electrode free from tarnish by polishing with aq.
NaHCO r CaC0 3 (1 + 1) paste. Before each analysis, rinse Ag
electrode with NH 4 OH (1 + 1) followed by H 2 0.
C. Reagents
(Use deionized H 2 thruout.)
(a) Alcoholic potassium hydroxide soln. — 0.5N. Dissolve
28.1 g KOH pellets in ca 600 mL alcohol and dil. to 1 L with
alcohol.
(b) Potassium chloride std soln. — 0.1N. Dissolve 7.456 g
KC1 in H 2 and dil. to 1 L with H 2 0.
(c) Silver nitrate std soln. — 0. IN. Dissolve 17.00 g AgN0 3
in 100 mL H 2 0, add 1 .7 mL HN0 3 , and dil. to 1 L with H 2 0.
To stdze, dil. 25 mL 0.1N KC1 to 200 mL with H 2 in 400
mL beaker. Adjust pH to 2.0±0.2, using NH 4 OH (1 + 4) and/
or HN0 3 (1+4), and dil. to 300 mL. Titr., using potentiometer
as in F. Plot mv against vol. 0AN KC1 and det. mL 0.LV
AgN0 3 at end point. Calc. normality of AgN0 3 std soln. Stdze
AgN0 3 std soln daily. (Equiv. wt dicofol = 370.5/3 = 123.5.)
(d) Thymol blue indicator soln. — 0.1%. Dissolve 100 mg
thymol blue in 100 mL alcohol (1 + 1).
D. Preparation of Sample
(a) Dicofol technical. — Fuse sample in loosely capped jar
in 100° oven and mix thoroly with glass rod. Accurately weigh
ca 4-6 g molten sample into 150 mL beaker. Add 50-75 mL
isopropanol and heat with occasional swirling until sample dis-
solves. Transfer quant, to 500 mL vol. flask, let cool to 25°,
and dil. to vol. with isopropanol. Pipet 25 mL sample soln
into 300 mL ¥ 24/40 Pyrex erlenmeyer. (Caution: See safety
notes on pipets.)
(b) Dicofol formulations . — (1 ) Dicofol MF and dicofol 35 . —
Accurately weigh ca 1 g sample into 300 mL ¥ 24/40 Pyrex
erlenmeyer. (2) Dicofol EC. — Proceed as in (7), using ca 2 g
sample.
E. Hydrolysis
Transfer 50 mL ale. KOH soln to erlenmeyer contg sample.
Attach condenser, seal with 2-3 drops alcohol, and reflux gently
on hot plate 1.5 hr. Let cool, and rinse condenser and tip witlj
25 mL alcohol. Quant, transfer soln to 400 mL beaker, using
50 mL alcohol and 100 mL H 2 0. Rinse erlenmeyer with addnl
portions H 2 to total vol. of 250 mL.
Add 10 drops thymol blue indicator soln to beaker and, with
stirring, add HN0 3 (1 + 1) dropwise to first pink color of
indicator. Adjust pH to 2 ± 0.2, using NH 4 OH (1 + 4) and/
or HNO3 (1 + 4). Adjust total vol. to 300 mL with H 2 0.
F. Determination
Place sample beaker on mag. stirrer, and adjust to rapid stir-
ring. Titr. with AgN0 3 std soln to same mv end point used
for stdzg AgN0 3 std soln. Titr. blank (unhydrolyzed sample).
% Active ingredient in tech. dicofol
= {[(V./SJ - (V h /S b )\ X N x v x x 0.1235 X 100}/V
% Active ingredient in dicofol formulations
= [(VVSa) - (Vt/Sb)] x N x 0.1235 x 100
where V s and V b = mL AgN0 3 std soln required to titr. sample
and blank, resp.; N = normality of AgN0 3 std soln; S a and S b
= g sample taken for hydrolysis and blank, resp.; V t = total
voL sample soln = 500 mL; and V - aliquot vol. sample soln
= 25 mL.
Ref.: JAOAC 59, 1109(1976).
CAS- 11 5-32-2 (dicofol)
190
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
986.06 Dicofol in Pesticide Formulations
Liquid Chromatographic Method
First Action 1986
A. Principle
DicofoJ is dissolved in MeOH, sepd by liq. chromatgy,
detd by comparison of peak hts of stds and samples.
and
B. Apparatus
(a) Liquid chromato graph. — Provided with pulseless, const
flow pump and 15 pU sample loop or auto- injector. Operating
conditions: mobile phase flow rate 2.0 mL/min; detector sen-
sitivity 0.5 AUFS; temp., 30°.
(b) Detector — UV spectrophtr or fixed wavelength UV de-
tector at 254 nm.
(c) Recorder. — Range to match output of LC detector.
(d) Liquid chromatographic columns. — Analytical: stainless
steel, 250 x 4.6 mm, packed with Zorbax C8, 6 |xm spherical
particles (DuPont Co., Instruments Div., Concord Plaza, Wil-
mington, DE 19898). Guard: stainless steel, 50 x 4.6 mm,
packed with LiChrosorb RP-18, 10 |xm particle size (Merck,
EM Science) (replacement model from EM Science is a car-
tridge system: LiChrospher RP-18, 5 |xm, prepacked guard
column No. 50803 and universal guard column holder No.
16217).
(e) Filter. — Millex-HV disposable filter assembly, 0.45 jxm
(No. SLHV025NS, Millipore Corp., or equiv.).
C. Reagents
(a) Mobile phase.— MeOH-H 2 0-HOAc (75 + 25 + 0.2),
components individually filtered thru 0.45 |xm filter. Mix 750
mL MeOH, 250 mL H 2 0, and 2 mL glacial HO Ac, and degas.
(b) Dicofol std soln. — Accurately weigh ca 35 mg pure 1,1-
bis(4-chlorophenyl)-2,2,2-trichloroethanol and ca 7.5 mg pure
1 -(2-chloropheny I)- 1 -(4-chlorophenyl)- 2,2, 2-trichloroethanol
into 1 oz vial and add 20 mL MeOH by pipet.
D. System Performance Check
Dissolve, in 1 oz viaJ, ca 50 mg Kelthane tech. in 20 mL
MeOH. Sample is very plastic; heat at 60° to liquefy.
Inject 15 |ulL onto liq. chromatgc column and det. sepn fac-
tor (a) for dicofol and p,p'~ and o,p f -DDE isomers as follows:
a = k'2/k'l
where k'2 and k'\ are column capacity ratios of isomer pairs,
defined as follows:
k' = (t T - Q/t
where t v — elution time of retained component and / = dead
vol. elution time. Sepn factors are 1.62 for dicofol o,p'- and
/^'-isomers, and 1.38 for DDE isomers. Sepn factors of 1.55
for dicofol pair and 1 .25 for DDE pair would be approx. limits
for proper performance. Performance check should also con-
firm that key components are adequately resolved. DDT iso-
mer pairs should be resolved from DDE isomers and should
fit between DDE isomers. Example chromatogram is shown
in Fig. 986. 06 A. Fig. 986. 06B is example of unacceptable
resolution where DDT isomer pair is not resolved from DDE
isomers.
E. Preparation of Sample
(a) Kelthane MF. — Accurately weigh ca 100 mg MF for-
mulation in 1 oz via) and add 20 mL MeOH by pipet. Shake
to dissolve. Filter thru 0.45 |mm filter.
(b) Kelthane EC. — Accurately weigh ca 250 mg EC for-
mulation in 1 oz vial and add 20 mL MeOH by pipet. Shake
to dissolve. Filter thru 0.45 (xm filter.
Fig. 986.06A — LC chromatogram of Kelthane technical
F Determination
Inject 15 |xL dicofol std soln. Det. peak hts of p,p'- and
o,p' -dicofol. Inject 15 julL sample soln.
%/?,// -dicofol = (PH/PH') x (W'/W) x 100
% o,p' -dicofol = (PH/PH 1 ) x (W'/W) x 100
% Active ingredient = % p,p' -dicofol + % o,p' -dicofol
where PH and PH' = peak ht of isomer in sample and std
solns, resp.; W and W r — wt of sample and std injected, resp.
Wt sample injected is calcd by wt = 15 x W v /20, where W v
= wt of sample in vial, u.g.
Ref.: JAOAC 69, 714(1986).
CAS-1 15-32-2 (dicofol)
983.07 Difiubenzuron
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1983
Final Action 1984
CIPAC-AOAC Method
A. Principle
Difiubenzuron is dissolved in 1 ,4-dioxane, sepd by LC, and
detd from peak areas vs linuron internal std. Identity is con-
firmed by retention time.
B. Apparatus
(a) Liquid chromato graph. — Provided with const flow pump
and 20 |ulL sample loop. Operating conditions: mobile phase
flow rate, 1.3 mL/min; detector sensitivity, 128 X 10~ 3 AUFS;
temp., ambient, should not fluctuate >2°; retention time of
difiubenzuron relative to internal std, ca 1.36.
(b) Detector. — UV spectrophtr or fixed wavelength UV de-
tector at 254 nm.
AOAC Official Methods of Analysis (1 990)
Organohalogens
191
1-
D
Q
Q.
p d
9 <*
Q ~
It < UJ
o d ,
7 * a
"q. Hi <£
*SS
Si O
E
1
££
CO
'a
a
0)
c
ra
£
0)
£
E
a
.§
i
oO
"a
J
op
cT
1
cc
0)
UJ
c
rtj
op
£
UJ
a
<u
d
*
a.
m |
o"
od
to I
CO
i
(-
II
cc
1
a.
< u"
1
O
1- (O n . \\ \
I
/ \
i-
Wl T^ ^
J U
JV
.w
-i
■z
Fig. 986.06B— LC chromatogram of Kelthane standards
(c) Recorder. — Range to match output of LC detector.
(d) Liquid chromatography column. — Stainless steel,
250 x 4.6 mm, packed with Zorbax BP-Q (E.I. DuPont de
Nemours & Co.), or equiv. (e.g., uJBondapak C t8 , 10 |xm,
Waters Associates, Inc.; Spherisorb ODS, 5 |xm, Phase Sep-
arations Ltd, Dee side Industrial Estate, Queen sferry Clwyd,
UK; Zorbax BP-ODS, 7 jam, E.I. DuPont de Nemours).
(e) Filter, — Acrodisc disposable filter assembly, 1-2 [xm
(Gelman Sciences, Inc.) or equiv.
C. Reagents
(a) Mobile phase. — Acetonitrile-H 2 0- 1 ,4-dioxane (45 +
45 + 10). Mix 450 mL acetonitrile, 450 mL H 2 0, and 100
mL 1 ,4-dioxane and degas.
(b) Solvent mixture. — Aeetonitrile-H 2 (45 + 55).
(c) Internal std soln, — Accurately weigh 25 mg linuron (No.
P14-04, Office of Reference Materials, Laboratory of the Gov-
ernment Chemist, Teddington, Middlesex, TW11 OLY, UK)
into 100 mL vol. flask, dil. to vol. with acetonitrile, and mix.
(d) Diflubenzuron std soln. — Accurately weigh ca 50 mg
pure diflubenzuron (>99.5% Duphar B.V., PO Box 2, 1380
AA Weesp, The Netherlands) into 100 mL vol. flask. Add 50
mL dioxane and dissolve by heating 30 min in 80° H 2 bath.
Swirl occasionally. Add 40 mL dioxane, cool, and dil. to vol.
with dioxane. Pipet 5 mL into 50 mL vol. flask, add 5.00 mL
internal std soln, (c), dil. to vol. with sol v. mixt., (b), and
mix.
(e) 1 ,3 -Di{4-chlorophenyl)urea . — Duphar B.V.
D. System Performance Check
Dissolve, in 100 mL vol. flask, 4.2 mg diflubenzuron and
1.8 mg 1 ,3-di(4-chlorophenyl)urea in 20 mL dioxane, dil. to
vol. with solv. mixt., (b), and mix. Filter thru 1.2 \xm filter
before injection.
Inject 20 |ulL onto column and det. resolution (R) by follow-
ing formula:
R = 2 d/(W } + W 2 )
where R — resolution; d — distance between peak maxima; W,
and W 2 = peak width at baseline of diflubenzuron and 1,3-
di(4-chlorophenyl)urea, resp. Resolution should be >1 . If nec-
essary, resolution can be improved by slightly increasing H 2
content of mobile phase.
E. Preparation of Sample
(a) Diflubenzuron pre-concentrate. — Accurately weigh
sample contg 1.0 g diflubenzuron into 200 mL vol. flask. Add
150 mL dioxane and heat 30 min in 80° H 2 bath. Swirl oc-
casionally. Add 40 mL dioxane, cool, and dil. to vol. with
dioxane. Pipet 10 mL into 100 mL vol. flask and dil. to vol.
with dioxane. Pipet 5 mL dild soln into 50 mL vol. flask, add
5.00 mL internal std soln, (c), dil. to vol. with solv. mixture
(b), and mix. Filter thru 1.2 |xm filter.
(b) Water dispersible powder. — Accurately weigh sample
contg 0.5 g diflubenzuron into 200 mL vol. flask. Add 150
mL dioxane and heat 30 min in 80° H 2 bath. Swirl occa-
sionally. Add 40 mL dioxane, cool, and dil. to vol. with diox-
ane. Immediately pipet 20 mL homogeneous suspension into
100 mL vol. flask and dil. to vol. with dioxane. Pipet 5 mL
dild soln into 50 mL vol. flask, add 5.00 mL internal std soln,
(c), dil. to vol. with solv. mixture, (b), and mix. Filter thru
1.2 [im filter.
F. Determination
Inject 20 jxL diflubenzuron std soln, (d). Det. peak areas
(or peak hts X retention times) of diflubenzuron and internal
std. Repeat injections until response ratio (area diflubenzuron
peak/area internal std peak) varies < 1 % for successive injec-
tions. Inject 20 |xL sample soln.
G. Caicuiation
% Diflubenzuron - (R/R r ) x (W /W) x V x 100
where R and R' = ratio of area of diflubenzuron peak to area
of internal std peak for sample and std, resp.; W and W — g
sample and std, resp.; V = diln factor (= 20 and 1.0 for pre-
conc. and H 2 0-dispersible powder, resp.).
Ref.: J AOAC 66, 312(1983).
CAS-35367-38-5 (diflubenzuron)
983.08 Endosulfan
in Pesticide Formulations
Gas Chromatographic Method
First Action 1983
Final Action 1984
C1PAC-AOAC Method
A. Principle
Sample is extd with toluene and a- and p-endosulfan iso-
mers are detd sep. by thermal conductivity or flame ionization
GC, using di(2-ethylhexyl)phthalate as internal std.
B. Apparatus
(a) Gas chromatograph. — Suitable for on-column injec-
tion; equipped with thermal conductivity detector. Flame ion-
ization detector may be used with proper diln of samples and
stds.
(b) Chromatographic column. — 3 mm id X 1.5 m (5 ft)
glass column packed with 10% OV-210 on 80-100 mesh
Chromosorb W-HP. Condition column >:16 h, at 250°, using
carrier gas at ca 25 mL/min. Operating conditions: injector
300°, detector 250°, column 230°, He carrier gas flow ca 60
mL/min.
192
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
C. Reagents
(a) Toluene. — GC quality.
(b) Internal std soln. — 15 mg/mL. Dissolve 15 g di(2-
ethylhexyl)phthalate, 99% + , in toluene and dil. to 1 L with
toluene.
(c) Endosulfan std soln. — Accurately weigh 0.300 g en-
dosulfan of known a- and p-isomer content (Riedel de Haen
Co., D-3016 Hannover-Seelze 1, GFR; U.S. and Canadian
distributor, Crescent Chemical Co., Inc., 1324 Motor Pkwy,
Hauppauge, NY 11788) into 25 mL g-s flask. Pipet in 10 mL
internal std soln, (b), mix, and store > 20 min at 60° to ensure
dissolution.
D. Preparation of Sample
(a) Technical endosulfan. — Accurately weigh 0.300 g sam-
ple into 25 mL g-s flask, pipet in 10 mL internal std soln, (b),
mix, and store >20 min at 60°.
(b) Emulsifiable concentrates. — Prep, as in (a), using wt
equiv. to 0.300 g endosulfan.
(c) W enable powder . — Accurately weigh sample contg 0.300
g endosulfan into 25 mL g-s flask, pipet in 10 mL internal std
soln, (b) mix, store ^20 min at 60°, and centrf.
E. Determination
Inject 2 [xL portions of std soln, (c), until response factor
for each component varies <1% for successive injections. In-
ject duplicate 2 u,L portions of sample soln followed by 2 \xL
portions of std soln. For each injection, calc. response ratio
a-isomer peak to internal std peak, and ratio of p-isomer peak
to internal std peak, either as peak ht or digital integrator count
for area. Retention time for each isomer and internal std should
be the same for sample and std solns.
F. Calculation
% a-isomer = C a = (RJK ) * (W /W) x P a
% p-isomer = C p = (/? p /*e> x (W /W) x p p
where R a and R' a - response ratios of a-isomer peaks to in-
ternal std peaks for sample and std soln, resp.; 7? p and R$ =
response of p-isomer peaks to internal std peaks for sample
and std soln, resp.; W and W - wt (g) of sample and endo-
sulfan std, resp.; P a and P p = % a- and p-isomers in std, resp.
Ref.: J AOAC 66, 999(1983).
CAS- 11 5-29-7 (endosulfan)
984.08 Fluazifop-Butyl
in Pesticide Formulations
Gas Chromatographic Method
First Action 1984
Final Action 1985
(Method is suitable for tech. and formulated fluazif op- butyl.)
A. Principle
Sample is dissolved in CH 2 C1 2 contg dibenzyl succinate as
internal std, and 1 |xL is injected into GC for flame ionization
detection. Peak areas are measured for fluazifop-butyl and di-
benzyl succinate and compared with those from std injection.
B. Apparatus
(a) Gas chromato graph. — With heated, glass-lined injec-
tion port and flame ionization detector. Temps (°) — column
230, injection port 250, detector 270; gas flows (mL/min) —
N carrier gas 30, H 60, air 240; sample size 1.0 |ulL; retention
times (min) — fluazifop-butyl 4.3, isomeric impurity 4.9, in-
ternal std 8.0. Adjust parameters to assure complete sepn of
peaks, particularly isomeric impurity from fluazifop-butyl, and
peak hts ca 60-80% full scale on chart at quoted retention
times.
(b) Column. — 1.8 m (6 ft) x 2 mm (id) glass column packed
with 3% OV-17 on 100-120 mesh Chromosorb WHP (Su-
pelco Inc., Cat. No. 1-1757). Precondition overnight at 250°
before use.
C. Reagents
(a) Dibenzyl succinate internal std soln.- — Weigh 7 g di-
benzyl succinate (ICN Pharmaceuticals, Inc., Cat. No. 13686),
dissolve in CH 2 C1 2 , and dilute to 1 L. Check internal std soln
for interfering components by injecting 1 julL into chromato-
graphy Store in tightly capped bottles to avoid evapn.
(b) Fluazifop-butyl std soln. — Accurately weigh ca 100 mg
fluazifop-butyl std of known purity (ICI Americas Inc.) into
vial. Pipet 10.0 mL internal std soln into vial, cap, and shake
to dissolve. Store in tightly capped bottles to avoid evapn.
D. Determination
(a) Liquid and technical samples. — Accurately weigh amt
sample contg ca 100 mg fluazifop-butyl into vial. Pipet 10.0
mL internal std soln into vial, cap, and shake to dissolve.
(b) Granular formulations . — Accurately weigh amt sample
contg ca 100 mg fluazifop-butyl into vial. Pipet 10.0 mL in-
ternal std soln into vial, cap, and shake on a wrist-action shaker
10 min. Let insoluble inerts settle 10 min before analysis.
Inject 2 or more aliquots of std soln to set integration pa-
rameters and stabilize instrument. Monitor response factor un-
til results agree within 2%. Inject 4 aliquots of std soln and 2
aliquots of sample soln in succession. Calc response factor, /?,
for each injection:
R = area fluazifop-butyl peak/area internal std peak
Fluazifop-butyl, % = (R/R f ) x (W /W) x P
where R and R f — av. response factor for sample and std solns;
W and W = mg sample and std; and P - purity (%) of std.
Ref.: J AOAC 67, 499(1984).
CAS-69806-50-4 (fluazifop-butyl)
977.03 Folpet in Pesticide Formulations
Liquid Chromatographic Method
First Action 1977
Final Action 1979
AOAC-CIPAC Method
(Applicable to dry formulations contg folpet as only active in-
gredient and to folpet combination formulations except those
contg propargite or Me parathion. Compds insol. in CH 2 C1 2 ,
e.g., maneb or inorg. salts, do not interfere.)
(Caution: See safety notes on pesticides.)
A. Apparatus
(a) Liquid chromato graph. — Equipped with 254 nm UV
detector. Typical operating conditions: chart speed, 0.2"/min;
flow rate, 2 mL/min (ca 800 psi); detector sensitivity, 0.16 A
unit full scale; temp., ambient; valve injection vol., 20 |x'L.
Adjust operating conditions to elute folpet peak in 4±1 min.
Factors such as different H 2 content in CH 2 C1 2 mobile phase
can change retention times. Folpet peak must be completely
resolved from dibutyl phthalate peak which normally elutes in
ca 7 min.
(b) Liquid chromatographic column. — Stainless steel, 300
AOAC Official Methods of Analysis (1990)
Organohalogens
193
X 4 mm id, packed with 10 fxm diam. silica gel particles (Waters
Associates, Inc., No. 27477, or equiv.).
B. Reagents
(a) Mobile phase. — Degassed CH 2 C1 2 .
(b) Internal std soln. — Accurately weigh ca 0.5 g dibutyl
phthalate (EM Science No. DX0605) into 200 mL vol. flask.
Dil. to vol. with CH 2 C1 2 and mix.
(c) Folpet std soln. — (100 |xg folpet + 250 |xg dibutyl
phthalate) /mL. Accurately weigh ca 20 mg folpet ref. std,
99+% pure (Chevron Chemical Co., PO Box 4010, Rich-
mond, CA 94804) into glass vial, pi pet 20 mL internal std soln
into vial, and shake to dissolve. Pipet 1 mL into 10 mL vol.
flask. Dil. to vol. with CH 2 C1 2 .
C. Preparation of Sample
Accurately weigh sample contg 20 mg folpet into vial. Pipet
20 mL internal std soln into vial and shake 30 min. Centrf. to
ppt solids. Pipet 1 mL supernate into 10 mL vol. flask, dil.
to vol. with CH 2 C1 2 , and mix. Sample contains ca (100 u,g
folpet + 250 |mg dibutyl phthalate)/mL.
D. Determination
Inject 20 |ulL folpet std soln onto column thru sampling valve
and adjust operating conditions to give largest possible on-scale
peaks with retention time of 4±1 min for folpet. Repeat in-
jections until ratio of folpet to dibutyl phthalate peak hts is
within ±1% of previous injection. Without changing condi-
tions, inject sample soln until its ratio is within ±1% of pre-
vious ratio for sample. Average last 2 peak ht ratios for sample
and for std, resp., and calc. % folpet.
% Folpet = (R/R r ) x (W'/W) x P
where R and /?' = av. peak ht ratios for sample and std, resp.;
W - mg folpet in std soln (ca 20 mg); W = mg sample extd
for analysis; and P — % purity of std.
Ref.: JAOAC 60, 1157(1977); 61, 384(1978).
CAS-133-07-3 (folpet)
962.07 Heptachlor
in Pesticide Formulations
Active Chlorine Method
First Action 1962
Final Action 1965
A. Reagents
(a) Dilute acetic acid.— 80%. Dil. 800 mL HO Ac to 1 L
with H 2 0.
(b) Silver nitrate-acetic acid std soln. — Dissolve 17 g AgN0 3
in 200 mL H 2 0, add 56 mL HN0 3 (1 + 1), and dil. to 1 L with
HO Ac. Stdze potentiometrically by adding 25 mL of this soln
to 600 mL beaker contg 250 mL 80% HO Ac. Immerse glass
and Ag electrodes in soln and stir with mag. stirrer. Titr. with
0.1/V NaCl soln, 962.05A(a), to end point (max. change in
mv/mL NaCl soln). Normality AgN0 3 = mL NaCl x nor-
mality NaCl/m'L AgN0 3 .
B. Preparation of Sample
(a) Emulsifiable concentrate formulations. — Accurately
weigh sample contg O.3I±0.O5 g heptachlor in 250 mL erlen-
meyer. Dissolve in 50 mL HOAc, and pipet in 25 mL 0.17V
AgN0 3 , (b). Attach reflux condenser and reflux 1 hr.
(b) Granular and dust formulations . — (Caution: See safety
notes on flammable solvents, pesticides, and pentane.) Ac-
curately weigh sample contg 0.3 ±0.05 g heptachlor into 80 x
25 mm Soxhlet extn thimble. Ext 2 hr with pentane and trans-
fer ext to 250 mL erlenmeyer. Attach short reflux column such
as 3 -ball Snyder or 12" (30 cm) Vigreux to flask and evap. to
dryness on steam bath. (Results will be low if reflux column
is not used.) Rinse down column with 50 mL HOAc, pipet in
25 mL 0AN AgN0 3 , (b), attach reflux condenser, and reflux
1 hr.
(c) Technical. — Accurately weigh 0.40 ±0.05 g heptachlor
and proceed as in (a).
C. Determination
Rinse tip of condenser or column with H 2 and cool soln
to room temp. Transfer quant, to 600 mL beaker, rinsing with
four 10 mL portions 80% HOAc. Immerse glass and Ag elec-
trodes in soln and stir with mag. stirrer. Titr. with 0.1/V NaCl
soln, 962.05A(a), to end point.
% Heptachlor = 37.33 x (25 x normality AgN0 3 soln
— mL NaCl soln x normality NaCl soln)/g sample
Ref.: JAOAC 45, 513(1962); 57, 424(1974).
CAS-76-44-8 (heptachlor)
968.04 Heptachlor
in Pesticide Formulations
Gas Chromatographic Method
First Action 1968
Final Action 1969
A. Apparatus
(a) Gas chromatograph. — Equipped with H flame ioniza-
tion detector; capable of accepting glass column and glass-lined
sample introduction system or on-column injection. Use fol-
lowing conditions: Temps (°): column 175, detector 175-190,
sample inlet 190; N carrier gas pressure 30 psig; recorder chart
speed 2.5 cm/min.
(b) Glass-stoppered tubes. — Approx. 25 and 75 mL capac-
ity.
(c) Microliter syringe. — 10 |jlL, Hamilton Co., 701 -N.
B. Reagents
(a) Heptachlor. — Ref. grade (Velsicol Chemical Corp. 5600
N. River Rd, Rosemont, IL 60018-5119).
(b) Aldrin. — Ref. grade (Sandoz, Inc., 480 Camino del Rio
South, Suite 204, San Diego, CA 92108).
C. Preparation of Column
To 9.5 g 100-120 mesh Gas Chrom Q in vac. flask add
0.50 g silicone GE Versilube F-50 (available from Applied
Science) dissolved in 50 mL CH 2 C1 2 . Shake slurry well to wet
solid thoroly. Connect flask to H 2 aspirator and evap. sol v.
with frequent shaking. When solids appear dry, complete drying
by placing flask in steam bath and connecting to vac. pump
until ca 4 mm pressure is attained. Remove flask from steam
bath and let cool under vac.
Fill 1.5 m (5') x Vs" od (0.067" id) Pyrex glass tube with
this packing, using vac. pump and gentle tapping. Plug ends
of column with glass wool. Condition column 24 hr in 190°
oven while purging with N. Let column cool while still purg-
ing with N; then install in chromato graph.
D. Preparation of Sample
(a) Liquids. — Weigh sample contg ca 750 mg heptachlor
into 75 mL g-s vial and add 500 mg ref. grade aldrin. Add 75
mL fresh CS 2 , stopper, and shake vigorously 2 min.
(b) Solids. — Transfer weighed sample contg ca 750 mg
194
Pesticide Formulations
AOAC Official Methods of Analysis (1 990)
heptachlor to Soxhlet and ext 2 hr with 75 mL pentane. Let
cool, add 500 mg ref. grade aldrin to soln, and swirl.
E. Calibration
Weigh 0.2500 g ref. grade heptachlor and 0. 1670 g ref. grade
aldrin into 25 mL g-s flask. Dissolve in 25 mL CS 2 . Chro-
matograph this soln under conditions given in 968*04A(a) 5
times to obtain accurate response correction factor. (On new
column, it is sometimes desirable to inject several 5 |xL ali-
quots of std soln to condition column before use.)
F. Determination
Let instrument equilibrate as in 968.04A(a). Inject ca 1 |xL
sample soln at sensitivity setting such that ht of heptachlor
peak is ca 3 / 4 full scale. For each analysis, allow 10-12 min
for heptachlor related components to elute. Components and
approx. retention times in min are: heptachlor 4.5, aldrin 5.9,
chlordene 3.1, and 7-chlordane 9.9.
G. Calculations
Calc. area of heptachlor and aldrin peaks by multiplying peak
ht in mm by width of peak at half ht in mm. Alternatively,
use integrator. Caic. response correction factor (/, ca 0.82)
for each of the 5 std injections as follows:
/ = (area of heptachlor peak X mg aldrin
x purity of aldrin) /(area of aldrin peak X mg heptachlor
X purity of heptachlor)
Average 5 replicates and use av. to calc. % heptachlor in sam-
ples.
% Heptachlor = (area of heptachlor peak
X mg aldrin X purity of aldrin x 100) /(area of aldrin peak
x mg sample x f)
Ref.: JAOAC 51, 565(1968).
CAS-76-44-8 (heptachlor)
980.07 MCPA Ester and Salt
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1980
{Caution: See safety notes on sodium hydroxide, and aceto-
nitrile.)
A. Principle
Esters of MCPA are saponified in situ and amine salts are
converted to H 2 0-sol. K salt of MCPA. Ion suppression in
reverse phase bonded microparticulate column seps isomers and
impurities. Ionic MCPA moiety is protonated by acidic mobile
solv., forming nonionic MCPA moiety, which greatly in-
creases partitioning into stationary phase. Small changes in
mobile solv. pH significantly affects retention time. Using pH
2.83 and flow rate of 3 mL/min, MCPA at 16.3 min retention
time will elute between impurities 2-Me-4-chlorophenol at 14.5
min and 2,6-diMe-4-chlorophenoxyacetic acid at 23.0 min.
B. Apparatus
See 978. 05B. Do not use column other than that specified.
C. Reagents
(a) Soponification-internal std soln (SISS). — Partly fill vol.
flask with isopropanol-H 2 (2+1). Dissolve salicylic acid in
the aq. isopropanol to produce final concn of 0.6 g/L. Add
KOH (1 + 1) of known normality (ca 10. IN) to produce final
soln of 0.2N KOH. Dil. to final vol., adjusting concn of sal-
icylic acid according to detector response to obtain peak ht
approx. equal to that of MCPA, avoiding off scale peak. Inject
different strength solns of salicylic acid in aq. isopropanol (2+1)
to det. appropriate concn.
(b) Eluant. — pH 2.83. Mix CH 3 CN (distd in glass, Burdick
& Jackson Laboratories, Inc., or equiv.) with H 2 (deionized,
0.4 [xm filtered) (15 + 85) contg NaOH added from (1 + 1) aq.
soln of known normality, 936.166(b), to concn in final vol.
of 0.3M. Adjust to pH 2.83 with H 3 P0 4 .
(c) MCPA std soln.— 300 mg/25 mL SISS. Accurately weigh
ca 300 mg MCPA anal. ref. std (99 + % isomer pure; available
from Dow Chemical Co.), previously dried 15 min at 95°, into
1 or 2 oz (30 or 60 mL) glass vial with polyethylene-lined
screw cap. Pipet in 25 mL SISS, and shake to dissolve. Prep.
MCPA std and sample solns at same time, using same pipet.
D. Preparation of Sample
Proceed as in 978. 05D, using sample contg ca 300 mg MCPA
and 25 mL SISS, but filter thru microfiber filter GF/C, or
equiv. For ester formulations, to eliminate baseline interfer-
ence from aromatic solvents, transfer saponified sample to 30
mL separator. Add ca 5 mL hexane, shake 1 min, let sep.,
and collect major portion of aq. phase for chromatgy.
E. Determination
Proceed as in 978. 05E, substituting MCPA samples and stds
for 2,4-D.
Ref.: JAOAC 63, 873(1980).
CAS-94-74-6 (MCPA)
982.03 Methazole
in Pesticide Formulations
Infrared Spectrophotometric Method
First Action 1982
(Applicable to wettable powder contg methazole as only active
ingredient)
A. Apparatus and Reagents
(a) Infrared spectrophotometer . — Capable of measuring A
from 700 to 900 cm" 1 , with matched 0.5 mm NaCl or KBr
cells.
(b) Methazole std soln. — Weigh, to nearest mg, 0.48-0.52
g ref. std methazole (available from Velsicol Chemical Corp.,
5600 N. River Rd, Rosemont, IL 6001 8-51 19) into 4 oz poly-
ethylene screw-cap bottle, pipet in 50.0 mL acetone, and mech.
shake 15 min to dissolve.
(c) Acetone. — Anal, reagent grade (Mallinckrodt, or equiv.).
B. Determination
Weigh, to nearest mg, 0.63-0.67 g sample into 4 oz poly-
ethylene screw-cap bottle, pipet in 50,0 mL acetone, and mech.
shake 1 h. Centrfg. 30 min to obtain clear supernate.
Fill both cells of spectrophtr with acetone, and place in in-
strument. Optimize gain; set 100% adjust to give 95-98% T
at 755 cm -1 . Set slit in program or manual mode for optimum
sensitivity and resolution. Fill sample cell with std soln, and
scan region from 860 to 700 cm -1 04'). Using same condi-
tions, fill same cell with sample soln and scan twice (A). Mea-
sure A and A' at 755 cm -1 , using min. at 845 cm -1 as baseline.
C. Calculation
% Methazole = (W x A x P x 100)/(W x A')
where W and W = g sample and std, resp.; and P ~ % purity
of std.
AOAC Official Methods of Analysis (1990)
Organohalogens
195
Ref.: JAOAC 64, 1185(1981).
CAS-20354-26-1 (methazole)
Ref.: JAOAC 68, 570(1985).
CAS-5 121 8-45-2 (metolachlor)
985.06 Metolachlor
in Pesticide Formulations
Gas Chromatographic Method
First Action 1985
Final Action 1988
AOAC-CIPAC Method
(Method is suitable for formulations where metolachlor is only
active ingredient.)
A. Principle
Metolachlor is extd with acetone and detd by flame ioni-
zation gas chromatgy, using dipentyl phthalate as internal std.
Identity is verified simultaneously by comparing retention times
with std.
B. Apparatus
(a) Gas chromatography — Equipped with flame ionization
detector.
(b) Chromatographic column. — 2 mm id X 1.83 m (6 ft)
glass column packed with 3% OV-101 on 80-100 mesh Gas-
Chrom Q, or equiv. Condition column >24 h at 240°, using
carrier gas at ca 20 mL/min. Operating conditions: injector
250°, detector 250°, column 180° ± 10°, He carrier gas flow
ca 25 mL/min. Retention times for metolachlor and internal
std are ca 8.8 and 15.6 min, resp.
C. Reagents
(a) Internal std soln. — 4 mg/mL. Dissolve 4.0 g dipentyl
phthalate in acetone and dil. to 1 L with acetone. Check in-
ternal std for interfering components by injecting aliquot into
chromatograph.
(b) Metolachlor std soln. — Accurately weigh 200 mg me-
tolachlor std of known purity (Ciba-Geigy Corp., Production
Technical Dept, PO Box 18300, Greensboro, NC 27419) into
4 oz bottle. Pipet 50.0 mL internal std soln into bottle, cap,
and shake 10 min.
D. Preparation of Sample
Accurately weigh amt sample contg ca 200 mg metolachlor
into 4 oz bottle. Pipet 50.0 mL internal std soln into bottle,
cap, and shake 10 min to ext.
E. Determination
Set integration parameters and stabilize instrument by in-
jecting 1-3 |ulL aliquots of std soln until area ratios of meto-
lachlor to internal std vary <2% for successive injections. Us-
ing same established injection vol. as for std, inject sample.
Sample area ratio should be ±10% of std area ratio. Inject 2
aliquots of std and 2 aliquots of sample followed by 2 aliquots
of second sample and 2 aliquots of std. Repeat sequence until
all samples are analyzed. Calc. response factor, R, for each
injection:
R = peak area (or ht) metolachlor/peak area (or ht) int. std
Metolachlor, % - (R/R') X (W f /W) x P
where R and R f — av. response factor for sample and std solns,
resp.; W and W = mg sample and std, resp.; and P = purity
(%) of std.
982.04 PCNB in Pesticide Formulations
Gas Chromatographic Method
First Action 1982
Final Action 1983
(Caution: See safety notes on pesticides.)
A. Principle
Sample is dissolved in CHCl 3 , o-terphenyl is added as in-
ternal std, and PCNB is detd by GC with flame ionization de-
tection.
B. Apparatus and Reagents
(a) Gas chromatograph with recorder. — With flame ioni-
zation detector and 1.8 m X 4 mm (id) glass column packed
with 5% SE-30 on 80-100 mesh Chromosorb W (dimethyl-
chlorosilane-treated) (Analabs, Inc.). Condition newly packed
column 24 h at 285° with low N flow. Operating conditions:
temps (°) — inlet 200, column 175-180, detector 250; carrier
gas flow to elute PCNB at ca 4.5 min; adjust H and air as
recommended for detector by manufacturer; sensitivity to give
peak hts 60-80% full scale.
(b) PCNB std soln,— 2.0 mg/mL CHC1 3 . Accurately weigh
ca 0.2 g PCNB (Olin Corp., Agriculture Products Dept., PO
Box 991, Little Rock, AR 72203) into 100 mL vol. flask and
dil. to vol. with CHC1 3 .
(c) Internal std soln. — 0.8 mg/mL CHC1 3 . Accurately weigh
ca 0.2 g o-terphenyl into 250 mL vol. flask and dil. to vol.
with CHCI3.
(d) Mixed std soln. — 1 .0 mg PCNB + 0.4 mg o-terphenyl/
mL. Pipet 25 mL each of PCNB and internal std solns into
vial and mix.
C. Preparation of Sample
(a) Solid formulations . — Grind 100 g well mixed sample to
pass 1 mm sieve. Accurately weigh portion of well mixed,
ground sample contg ca 0.2 g PCNB into 250 mL g-s erlen-
meyer and add 100 mL CHC1 3 ; stopper and shake 2 h on rotary
shaker. Let insol. matter settle.
(b) Wettable powders. — Accurately weigh portion of well
mixed sample contg ca 0.2 g PCNB into 250 mL g-s erlen-
meyer and proceed as for solids.
(c) Liquids. — Accurately weigh portion of well mixed sam-
ple contg ca 0.2 g PCNB into 100 mL vol. flask and dil. to
vol. with CHCI3.
(d) Soln for analysis. — Pipet 10 mL sample ext above and
10 mL internal std soln into vial, cap, and mix.
D. Determination and Calculation
Inject 4 |ulL aliquots of mixed std soln until variation in re-
sponse ratio (area or peak ht) for PCNB (first peak) to o-ter-
phenyl (second peak) is ca 1%. Inject mixed std, inject sample
twice, and repeat injection of mixed std. Retention times must
be the same for sample and std. Calc. av. ratios of PCNB to
o-terphenyl for the 2 mixed std and sample injections, and calc.
% PCNB.
% PCNB - (R/R') x (W'/W) x P
where R and R ( = av. response ratios for sample and mixed
std, resp.; W = g PCNB/ 100 mL std soln; W = g sample
extd; and P = purity (%) of PCNB std.
Refs.: JAOAC 65, 110(1982); 66, 410(1983).
CAS-82-68-8 (pentachloronitrobenzene)
196
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
976.03 Picloram and 2,4-D
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1976
Final Action 1978
A. Apparatus
(a) Liquid chromato graph. — Equipped with 280 nm UV
detector and injection valve. Alternatively, septum injection
head may be used; however, stop-flow injection is recom-
mended. Operating conditions: flow rate, 0.7 mL/min (ca 1000
psi); detector sensitivity, 0.08 A unit full scale; temp., am-
bient, but within ±2.5°.
(b) Liquid chromatographic column. — No. 316 stainless
steel, 1000 X 2.1 mm id, with Varian Instrument Group, No.
96-000075-00 reducing union (Vs" x Vie") contg 2 \x,m frit
(regular reducing union packed with glass wool may be used
instead) packed with DuPont No. 820960005 Zipax® SAX
(strong anion exchange) resin. Preclean column with few mL
each of CHC1 3 , acetone, and MeOH, and vac, -dry. Pack in
small increments over 40 min period while tapping column on
hard surface.
B. Reagents
(a) Mobile phases. — Prep. sep. solns of 0.0 1M Na 2 B 4 7 .
10H 2 O (3.8 g/L) and 0.002Af NaC10 4 .H 2 (0.28 g/L) in pre-
viously boiled and cooled deionized H 2 0.
(b) Salicylic acid internal std soln. — Accurately weigh ca
3.6 g USP Ref. Std Salicylic Acid into 1 L vol. flask, dil. to
vol. with 0.05N NaOH in isopropanol-H 2 (1 + 1), and mix.
(c) Picloram-2,4-D std soln. — (4 mg picloram +12 mg
2,4-D ~f~ 3.6 mg salicylic acid)/mL. Accurately weigh ca 100
mg picloram ref. std, 99+% pure (Dow Chemical Co.), and
ca 300 mg 2,4-D ref. std, 99 + % pure (Dow Chemical Co.),
into glass vial, pipet in 25 mL salicylic acid internal std soln,
and shake to dissolve,
C. Preparation of Sample
Accurately weigh ca 1.6 g sample into ca 10 dram glass
vial, pipet in 25 mL salicyclic acid internal std soln, and shake
to dissolve.
D. Determination
Inject 2 |xL picloram-2,4-D std soln onto column and adjust
attenuation to give largest possible on-scale peaks. Repeat in-
jections until peak ht ratios of herbicide: internal std vary <1%
for successive injections. Without changing conditions, inject
2 (xL aliquots sample soln until peak ht ratios vary <1%. Av-
erage last 2 peak ht ratios for picloram and 2,4-D and calc,
% herbicide.
% Herbicide - (/?//?') x (W /W) x P
where R and R' = av. peak ht ratios of each herbicide to the
internal std for sample and std, resp.; W = mg herbicide in
std; W = mg sample; and P — % purity of std.
Ref.: J AOAC 59, 748(1976).
CAS-94-75-7 (2,4-D)
CAS-1918-02-1 (picloram)
986.05 Propachlor
in Pesticide Formulations
Gas Chromatographic Method
First Action 1986
Final Action 1988
AOAC-CiPAC Method
A. Principle
Sample is dissolved in acetone contg diisobutyl phthalate as
internal std, analyzed by gas chromatgy with flame ionization
detection, and measured by comparison with internal std on
basis of integrated peak areas.
B. Safety
LD 50 of propachlor has been found to be 1800 mg/kg in rat
acute oral studies (Monsanto). Material is classified as slightly
toxic. Avoid excessive exposure; wear protective clothing.
C. Apparatus
(a) Gas chromatograph. — With flame ionization detector
and on-column injection ports. Temps — column oven 200°
(isothermal), injection port 250°, detector 250°; gas flows (mL/
min) — He carrier gas 30, H 34, air 430; sample size 1.0 |xL;
run time 25 min.
(b) Column. — 6 ft X 2 mm (id) glass column (on-column
configuration) packed with 10% SP-2250 on 100-120 mesh
Supelcoport (Supelco, Cat No. 1-2132), or equiv. SP-2250 is
methyl-phenyl silicone (50 + 50). Precondition overnight at
250° before use. Retention times for propachlor and internal
std are ca 5.29 and 11.51 min, resp.
D. Reagents
(a) Acetone. — Pesticide grade (Fisher Scientific Co., or
equiv.).
(b) Diisobutyl phthalate internal std soln. — Accurately weigh
6.4 g diisobutyl phthalate (Eastman Kodak Co,) into 1 L vol.
flask. Dissolve in and dil. to vol. with acetone.
(c) Propachlor std soln. — Accurately weigh 0.2 g propa-
chlor (recrystd from MeOH; Monsanto Chemical Co.) into small
flask. Add by pipet 25 mL internal std soln and shake to dis-
solve.
E. Determination
Accurately weigh sample contg ca 0.2 g propachlor into small
flask. Add by pipet 25.0 mL internal std soln and shake 5:5
min to ext propachlor. For flowable formulations, use 0.5 g
sample; for Ramrod® /Atrazine flowable formulation, use 0.6
g sample; for granular formulations (20 G), use 1.0 g sample.
Thoroly mix solns by mech. shaking. Vigorously mix granular
samples 15 min.
Make replicate 1 juiL injections of propachlor std soln and
measure response ratio, R (area propachlor peak/area internal
std peak), for each injection. Repeat until consecutive re-
sponse ratios agree ±0.5%.
Make duplicate injections of sample soln and det. av. R.
Follow with injection of propachlor std soln; average R' for
std before and after sample injection
Propachlor, % = (R/R f ) x (W'/W) x P
where R and R' = av. response ratios for sample and std, resp.;
W and W = wt (g) of sample and std, resp.; P = % purity
of std.
Ref.: JAOAC 69, 723(1986).
CAS-1918-16-7 (propachlor)
980.08 2,4,5-T in Pesticide Formulations
Liquid Chromatographic Method
First Action 1980
(Method is modification of LC method for 2,4, -D, 978.05.)
(Caution: See safety notes on pesticides, and acetonitrile.)
A. Apparatus
See 978.05B.
B. Reagents
(a) Eluant.—pR 2.95. 18% CH 3 CN-82% H 2 0, v/v, contg
NaOH added from (1 + 1) aq. soln of known normality,
AOAC Official Methods of Analysis (1990)
Organophosphates
197
936.16B(b), ca 17.77V, to concn in final vol. of 0.3M. Adjust
to pH 2.95 with H 3 P0 4 . Use std buffers at pH 3.0 or 4.0 as
ref.
(b) Saponification- internal std soln. — See 978.05C(b).
(c) 2,4,5-T std soln.— 240 mg/25 ml. Prep, as in
978.05C(c), using 240 mg 2,4,5-T instead of 300 mg 2,4-D.
C. Preparation of Sample
Prep, sample as in 978. 05D, using sample contg ca 240 mg
2,4,5-T, and using GF/C filter. Ext with hexane as in 980.07D,
D. Determination
Proceed as in 978.05E, substituting 2,4,5-T samples and stds
for 2,4-D.
Ref.: JAOAC 63, 873(1980).
CAS-93-76-5 (2,4,5-T)
981.02 Tetradiffon (Technical)
and Pesticide Formulations
Gas Chromatographic Method
First Action 1981
Final Action 1982
CIPAC-AOAC Method
A. Principle
Tetradifon is detd by flame ionization GC, using n-hexa-
cosane as internal std.
B. Apparatus
(a) Gas chromato graph. — Suitable for on -column injec-
tion; equipped with flame ionization detector.
(b) Gas chromatographic column. — 3 mm id x 6 ft glass
column packed with 3% SE-52 on 100-120 mesh Chromosorb
W-HP. Operating conditions: injector and detector 250°, col-
umn 230°, N or He carrier gas flow ca 35 mL/min. Approx.
retention times for tetradifon and w-hexacosane = 8.6 and 10
min, resp.
C. Reagents
(a) Internal std soln. — Accurately weigh 0.30 g pure n-
hexacosane into 100 mL vol. flask. Dil. to vol. with 1,2-di-
chloroethane and mix.
(b) Tetradifon std soln. — Accurately weigh 0.100 g pure
tetradifon (>99.5%, Duphar B.V., PO Box 2, 1380 Weesp,
The Netherlands; or Chemical and Biological Investigations,
Environmental Protection Agency, Belts ville, MD 20705) into
100 mL vol. flask, pipet 20.0 mL internal std soln, (a), into
flask, dil. to vol. with dichloroethane, and mix.
D. Preparation of Sample
(a) Technical tetradifon. — Accurately weigh 100 mg sam-
ple into 100 mL vol. flask. Pipet 20.0 mL internal std soln,
(a), into flask, dil. to vol. with dichloroethane, and mix tho-
roly.
(b) Wettahle powder. — Accurately weigh sample contg 100
mg tetradifon into 250 mL g-s flask. Pipet in 20.0 mL internal
std soln, (a), and 80.0 mL dichloroethane. Heat on H 2 bath
5 min, cool, and mix thoroly. Transfer ca 40 mL soln to cen-
trif. tube, and centrif. 10 min.
(c) Emulsifiable concentrates. — Accurately weigh sample
contg 100 mg tetradifon into 100 mL vol. flask. Pipet 20.0
mL internal std soln, (a), into flask. Dil. to vol. with dichlo-
roethane and mix thoroly.
E. Determination
Adjust chromatographic conditions to give ca 1 / 2 FSD for 2
|jLg tetradifon. Inject 2 jjlL portions of std soln, (b), until re-
sponse factor ( F) varies <l% for successive injections. Inject
2 |xL sample soln, and measure peak hts and retention times
for both std and sample.
F. Calculation
F=(/'xrx?x 5)/(/ x S x 100)
where /' and / ~ peak ht x retention time of internal std and
tetradifon, resp.; T = g tetradifon in calibration soln; P = %
purity of std; S = g internal std. (Response factor is ca 1.5.)
% Tetradifon - (/ x S X F x 100)/(/' x W x 5)
where W — g sample.
Ref.: JAOAC 64, 829(1981).
CAS- 11 6-29-0 (tetradifon)
962.08 Sodium TCA
in Pesticide Formulations
Titrimetric Method
First Action 1962
Final Action 1966
(Caution: See safety notes on distillation, flammable solvents,
pesticides, and peroxides.)
A. Apparatus and Reagent
(a) Reflux apparatus. — 250 mL erlenmeyer attached thru ¥
24/40 joint to 50 cm water-cooled condenser.
(b) Dioxane. — Freshly distd.
8. Determination
Dissolve 25 g sample in H 2 and dil. to 1 00.0 mL. Pipet
aliquot (usually 10 mL), titrg ca half that of blank, into 250
mL re fluxing flask, add 1 drop Me red, and neutze with ca
IN H 2 S0 4 to distinct orange-pink. pH is 5.3-5.5; usually <0. 15
mL is required. If soln is acid, titr. with ca \N NaOH. Add
25.00 mL 1W H 2 S0 4 , 35 mL dioxane, and few glass beads.
Boil vigorously under reflux >60 min. Cool, add 2 drops Me
red, and titr. with std 1/V NaOH to sharp change from orange
to yellow end point. Perform blank detn, omitting sample.
% Na trichloroacetate = Net mL IN acid x 0.1854
x 1.00/g sample in aliquot
Refs.: Anal. Chem. 27, 1774(1955). JAOAC 43, 382(1960);
45, 522(1962).
CAS-650-51-1 (sodium TCA)
Trifluralin
Final Action
See 973.13.
THIOPHOSPHORUS AND OTHER
ORGANOPHOSPHORUS PESTICIDES
980.09 Azinphos-ftflethyl
in Pesticide Formulations
Infrared Spectrophotometry Method
First Action 1980
Final Action 1981
(Applicable to 50% wettahle powders and 2 lb/gal. liq. concns
where azinphos-methyl is the only active ingredient.)
(Caution: See safety notes on pesticides.)
198
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
A. Apparatus
(a) Infrared spectrophotometer . — Capable of making mea-
surements in 600-700 cm" 1 region, such as Perkin-Elmer Corp. ,
Models 421 and 521.
(b) Cells. — Perkin-Elmer sealed cells, 0.5 mm KBr for 2
lb /gal. liq. cones and 0.2 mm KBr for 50% wettable powders.
(c) Disposable pipets. — Scientific Products Inc., No. P5205-
1 with bulb No. P2515, or equiv.
(d) Mechanical shaker. — Eberbach Model 6000 with 6040
carrier tray, or equiv.
(e) Centrifuge. — International centrifuge Model UV, or
equiv., with head for 15 mL tubes.
B. Reagents
(a) Azinphos-methyl. — Purified material, available from
Mobay Corp.,
(b) Azinphos-methyl std soln. — Weigh, to nearest 0.1 mg,
0.18-0.19 g purified azinphos-methyl (for 2 lb/gal. liq. cones)
into 50 mL vol. flask or 0.245-0.255 g (for 50% wettable
powder) into 25 mL vol. flask. Dil. to vol. with 1.2-dime-
thoxyethane and shake well.
(c) 1 ,2 -Dimethoxy ethane — Bp 83-85° (Eastman 4639, bp
83-85°, has been found satisfactory).
C. Preparation of Sample
Weigh, to nearest 0.1 mg, 0.77-0.. 79 g 2 lb/gal. liq. cone,
or 0.85-0.95 g 50% wettable powder sample into 50 mL vol.
flask. Add 25 mL 1 ,2-dimethoxy ethane into flasks and shake
on mech. shaker 10 min. Dil. to vol. with 1,2-dimethoxy-
ethane. Mix thoroly. Let wettable powder samples stand 15
min to allow clay to settle. Do not mix again before analyzing.
If soln remains turbid, centrf. 10 mL aliquot in stoppered tube
15 mm at 650-700 X g.
D. Determination
Fill appropriate KBr cell with corresponding std soln from
clean disposable pipet. Adjust gain to optimum setting (3-4)
at 654 cm" 1 . Set 100% adjust at 690 cm" 1 to give 95-98% T,
auto suppression — 0, scale change selector — linear, scan gear —
A-18, B-72, scan time — 1.5-1.8 min, slit program 950 X 2
(421), 750 manual (521), and speed change knob — fast gear.
Scan std soln from 700 to 630 cm"" 1 using air as blank. Using
same instrument settings, scan sample soln twice, filling cell
each time using clean pipet, in same manner as std. Repeat
scan with std soln. Draw baseline thru minima near 690 and
630 cm -1 and measure ht of 654 cm" 1 peak above this line in
A units. Record sample as A and std as A' . If A of sample and
std differ by > 0.030, repeat analysis using adjusted sample wt
{W\)j where
W.
W X A 1 1 A
If difference between replicate std A' or replicate sample A
is >2% of their average, repeat analysis:
(a) For 2 lb/gal. liq. cones:
K - Wj x p/A'
(b) For 50% wettable powder:
K = Wj x P x 2/A'
% (Azinphos-methyl) = K x A/W (or W t )
where W (or W^) and W refer to wts sample and std, resp.;
and P = % purity of std.
Ref.: JAOAC64, 628(1981), corr. 1269.
CAS-86-50-0 (azinphos-methyl)
989.01 Azinphos-Methyl
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1989
AOAC-CtPAC Method
(Applicable to wettable powder and liq. formulated products
contg azinphos-methyl as only active ingredient)
Method Performance
50% wettable powder:
s r = 0.19; s R = 0.53; RSD r = 0.40%; RSD R = 1.11%
2L emuisifiable cone. (2 lb /gal.):
s r = 0.41; s R = 0.56; RSD r = 1.79%; RSD R - 2.47%
2S emuisifiable cone. (2 lb/gal.):
s r = 0.17; s R = 0.25; RSD r = 0.79%; RSD R = 1.17%
A. Principle
Azinphos-methyl is detd by liq. chromatgy. Peak hts of
sample and anal, std are compared using w-butyrophenone as
internal std.
B. Apparatus
(a) Liquid chromato graph. — Able to generate >7 MPa
(>1000 psi). Equipped with spectrophtr to measure A at 285
nm and peak ht integrator or recorder. Operating conditions:
column temp, ambient; flow rate ca 1.5 mL/min (ca 1000 psi);
recorder speed 0.5 cm/min; recorder range 10 mV; injection
vol. ca 10 fxL; A range 0.16 AUFS. Retention times: azinphos-
methyl ca 4.0 min, internal std ca 4.8 min. Pump LC mobile
phase thru column until system is equilibrated (flat baseline).
After each injection, allow 10 min after internal std for elution
of formulation excipients.
(b) Chromatographic column. — 250 X 4.7 mm id packed
with <10 (xm C )8 bonded silica gel (Du Pont Zorbax ODS, or
equiv.).
(c) Filters. — 0.45 \wx\ porosity (Gel man Acrodisc-CR, or
equiv.)
C. Reagents
(a) Acetonitrile. — LC grade or distd in glass (Burdick &
Jackson, or equiv.)
(b) Water. — LC grade or distd in glass (Burdick & Jackson,
or equiv.)
(c) LC mobile phase.— CH 3 CN-H 2 (65 + 35).
(d) n-Butyrophenone internal std soln. — 10% n-butyro-
phenone (Aldrich No. 12,433-8, or equiv.) (v/v) in CH 3 CN.
(e) Azinphos-methyl reference std. — (Mobay Corp., Agri-
cultural Chemicals Div., PO Box 4913, Hawthorne Rd, Kan-
sas City, MO 64120-0013). Store ref. std at refrigeration temp.
(4-8°).
(f ) Azinphos-methyl std soln. — Accurately weigh ca 220 mg
azinphos-methyl ref. std into 100 mL vol. flask. Pipet 10.0
mL internal std soln into flask, dil. to vol. with CH 3 CN, and
mix thoroly. Filter portion of final soln and hold for LC anal-
ysis.
D. Preparation of Samples
Accurately weigh amt sample contg ca 220 mg azinphos-
methyl into 100 mL vol. flask. Pipet 10.0 mL internal std soln
into flask, dil. to vol. with CH 3 CN, and mix thoroly. Filter
portion of final soln and hold for LC analysis.
E. Determination
Adjust operating parameters so that azinphos-methyl elutes
in 3.8-4.2 min. Adjust injection vol. and attenuation to give
AOAC Official Methods of Analysis (1990)
Organophosphates
199
largest possible on-scale peaks. If peaks cannot be brought on
scale at 0.32 AUFS setting with 10 jjlL injection, further dil.
std and sample solns by pipetting 10 mL of each into 100 mL
vol. flasks, dilg to vol. with CH 3 CN, and mixing thoroly.
Readjust injection vol. and attenuation to give largest possible
on-scale peaks.
Make repetitive injections of std and calc. response ratios
by dividing azinphos-methyl peak ht by internal std peak ht
(area measurements are not acceptable). Response ratios must
agree within ±1% before continuing. Inject duplicate aliquots
of each sample soln (no more than 2 samples [4 injections]
between std injections). Response ratios of sample injections
must agree within ±1%. If not, repeat detn, starting with std
injections. Reinject std soln. Average response ratios of std
injections immediately preceding and following sample injec-
tions. These must agree within ±1%. If not, repeat detn.
F. Calculation
For each injection: Response ratio (R) = (azinphos-methyl
peak ht/internal std peak ht).
Azinphos-methyl, wt % = (R/R') x (W /W) x P
where R and/?' — av. response ratios for sample and std solns,
resp.; W and W = wt (mg) of azinphos-methyl in std and
sample solns, resp.; P — % purity of azinphos-methyl std.
Ref.: JAOAC 71, 988(1988).
CAS-86-50-0 (azinphos-methyl)
981.03 Chlorpyrifos
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1981
Final Action 1982
{Caution: See safety notes on acetic acid and acetonitrile.)
A. Principle
CH 3 CN contg internal std is added to solid or liq. sample
to ext chlorpyrifos. Aliquot is subjected to reverse phase LC.
Small amt of HO Ac is added to mobile phase to suppress non-
reproducible ionization of 3,5, 6-trichloro-2-pyridinol, which
might otherwise interfere.
B. Apparatus
(a) Liquid chromatograph.— Modular apparatus, LC-55,
variable wavelength detector (LC-75 can be substituted) (Per-
kin-Elmer Corp.), or equiv.; Altex 100 pump (replacement
Model 116 Programmable Solvent Module, Beckman Instru-
ments, Inc., 2350 Camino Ramon, PO Box 5101 , San Ramon,
CA 97583-0701), or equiv.; Model 728 Autosampler (Alcott
Chromatography, Inc., One Micromeritics Dr); column heat-
ing unit, LC-22A temperature controller, and LC-23A column
heater (Bioanalytical Systems, Inc., 2701 Kent Ave, West La-
fayette, IN 47906), or equiv. For manual injections, Model
7125 valve is recommended (Rheodyne, Inc., PO Box 996,
Cotati, CA 94928).
Operating conditions: FJow rate, 2 mL/min (1100 psi);
wavelength, 300 nm; 1.0 AUFS; injection volume, 10 jjlL;
temp., ambient (if temp, control is available, 30° is recom-
mended) .
(b) Liquid chromatographic column. — Zorbax® ODS, 4.6
mm X 25 cm (E. I. DuPont de Nemours & Co., Instrument
Products Div,); 2 |mm column filter, Cat. No. 7302 (Rheodyne,
Inc.).
C. Reagents
(a) Eluant.— CH 3 CN-H 2 0-HOAc (82 + 17.5 + 0.5). Mix
820 mL CH 3 CN, 175 mL H 2 0, and 5 mL glacial HOAc and
degas .
(b) Internal std soln. — 37.5 trig/25 mL. Weigh 1.5 ± 0.1
g 1 ,4-dibromonaphthalene (Eastman Organic Chemicals P7595)
into 1 L vol. flask, dil. to vol. with CH 3 CN, and mix.
(c) Chlorpyrifos std soln. — Accurately weigh ca 80 mg
chlorpyrifos ref. std, 99.7 + % pure (Dow Chemical Co.), into
glass vial, pipet in 25,0 mL 1 ,4-dibromonaphthalene internal
std soln, and mix.
D. Preparation of Sample
(a) Liquid formulations . — Accurately weigh into glass vial
amt sample contg ca 80 mg chlorpyrifos. Pipet in 25.0 mL
1 ,4-dibromonaphthalene internal std soln and mix.
(b) Solid formulations. — Accurately weigh into glass vial
amt sample contg ca 80 mg chlorpyrifos. Pipet in 25.0 mL
1 ,4-dibromonaphthalene internal std soln and place on wrist-
action shaker 5 min. (For Lorsban 15G insecticide, use 1.2 g
sample and 50 mL internal std soln.) Filter sample thru 1 jjim
filter before injection.
E. Determination
Inject 10 jjlL chlorpyrifos std soln onto column and adjust
attenuation to give largest possible on-scale peaks (ca 1.0
AUFS). Repeat injections until ratio of chlorpyrifos peak ht
(or area) to internal std peak ht (or area) varies ^0.5% (/?').
Without changing conditions, inject 10 (xL aliquots of sample
soln until ratio varies <0.5%. Average last 2 ratios for sample
soln (R) and calc. % chlorpyrifos.
% Chlorpyrifos = (R/R') x (W /W) x P
where W - mg chlorpyrifos ref. std (— 80.0 mg); W = mg
sample; and P — % purity of std.
Ref.: JAOAC 64, 628(1981); corr. 1269.
CAS-2921-88-2 (chlorpyrifos)
964.04 DDVP in Pesticide Formulations
infrared Spectrophotometric Method
First Action 1964
Method I
(Applicable to sand/sugar base fly bait contg ca 0.5% and 4
lb/gal. DDVP emulsifiable cones.)
A. Apparatus and Reagent
(a) Infrared spectrophotometer . — Capable of recording in
region 2-15 |xm. Slit width must be adjustable to give signal-
to-noise ratio of ca 1 00: 1 ; with sealed liq . absorption cell , NaCl
windows, and 0.2 mm path length.
(b) Hypodermic syringe. — Luer type, glass, 1.0 mL. Use
18 gage (Stubbs), 2" slip-on needle.
(c) 2 ,2-Dichlorovinyl dimethyl phosphate . — Use std DDVP
of known purity.
0. Calibration of Apparatus
Into each of five 10 mL vol. flasks, weigh, to nearest 0.1
mg, 25, 75, 100, 150, and 200 mg DDVP std, and dil. to vol.
with CHC1 3 . Calibration solns contain ca 2.5, 7.5, 10, 15, and
20 g DDVP/L.
Fill sealed liq. absorption cell with CH'C1 3 , adjust spec-
trophtr to optimum settings, and scan over 10.7—9.9 |xm.
Without changing settings, fill cell in turn with each of prepd
calibration solns, starting with most dil., and scan each soln
over 10.7-9.9 ixm.
200
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
For each scan, construct baseline thru absorption min. at ca
10.0 u>m parallel to radiation line. Draw perpendicular to
radiation line thru absorption max. of calibration soln at ca
10.2 |xm and measure radiant power P (at 10.0 |xm) and P
(at 10.2 juim), in any convenient units but keeping same units
thruout. Calc. A as 1og(P /P). Repeat calcns, using absorp-
tion min. at ca 10.5 jim as ref. point.
Subtract A of cell and CHC1 3 obtained above from A of cell
and calibration solns. Plot A A of DDVP as ordinate against
g/L DDVP as abscissa for each ref. point (10.0 and 10.5 fxm),
C. Preparation of Sample Solution
(a) Sand /sugar base fly baits. — Prep. 25 X 400 mm extn
column by adding enough diat. earth (Hyflo Super-Cel) to make
layer 5 cm high when gently packed. Place 250 mL vol. flask
under outlet. Accurately weigh sample contg 0.2-1 .0 g DDVP.
Transfer sample to extn column with CHC1 3 , and rinse sample
container with CHC1 3 .
Working in well -ventilated hood, add 50 mL CHC1 3 to col-
umn. Using stirring device, vigorously agitate sample and top
half of adsorbent layer to form slurry with solv. Withdraw stir-
ring device, and rinse it and column with addnl CHC1 3 from
wash bottle. Let solv. percolate thru column until level is few
mm above diat. earth-sample layer.
Add ca 50 mL CHC1 3 to column, agitate sample and diat.
earth with stirrer as above, and let solv. percolate thru column
until upper level approaches sample layer. Repeat with two
addnl 50 mL portions CHC1 3 . When solv. ht has diminished
to 2—3 mm, rinse column with three 10 mL portions CHC1 3 ,
letting each portion enter diat. earth layer before adding next.
Let column drain and rinse outlet tip with CHC1 3 , collecting
rinse in 250 mL vol. flask.
Transfer CHC1 3 eluate to evapg dish (125 mm diam.) marked
at 40-50 mL. Evap. on steam bath to 40-50 mL. Remove
dish and continue evapn at room temp, to 10-15 mL. Using
CHC1 3 , quant, transfer to vol. flask of such size to give DDVP
concn of 0.5-1.0 g/100 mL when soln is dild to vol.
(b) Emulsifiable concentrates. — Weigh enough sample, to
nearest 0.2 mg, to give ca 1 g DDVP/100 mL CHC1 3 when
dild to vol. in 10, 25, or 50 mL vol. flask.
D. Determination
Dil. CHC1 3 soln of DDVP to vol. with CHC1 3 , mix thoroly,
and fill calibrated liq. absorption cell with sample soln. Using
same instrument settings as for calibration, scan sample soln
over 10.7-9.9 fxm.
Examine spectra for possible interference and use appropri-
ate absorption min. as ref. point. (If solvs or other ingredients
interfere at one of ref. points, use alternative ref. point.) For
example, p-naphthol, often used as stabilizer in fly baits, exts
with CHC1 3 and absorbs at ca 10.5 |xm, requiring use of 1.0.0
|xm ref. point.
Calc. A of sample soln as in 964. 04B.
From calcd A, read g DDVP/L from calibration curve.
% DDVP by wt
= [(g DDVP/L) x mL sample soln] /( 10 x g sample)
Ref.: JAOAC 47, 268(1964).
CAS-62-73-7 (2,2-dichlorovinyl dimethyl phosphate)
966.07 DDVP in Pesticide Formulations
Infrared Spectrophotometric Method
First Action 1966
Method II
(Applicable to ca 0.5% (w/w) spray soln and ca 1.0% (w/w)
cattle spray in hydrocarbon solvs)
A. Apparatus and Reagent
(a) Infrared spectrophotometer , — Double beam instrument
with specifications as in 964.04A(a).
(b) 2,2-D ichlorovinyl dimethyl phosphate . — See 964 .04A(c) .
B. Preparation of Compensating Solvent
Transfer ca 30 mL sample to 125 mL separator and ext (2-
3 min per extn) with 4 ca 30 mL portions 0.57V NaOH. Dry
DDVP-free hydrocarbon phase by passing it thru 2-3 g anhyd.
Na 2 S0 4 . Reserve dried solv. for prepn of DDVP std soln and
as compensating solv. in ref. cell.
C. Determination
Prep, std DDVP soln in compensating solv. that approxi-
mates (on wt basis) DDVP content of sample. Calc. DDVP
content of std soln to nearest 0.01% by wt.
After detg optimum instrument parameters for compensation
technic, scan std soln over 9.9-10.7 |xm (1010-935 cm" 1 )
region with ref. cell contg compensating solv. in ref. beam of
spectrophtr. Scan sample against compensating solv. in same
manner. Always use same cell in ref. beam.
D. Calculations
From differential spectra, det. A of DDVP at 10.2 fjim (980
cm™ 1 ) of std, A', and sample, A, measured from baseline drawn
between minima near 10.0 and 10.6 juim. Calc. DDVP as fol-
lows:
% DDVP by wt = % DDVP in std x A/A'
Ref.: JAOAC 49, 251(1966).
CAS-62-73-7 (2,2-dichlorovinyl dimethyl phosphate)
Diazinon in Pesticide Formulations
Gas Chromatographic Method
First Action 1982
Final Action 1988
See 971.08.
982.06 Diazinon in Microencapsulated
Pesticide Formulations
Gas Chromatographic Method
First Action 1982
A. Principle
Sample is ground in tissue grinder and extd with CH 3 CN,
dibutyl phthalate is added as internal std, and diazinon is detd
by GC with flame ionization detection.
B. Apparatus and Reagents
(a) Gas chromato graph. — Equipped with flame ionization
detector (Perkin-Elmer 900, or equiv.).
(b) GC column. — 6 ft X 1 / 4 in. od, 2 mm id, glass, packed
with 3% OV-17 on 80-100 mesh Supelcoport. Operating con-
ditions: injection port 200°; column 190° (isothermal); detector
250°; He flow 35 mL/min; H flow optimum for instrument
detector; chart speed 0.2 in. /min; sample size: 1 |xL.
(c) Tissue grinder. — 40 mL capacity (Corning Glass Works,
No. 441969 or 7726-L), or equiv.
(d) Internal std soln. — Accurately weigh ca 2.0 g dibutyl
phthalate into 100 mL vol. flask. Dissolve in and dil. to vol.
with CH 3 CN.
(e) Diazinon std soln. — Accurately weigh ca 0.2 g diazinon
(W) into 50 mL vol. flask. Pipet in 10.0 mL internal std soln,
dil. to vol. with CH 3 CN, and mix well.
AOAC Official Methods of Analysis (1990)
Organophosphates
201
C. Determination
Mix sample thoroly. With medicine dropper or disposable
pipet, transfer ca 2 g sample to Al weighing dish and weigh
accurately (W). Transfer to tissue grinder, add 30 mL CH 3 CN,
and grind 3 min. When sample is thoroly ground, quant, trans-
fer to 100 mL vol. flask, wash grinder with CH 3 CN, and add
washings to vol. flask. Pipet in 20.0 mL internal std soln and
dil. to vol. with CH 3 CN. Using 10 |jlL syringe, make duplicate
1 |xL injections of sample and std solns.
Measure peak hts of first peak, diazinon, and second peak,
dibutyl phthalate, in sample and std soln and det. ratios of
diazinon to dibutylphthalate peaks for each.
Diazinon, wt% = (R/R') x (W /W) x P
where R and R' = peak ht ratios for sample and std solns,
resp.; W and W = g sample and diazinon std resp.; P = %
purity of diazinon std.
Ref.: JAOAC65, 115(1982).
CAS-333-41-5 (diazinon)
980.10 Disulfoton
in Pesticide Formulations
Gas Chromatographic Method
First Action 1980
Final Action 1981
(Caution: See safety notes on pesticides.)
A. Principle
Concn disulfoton in sample is detd by gas-liq. chromatgy,
employing di-n-butyl phthalate as internal std and flame ion-
ization detection. Response is compared to that of std of known
purity.
B. Apparatus and Reagents
(a) Gas chromato graph. — With on-column injection sys-
tem and flame ionization detector. Conditions given are for
use with Varian Model 3700. Other instruments may require
changing operating parameters to obtain good peak response
and proper resolution. Temps (°) — column 190, injection port
220, detector base 250; gas flow rates (mL/min) — carrier 30,
H 2 30, air 300; attenuation— -4 x 10~ 10 ; sample size— 3.0 (xL;
retention time (min) — disulfoton peak 5.5, internal std peak
9.25. Parameters ensure complete peak sepn with app. de-
scribed. Adjust conditions to obtain peaks ca 60-80% full scale
on chart at retention times given.
(b) Column. — 1.8 m (6') X 0.25" (od) x 2 mm id glass
column packed with 10% silicone SE-30 on 80/100 Chro-
mosorb W-HP (available from Alltech Associates, 2051 Wau-
kegan Rd, Deerfield, IL 60015). Condition new columns 16
hr at 275° before use.
(c) Syringe. — Precision syringe, 10 pJL.
(d) Di-N -butyl phthalate internal std soln. — 1.8%. Y)\\. ap-
propriate amt internal std (Eastman Kodak, or equiv.) with
acetone. Adjust concn, if needed, to obtain peaks nearly equiv.
to disulfoton peak. Keep refrigerated in suitable container to
avoid evapn. Internal std soln must contain no peaks eluting
near disulfoton peak on chromatogram.
(e) Disulfoton std solns. — Approx. 3, 4, and 5 mg/mL.
Weigh to nearest 0.1 mg ca 75, 100, and 125 mg disulfoton
std of known purity (available as Di-Syston from Mobay Corp.)
into pre- weighed 25 mL flasks. Pipet into each flask 5.0 mL
internal std soln and dil. to vol. with acetone. Label these solns
A, B, & C, resp. Soln B is working std. Use stds A and C
for linearity check.
C. Linearity Check
Perform when new working std is prepd, or when new col-
umn is installed, to check for weighing errors and instrument
difficulties. Inject triplicate aliquots std solns A, B, and C and
det. "response ratio" for each by dividing peak ht (or area)
disulfoton peak by internal std peak. Obtain "response factor"
by dividing av. response ratio by disulfoton content. Factors
should agree within 2%.
D. Preparation of Sample
(a) Liquid samples and technical material. — Accurately
weigh amt sample contg ca 100 mg active ingredient into 25
mL vol. flask. Pipet in 5.0 mL internal std soln, dil. to vol.
with acetone, and mix.
(b) Granular formulations . — Accurately weigh amt sample
contg ca 100 mg active ingredient into 2 oz (60 mL) bottle.
Pipet in 5.0 mL internal std soln and 20.0 mL acetone. Cover
and shake 30 min. Let settle or centrf . before removing aliquot
for injection.
E. Determination
Inject 2 aliquots working std soln B. Response ratios should
agree within 2%. If they do not, some difficulty is present.
Correct before continuing.
If std response is satisfactory, make duplicate injections of
each of the following:
Std B, Sample 1, Sample 2, Std B, Sample 3, Sample 4,
Std B, etc.
For each injection
R = response ratio
ht (area) disulfoton peak
ht (area) internal std peak
% Disulfoton - (R/R f ) (W /W) x P
where R = av. response ratio for sample soln, R' = av. re-
sponse ratio for ref. std B, W = mg sample, W — mg std,
and P - purity (%) of std.
For R' use av. response ratios of duplicate std injections just
before and after peaks. Average these ratios to det. R f .
Ref.: JAOAC 63, 869(1980).
CAS-298-04-4 (disulfoton)
979.04 Ethion in Pesticide Formulations
Liquid Chromatographic Method
First Action 1979
Final Action 1980
(Applicable to dry and liquid formulations contg ethion as only
active ingredient.)
A. Apparatus
(a) Liquid chromatograph. — Waters Associates with Model
6000 A pump, or equiv., with 254 nm UV detector (Waters
Associates, Inc). Typical operating conditions: eluant flow rate
1 mL/min (ca 1100 psi), chart speed 0.25 in. /min, detector
sensitivity 0.2 A unit full scale, ambient temp, injection vol.
10 \iL. Adjust operating conditions to elute ethion peak in 6
± 2 min. Column condition and H 2 content of MeOH eluant
can change retention times. Ethion peak must be sepd com-
pletely from internal std peak which normally elutes in ca 7
min (Waters C 18 column).
(b) Liquid chromatographic column. — Either (7) Waters
luuBondapak C, 8 , 300 X 3.9 mm id; or (2) DuPont ODS Per-
maphase, 0.5 m x 2.1 mm id.
202
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
B. Reagents
(a) Eiu.ant.~~ Either (7) degassed MeOH~H 2 (90 + 10), UV
cutoff <230 nm, or (2) degassed acetonitrile-H 2 (40 + 60),
UV cutoff <230 nm.
(b) Light mineral oii. — USP, viscosity 38.1 centistokes at
37.8°.
(c) Internal std soln. — (I) For Waters column. — Accu-
rately weigh ca 0.24 g pentachloronitrobenzene (PCNB), ref.
grade, with no interfering peaks on LC, into 200 mL vol. flask.
Dil. to vol. with MeOH and mix. (2) For DuPont column. —
Using CH 3 CN as solv., vary amt PCNB in internal std to give
peak ht approx. same as ethion peak.
(d) Ethion std solns. — (I) For Waters column. — Stock
soln. — Accurately weigh amt of std equiv. to 250 mg ethion,
95 + % pure (available from Chemical and Biological Investi-
gations, Environmental Protection Agency, Belts ville, MD
20705) into 25 mL vol. flask, dil. to vol. with MeOH, and
mix. Working soln. — Pipet 10 mL stock soln into 50 mL vol
flask, pipet 10 mL internal std soln, (c)(7), into flask, dil. to
vol. with MeOH, and mix. Prep, std and samples daily. (2)
For DuPont column.— Prep, as above, using CH 3 CN instead
of MeOH. (3) For oil formulations . — Pipet 10 mL 1% stock
soln (7) or (2) into 50 mL vol. flask contg ca same wt of light
mineral oil as sample. Add 20 mL MeOH (or CH 3 CN) and
proceed as in 979.04C(c) beginning with "Stopper and agitate
C. Preparation of Sample
(a) Dry powder. — Accurately weigh sample contg ca 100
mg ethion into 250 mL g-s flask. Pipet in 40 mL MeOH (or
CH 3 CN) and 10 mL internal std soln. Shake 30 min on mech.
shaker and centrf. to sep. phases.
(b) Liquid concentrates. — Prep, sample as in 979.04B(d).
(c) Oil formulations. — Accurately weigh sample contg ca
100 mg ethion into 50 mL vol. flask. Add 30 mL MeOH (or
CH 3 CN). Stopper and agitate vigorously 1 min, with side to
side action, keeping mixt. in main body of flask. Pipet in 4
mL H 2 and repeat vigorous mixing 1 min. Dil. to approx.
vol. with MeOH (or CH 3 CN). Cool to ambient temp and dil.
to vol. Mix thoroly by inverting 10 times and swirling vig-
orously each time. Centrf. to sep. phases.
D. Determination
Use high-pressure liq. syringe or sample injection loop to
inject 10 (jlL portions of std until 2 peak ht ratios agree within
±1%. Alternately inject two 10 |xL portions each of sample
and std solns. Measure peak hts and calc. av. peak ht ratios
for both std and sample. Adjust attenuation or amt injected for
convenient size peaks (60-80% full scale). Measure peak hts
from baseline between ethion and internal std peaks.
% Ethion - (R/R 1 ) x (W /W) x P
where R and R' = av. peak ht ratios for sample and std, resp.;
W' — mg ethion in working std soln (ca 100 mg); W — mg
sample in final diln; and P — % purity of std.
Refs.: JAOAC62, 11(1979); 63, 302(1980).
CAS-563-12-2 (ethion)
985.07 Fenitrothion Technical
and Pesticide Formulations
Gas Chromatographic Method
First Action 1985
A. Principle
Samples of fenitrothion tech. and formulations are dissolved
in CHCl 3 with fluoranthene added as internal std. Fenitrothion
content is detd by gas chromatgy with flame ionization detec-
tion.
B. Apparatus and Reagents
(a) Gas chromatography — Suitable for on-column injection
and equipped with flame ionization detector.
(b) Gas chromatographic column. — 2 mm id X 1.83 m glass
column packed with 3.0% PPE-6R (polyphenylether, Alltech
Associates, Inc., 2051 Waukegan Rd, Deerfield, 1L 60015)
on 100-120 mesh Chromosorb W-HP. Operating conditions:
temps: injector, 200°; detector, 250°; column, 195°; N carrier
gas flow ca 30 mL/min.
Approximate retention times for fenitrothion and internal std
are 16 and 26 min, resp.
(c) Internal std soln. — Accurately weigh ca 1.5 g fluoran-
thene into 500 mL vol. flask, dil. to vol. with CHC1 3 , and
mix.
(d) Fenitrothion std soln. — Accurately weigh amt of std
fenitrothion (Sumitomo Chemical Co., Ltd, Plant Protection
Div. International, 15-5 Chome, Kitahama, Higashi-Ku, Osaka,
Japan) contg ca 200 mg active ingredient into 50 mL screw -
cap bottle. Add by pipet 25.0 mL internal std soln and mix to
dissolve fenitrothion.
C. Preparation of Chromatographic Column
Clean glass column by passing H 2 S0 4 thru column, and rinse
with H 2 0. Draw ca 50 mL acetone thru column followed by
50 mL MeOH. Pass N thru column until it is dry. Treat col-
umn with 5% soln of dichlorodimethylsilane in toluene; rinse
with toluene followed by MeOH. Pass N thru column to dry.
Attach 7.6 cm funnel to exit end of column. While tapping
column with glass rod fitted with short length of heavy rubber
tubing, add prepd packing in small quantity until exit end of
column is filled to ca 0.5 cm from end of tube. Move funnel
to entrance of column. Insert pledget of silane-treated glass
wool in exit end of column, and attach a source of moderate
vac. to exit end. Continue to add packing slowly with vigorous
tapping until tube is filled to ca 0.5 cm from entrance end.
Insert pledget of glass wool in entrance end; compress glass
wool only enough to hold packing in place.
Condition column overnight at 230°. This step should be
conducted with exit end of column disconnected from detector
but with carrier gas flowing at recommended rate.
D. Preparation of Standard and Sample Solutions
Accurately weigh samples of fenitrothion tech. , emulsifiable
cone, and H 2 0-dispersible powder, each contg ca 200 mg ac-
tive ingredient, into sep. 50 mL screw-cap bottles. To each
bottle add by pipet 25.0 mL internal std soln and shake 30 s.
Filter or centrf. H 2 Odispersible powder to remove particu-
lates.
E. Determination
Inject 2 julL portions of std soln until response ratios (area
or peak ht) of fenitrothion to internal std agree ±2%. Make
duplicate injections of std soln, followed by duplicate injec-
tions of sample solns (see Note 1). Recalibrate after not more
than 4 injections of sample solns. (Note 1: To avoid interfer-
ence from late -emerging impurity (retention time, ca 45 min),
subsequent samples must be injected not earlier than 7 min
after elution of internal std. Thus, total analysis time for each
sample is ca 35 min.)
F. Calculation
For each injection, response ratio (R) = area (or ht) of fen-
itrothion peak/area (or ht) of internal std peak.
Fenitrothion, wt % = (R/R f ) x (W f /W) X P
AOAC Official Methods of Analysis (1990)
Organophosphates 203
where R f and R = av. response ratio for std and sample solns,
resp.; W' and W - wt (rag) of fenitrothion std and sample,
resp.; and P = purity (%) of fenitrothion std.
Ref.: J AOAC 68, 576(1985).
CAS-122-14-5 (fenitrothion).
989.02 Fenitrothion Technical
and Pesticide Formulations
Alternative Gas Chromatographic Method
First Action 1989
(Applicable to fenitrothion tech. and its emulsifiable cone, and
H 2 0-dispersible powder formulations)
Method Performance:
Technical:
s r = 0.50; s R = 0.51; RSD r = 0.53%; RSD R - 0.54%
Wettable powder:
s r = 0.19; s R = 0.38; RSD r = 0.50%; RSD R = 1.00%
Emulsifiable cone.:
s r = 0.25; s R = 0.81; RSD r - 0.48%; RSD R = 1.56%
A. Principle
Samples of fenitrothion tech. and formulations are dissolved
in CHCI 3 with dibutyl sebacate is added as internal std. Fen-
itrothion content is detd by gas chromatgy with flame ioni-
zation detection using peak area measurements. Method is al-
ternative to 985.07 which uses PPE-6R column packing.
B. Apparatus and Reagents
(a) Gas chromato graph. — Suitable for on-column injection
and equipped with flame ionization detector.
(b) Chromatographic column. — 2 mm id x 1.83 m glass
column packed with 7.5% OV-210 (Alltech Associates, Inc.,
2051 Waukegan Rd, Deerfield, IL 60015) on 100-120 mesh
Chromosorb W-HP. Operating conditions: temps — injector,
190°; detector, 250°; column, 165°. N carrier gas flow ca 40
mL/min.
Approx. retention times for fenitrothion and internal std are
16.9 and 19.5 min, resp.
(c) Internal std soln. — Accurately weigh ca 3.0 g dibutyl
sebacate into 500 mL vol. flask, dil. to vol. with CHC1 3 , and
mix.
(d) Fenitrothion std soln. — Accurately weigh amt std feni-
trothion (Sumitomo Chemical Co., Ltd, Osaka, Japan) contg
ca 200 mg active ingredient into 50 mL screw-cap bottle. Add
by pipet 25.0 mL internal std soln and mix to dissolve feni-
trothion.
C. Preparation of Sample Solutions
(Acute oral LD 50 of fenitrothion tech. for rats is 250-500
mg/kg.) Accurately weigh samples of fenitrothion tech.,
emulsifiable cone, and FLO-dispersible powder, each contg
ca 200 mg active ingredient, into sep. 50 mL screw-cap bot-
tles. To each bottle add by pipet 25.0 mL internal std soln and
shake 30 s. Filter orcentrf. H 2 0-dispersible powder to remove
particulates.
D. Determination
Inject 2 jjlL portions of std soln until response ratios (peak
area) of fenitrothion to internal std agree ±2%. Verify that
small peak just preceding that of fenitrothion [due to isomeric
impurity O,0-dimethyl-0-(4-methyl-3-nitrophenyl) phospho-
rothionate] is effectively sepd from fenitrothion peak. Make
duplicate injections of std soln followed by duplicate injections
of sample solns. At end of each run raise column temp, to
230° at 20°/min and hold 5 min to allow rapid elution of late-
eluting peaks before next detn. Recalibrate after no more than
4 injections of sample solns.
E. Calculation
For each injection, response ratio (R) = area of fenitrothion
peak /area of internal std peak.
Fenitrothion, wt % - (R/R') x (W'/W) x P
where /?' and R — av. response ratio for std and sample solns,
resp.; W' and W = wt (mg) of fenitrothion std and sample,
resp.; and P = purity (%) of fenitrothion std.
Ref.: JAOAC 71, 991(1988).
CAS-122-14-5 (fenitrothion).
986.07 Fensulfothion
in Pesticide Formulations
Gas Chromatographic Method
First Action 1986
(Method is suitable for tech. and liq. formulations of fensul-
fothion.)
A. Principle
Sample is dissolved in CH 2 C1 2 contg 4-chlorophenyl sulf-
oxide as internal std, and fensulfothion is detd by gas chro-
matgy.
B. Apparatus
(a) Gas chromato graph. — Equipped with flame ionization
detector (FID). Temps — column 225°, injection port 250°, de-
tector 250°; carrier gas 30-40 mL/min (either He or N); air
and H flows as recommended for FID; sample size 2.0 |ulL;
retention times (min) — internal std 4.0, fensulfothion 5.5. Ad-
just parameters to cause fensulfothion to elute in 5-6 min, but
do not use column temp. >240°. If internal std and fensul-
fothion peaks are not completely sepd, repack column.
(b) Column. — 0.9 m (3 ft) or 1 m X 2 mm (id) glass column
packed with 5% OV-330 on 80-100 mesh Chromosorb WHP
(Supelco). Condition newly packed columns 8—16 h at 240°
before use.
C. Reagents
(a) 4-Chlorophenyl sulfoxide. — Aldrich Chemical Co., Cat.
No. 12,104-5, or equiv. that contains no impurities eluting at
retention time of fensulfothion.
(b) Internal std soln. — Weigh 1.0 g 4-chlorophenyl sulf-
oxide, dissolve in 1 L CH 2 C1 2 , and mix well. Keep tightly
stoppered.
(c) Fensulfothion reference std soln. — Accurately weigh amt
of ref. std (Mobay Corp.) contg ca 100 mg fensulfothion into
ca 100 mL glass bottle. Add by pipet 50.0 mL internal std
soln. Stopper and mix well.
D. Preparation of Sample
Accurately weigh sample contg ca 100 mg fensulfothion into
glass bottle (ca 100 mL). Pipet in 50.0 mL internal std soln.
Stopper and mix well.
E. Determination
Make repetitive 2 |xL injections of fensulfothion ref. std soln
until response is stable and ratios of fensulfothion peak area
to internal std peak area for successive injections agree within
1% of their mean. Peak ht may be substituted for peak area.
Make duplicate 2 julL injections of each sample. Response
204
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
ratios (R) for fensulfothion internal std for 2 sample injections
must agree ±1% of their mean, if not, repeat detn, starting
with std injections.
After every 4-6 sample injections and after last sample in-
jection, make 2 injections of fensulfothion std soln. Av. std
soln ratios preceding and following sample must be ±1 .0% of
mean; otherwise, repeat series of injections.
F. Calculation
Calc. ratios for each injection. Average 2 sample ratios and
4 std ratios (std injections immediately before and after sample
injections).
Fensulfothion, % - (R/R f ) x (W /W) x P
where R and R' — av. sample and std ratios (fensulfothion
peak /internal std peak), resp.; W and W = mg sample and
std, resp.; and P ~ % purity of fensulfothion std.
Ref.: J AOAC 69, 488(1986).
CAS-1 15-90-2 (fensulfothion)
983.09 Fensulfothion
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1983
Final Action 1984
A. Principle
Sample is dissolved in or extd with MeOH, benzophenone
is added as internal std, and fensulfothion is detd by liq. chro-
matgy and UV detection at 230 nm,
B. Apparatus
(a) Liquid chromato graph. — Able to generate >1000 psi
and equipped with detector able to measure A at 230 nm. Typ-
ical operating conditions: temp., ambient; flow rate, 0.8 mL/
min; wavelength, 230 nm; chart speed, 2 mm/min; sample
size, 10 |xL. Conditions may be varied to accommodate in-
strument and column differences.
(b) Column.— Whatman Partisil PXS 10/25 ODS-2, stain-
less steel 25 cm x 4.6 mm id (Whatman, Inc.) or equiv.
(c) Filter. — 10 jxm Teflon, or similar type.
C. Reagents
(a) Methanol. — Distd in glass (Burdick & Jackson Labo-
ratories, Inc., or equiv.).
(b) Phosphoric acid. — 85% (Fisher Scientific Co.).
(c) Internal std soln.— 0.25 mg benzophenone /mL. Ac-
curately weigh ca 250 mg benzophenone (Eastman Kodak Co.)
into small flask. Transfer to 1 L vol. flask and dil. to vol. with
same MeOH to be used in mobile phase. Concn may be varied
so that when std soln (d) is injected, peak ht of benzophenone
matches peak ht of fensulfothion within 20%.
(d) Std soln. — 0.3 mg fensulfothion /mL, within optimum
linearity range. Accurately weigh ca 150 mg fensulfothion
(Mobay Corp.) into 125 mL flask. Pipet in 100 mL MeOH,
shake to mix. Pipet 10 mL aliquot of soln into 125 mL flask
with screw cap, add exactly 40 mL internal std soln, and shake
to mix. Prep, fresh std daily. Keep ref. std in freezer.
(e) Mobile phase.— MeOH-H 2 (80 + 20) buffered to
0.0025M with H 3 P0 4 . Mix 800 ml MeOH + 200 mL H 2 +
156 |xL H3PO4, and degas. If using column other than ODS-
2, adjust MeOH-H 2 ratio as necessary.
D. Preparation of Sample
(a) Spray concentrate . — Accurately weigh sample contg ca
150 mg fensulfothion into 125 mL flask. Pipet in 100 mL MeOH
and shake to mix. Pipet 10 mL aliquot into 125 mL flask with
screw cap, add exactly 40 mL internal std soln, and shake to
mix.
(b) Granular formulations. — Pour sample into 400 mL
beaker and thoroly mix, turning granules over >10 strokes with
wide spatula. Take weighed amt from beaker before sample is
poured back into sample container. Accurately weigh sample
contg ca 150 mg fensulfothion into 125 mL flask. Pipet in 100
mL MeOH and place on mech. shaker 15-30 min. Filter thru
10 jJim Teflon or similar type filter. Place 10 mL aliquot of
filtrate in 125 mL flask with screw cap, add exactly 40 mL
internal std soln, and shake to mix.
E. Determination
Adjust liq. chromatgc operating parameters to elute fensul-
fothion in 4-7 min. Maintain all parameters const thruout
analysis. Benzophenone will elute 2-4 min after fensulfo-
thion.
Adjust injection size and attenuation to give 60-80% on-
scale peaks. Make repetitive injections of std until response is
stable, and ratios of fensulfothion peak ht to benzophenone
peak ht for successive injections vary <1%. Then make du-
plicate injections of sample followed by injection of std. Calc.
av. ratio of fensulfothion peak ht to benzophenone peak ht for
each set of duplicate injections and calc. % fensulfothion.
Fensulfothion, % = (R/R r ) x (W /W) x P
where R and R f = av. peak ht ratios for sample and std, resp.;
W = mg sample; W = mg fensulfothion anal, std; and P =
% purity of fensulfothion anal. std. Integrator area ratios may
be substituted for peak ht ratios.
ReL: JAOAC 66, 801(1983).
CAS-1 15-90-2 (fensulfothion)
974.03 Formothion
in Pesticide Formuiations
Gas Chromatographic Method
First Action 1974
Final Action 1978
CIPAC-AOAC Method
A. Reagents
(a) Solvent I. — Toluene contg 2% Ac 2 0.
(b) Solvent II. — Hexane-acetone (2 + 1) plus 2% Ac 2 0.
(c) Internal std soln. — Prep, soln contg ca 100 mg, accu-
rately weighed, of ethion/mL solv. I. Ethion must be >95%
pure and contain no impurities interfering at formothion re-
tention time.
(d) Reference std soln. — Accurately weigh ca 500 mg For-
mothion Ref. Std (Sandoz Ltd, Agro Division, Development,
CH4002 Basel, Switzerland) into 50 mL vol. flask, add 5.0
mL internal std soln, and dil. to vol. with solv. I.
B. Apparatus
(a) Gas chromato graph. — (Varian Model 1520, or equiv.)
With flame photometric detector (Tracor Instruments, Inc., FPD
100AT, or equiv.), automatic injector (Hewlett-Packard 7600
A, or equiv.), integrator, and effluent splitter at column end
with ratio 1:100—1: 1000 in favor of outlet. Use glass spiral
column, 1.0 m x 3.6 mm id, packed with 3% OV 225 on 80-
100 mesh Chromosorb W-HP. Operating conditions: temps (°) —
oven 210, injector and detector 220; N carrier gas 60 mL/min;
no. theoretical plates for ethion is ca 2000. Alternatively, flame
ionization may be used. Conditions are same, except effluent
splitter is not necessary.
(b) Bottles. — 50 mL with Mininert valve, or equiv. inert
system for closure (Pierce Chemical Co.).
AOAC Official Methods of Analysis (1990)
Organophosphates
205
C. Determination
Accurately weigh well mixed sample contg ca 500 mg for-
mothion into bottle, (b). Add 5.0 mL internal std soln, (c),
and dil. to 50 mL with sol v. I. Close tightly and shake. Trans-
fer 6 |xL soln to vial contg 1 mL sol v. II. Seal vial with inert
valve system. (For automatic injections with Hewlett-Packard
sampler, dil. in A] foil -sealed vials and use Teflon rubber lam-
inated disks as septa.) Keep tightly closed. Inject 1.0 (xL dild
mixt. into column, bypassing sol v. around detector by using
splitter to avoid contamination and deterioration. Det. appro-
priate time for splitting by test chromatogram . Compds may
be identified by retention times relative to ethion as 1.00 (ca
4.4 min): formothion 0.50, dimethoate (by-product) 0.36.
Inject 1 jjiL aliquots of reference std soln, (d), dild as above,
until ht or area ratio of formothion to ethion varies <2% for
successive injections. Precede and follow each sample by ref-
erence std soln and make 3 sep. detns with all peak area ratios
of reference std solns within ±2% of first accepted values.
% Formothion = W x H x / x P/W x H f
where W and W - mg sample and internal std, resp.; H and
H' = peak his or areas of formothion and internal std, resp.;
P = % formothion in reference compd;
/ = correction factor = w x ti /w' x h
where w and w' ~ mg formothion ref. std and internal std,
resp. , and h and ti = peak hts or areas of formothion and
internal std, resp.
Ref.: JAOAC57, 771(1974).
CAS-2540-82-1 (formothion)
983.10 GByphosate (Technical)
and Pesticide Formulations
Liquid Chromatographic Method
First Action 1983
Final Action 1984
A. Principle
Samples are dissolved in phosphate buffer mobile phase and
injected directly into ion exchange chromatgc system using fixed
vol. loop. Peak area response as measured by UV detector is
quantitated by external std technic.
B. Apparatus and Reagents
(a) Liquid chromato graph. — Able to generate over 1000 psi
and measure A at 195 nm.
(b) Loop injector. — Rheodyne Model 7125 syringe load-
ing, or equiv.
(c) Strip chart recorder. — Houston Instrument 10 mV full
scale (Industrial Scientific, Inc., PO Box 60002, Houston, TX
77060), or equiv.
(d) Electronic integrator. — Capable of handling detector
output.
(e) Chromatographic column. — 25 cm x 4.6 mm id, 1 / 4 in.
od, strong anion exchange, e.g., Partisil 10 SAX (available
from Whatman, Inc., 9 Bridewell PI, Clifton, NJ 07014).
(f) Methanol. — LC grade (available from Burdick & Jack-
son Laboratories, Inc.).
(g) Water. — LC grade (available from Burdick & Jackson
Laboratories, Inc.). Use thruout.
(h) Potassium dihydrogen phosphate. — Primary std grade
(available from Fisher Scientific Co.).
(i) Phosphoric acid. — 85%, reagent grade (available from
Fisher Scientific Co.).
(j) Glyphosate std. — Monsanto Agricultural Products Co.,
PO Box 174, Luling, LA 70070.
(k) Mobile phase. —Dissolve 0.8437 g KH 2 P0 4 in 960 mL
H 2 0. Add 40 mL MeOH and mix well. Using pH meter buff-
ered at pH 2.0, adjust pH to 1.9 with 85% H 3 P0 4 . Filter and
degas before use.
C. Preparation of Standard
Accurately weigh ca 400 ± 10 mg glyphosate std (dried 2
h at 105°) into 100 mL vol. flask. Dil. to vol. with mobile
phase and stir to dissolve (30 min may be required to dissolve
std). Soln is stable >1 week.
D. Preparation of Sample
Accurately weigh sample contg ca 400 mg glyphosate into
100 mL vol. flask contg ca 50 mL mobile phase. Dil to vol.
with mobile phase and mix well.
E. Determination
Adjust operating parameters so that glyphosate elutes at 2.5-
4.0 min. Maintain all parameters consistent thruout std and
sample analysis. Typical values are as follows: flow rate 2.3
mL/min; pressure ca 1200 psi; chart speed 0.5 cm/min; A
range 0.2 AUFS; column temp, ambient; injector vol. 50 p,L.
Let mobile phase flow thru system until steady baseline is
obtained; I h may be required for new column. When new
columns are installed or instrument has not been used for 24
h, make at least 6 rapid injections of std soln; then inject std
soln until peak areas for successive injections agree ±1%. Then
inject sample soln until peak areas for successive injections
agree ±1%. Let all components from samples elute (ca 10-
12 min) before making next injection.
F. Caicuiation
Average peak areas from 2 successive injections that agreed
±1% from both std and sample solns.
% Glyphosate - (R/R') x (W /W) x P
where R = av. peak area of sample; R' — av. peak area of
std; W = mg sample; W — mg std; and P — % purity of std.
To convert % glyphosate to isopropylamine salt, multiply by
1.3496.
Ref.: JAOAC66, 1214(1983).
CAS- 107 1-83-6 (glyphosate)
987.01 Isofenphos Technical and
in Pesticide Formulations
Gas Chromatographic Method
First Action 1987
AOAC-CIPAC Method
(Method is suitable for tech. isofenphos and formulations with
isofenphos as only active ingredient.)
A. Principle
Isofenphos is extd with MeOH contg diisobutyl phthalate as
internal std, analyzed by gas chromatgy with flame ionization
or thermal conductivity detection, and quantitated by compar-
ing peak areas (or hts) of sample and internal std.
B. Apparatus and Reagents
(a) Gas chromato graph. — Equipped with thermal conduc-
tivity (TC) detector or flame ionization detector or flame ion-
ization detector (FID). Operating conditions: Temps — inlet
250°, column 190°, detector 250°; carrier gas 20-30 mL/min
(He for TC detector, either He or N for FID); bridge current
1 80 mA or as recommended for TC detector; air and H flows
as recommended for FID; chart speed 1.0 cm/min; range 1
(10 for FID); attenuation (x 2) (x 8 for FID). Retention times:
206
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
interna] std ca 1.7 min, isofenphos ca 3.5 min. Let chroma-
tograph stabilize (flat baseline) before beginning injections and
allow ca 5 min run time for each injection.
(b) Chromatographic column. — 0.5 m x 2 mm id stainless
steel or glass column packed with 10% SP-2100 on 80-100
mesh Supelcoport (Supelco Inc., No. 1-2140) or equiv. sup-
port.
(c) Diisobutyl phthalate. — Kodak Laboratory Chemicals
(Eastman Kodak) No. 6830 or equiv. that contains no impur-
ities eluting at retention time of isofenphos.
(d) Isofenphos reference std. — Mobay Corp.
(e) Internal std soln. — Pipet 10 mL diisobutyl phthalate into
1 L vol. flask, dil. with MeOH, and mix well. If necessary,
adjust concn so that peak hts of isofenphos and internal std
are within 20% (± 10%).
(f) Filters. — 0.45 |xm porosity (Gelman Acrodisc-CR, Gel-
man Scientific, Inc.) or equiv.
C. Preparation of Standard
Accurately weigh ca 250 mg ref. std isofenphos into glass
bottle (ca 50 mL for TC detector or 150 mL for FID). Pipet
in 15 mL internal std soln. For FID, add addnl 100 mL MeOH.
Cap securely and mix well.
D. Preparation of Sample
Accurately weigh sample contg ca 250 mg isofenphos into
glass bottle (ca 50 mL for TC detector or 150 mL for FID).
Pipet in 15 mL internal std soln. For FID, add addnl 100 mL
MeOH. Cap securely and mix well to ext.
E. Determination
Adjust operating parameters so that isofenphos elutes in 3.2-
3.7 min. Adjust injection vol. and attenuation to give largest
on-scale peaks. Make successive 2 u.L injections of std soln
until response is stable and response ratios (R) of isofenphos
peak area (ht) to internal std peak area (ht) agree within ±1%
of their mean.
Make duplicate 2 \iL injections of each sample. Ratios of
isofenphos to internal std peak area (ht) must agree within ±1%
of their mean. If not, repeat detn, starting with std injections.
After every 4-6 sample injections, and /or after last sample
injection, make 2 successive injections of ref. std soln to bracket
samples. Av. std ratios preceding and following bracketed
samples must lie within ±1% of mean; otherwise, repeat series
of injections.
F. Calculation
Isofenphos, wt % = (/?//?') x (W /W) x P
where R and R' = av. response ratios for sample and ref. std
solns, resp.; W and W = wt (mg) of isofenphos std and sam-
ple solns, resp.; and P ~ purity of isofenphos std (%).
Ref.: JAOAC70, 55(1987).
CAS-253 11-71-1 (isofenphos)
979.05 Malathion
in Pesticide Formulations
Gas Chromatographic Method
First Action 1979
Final Action 1980
A. Apparatus
(a) Gas chromatography — With glass column, on-column
injection system, flame ionization detector, and electrometer
with sensitivity of >10~ n amp driving 1 mv recorder. Drift
should be <l%/hr. Totally solid state amplifier with FET in-
put is recommended. Electronic digital integrator or computer
calcd area measurements must be used. Integrator should have
independent controls for selection of up and down slope sen-
sitivities so that start and stop integration points can be se-
lected. Automated sample injection system contributes signif-
icantly to precision. Hewlett-Packard Model 7600 is suitable
when equipped as described. Equiv. instrumentation may be
used but may require modification of operating conditions to
obtain good peak shape, adequate resolution, and appropriate
retention times.
Typical conditions for Hewlett-Packard Model 7600 (instru-
ment may have to be adjusted to give complete resolution of
well shaped peaks): Cycle timers (min): analysis and stop in-
tegrate, 16; range, 10 3 ; temps (°): oven 180, injection port 200,
flame detector 300; gas flow rotameters (mL/min): H 35, air
425, He carrier gas 30; integrator settings (adjusted so that
deflections on slope meter do not exceed ±50% before injec-
tion): noise suppression max., slope sensitivity up and down
0.1, BL reset delay 0.15, area threshold 1000; retention times
(min): malathion 10, internal std 6, min. time between mal-
athion and internal std 3.5.
(b) Column. — Borosilicate glass tube 1.22 m (6') x 4 mm
id, 6 mm od, bent to fit chromatograph and packed with 5%
SP-2401 or OV-210 on Supelcoport (100-120 mesh). Can be
purchased as prepd packing from Supelco, Inc. (specify "Pes-
ticide Grade"); Alltech Associates, 202 Campus Dr, Arlington
Heights, IL 60006; and Applied Science. Use exclusively for
malathion analysis.
(c) Glass wool. — Silane treated (No. 14502, Applied Sci-
ence).
(d) Syringes. — 10 u,L, Series 700, Hamilton Co.
(e) HI-EFF Fluidizer. — Applied Science.
B. Reagents
(a) Internal std soln. — 1 .2% m-Diphenoxybenzene in CHC1 3 .
Must not contain any impurities which elute at or near mala-
thion peak. Bring soln to consistent temp, above ambient (e.g.
25°) before taking aliquots.
(b) Malathion std solns. — Accurately weigh ca 170, 200,
and 230 mg malathion std (anal, grade, available from Amer-
ican Cyanamid Co.) into sep. preweighed 25 mL vol. flasks.
Add by pipet 5 mL internal std soln and dil. to vol. with CHC1 3 .
Label A, B, and C. Soln B is working std soln for detn; solns
A and C are used for linearity check and to guard against
weighing error in prepn of working std soln. Solns are stable
ca 4 weeks if kept tightly sealed in refrigerator. Warm to room
temp, before use. Soln B can be prepd independently of solns
A and C, if conditions of linearity check are met.
C. Preparation and Conditioning of Column
Weigh 6.25 g of trifluoropropylsilicone (SP-2401 or OV-
210) in 250 mL beaker and dissolve in 125 mL EtOAc. Stir
to obtain vortex and add 25 g solid support (Gas-Chrom Q or
Supelcoport, 100-120 mesh) with continued agitation. Filter
slurry thru Whatman No. 1 paper, or equiv., on buchner, using
gentle vac. to minimize evapn of sol v. Continue filtration until
drop rate is ca 1/sec. Transfer packing to HI-EFF Fluidizer,
connect source of N thru pressure reducer to base, and place
fluidizer on controlled temp, hot plate set for 75°. Continue
gas flow until solv. vapors can no longer be detected by odor,
taking care that packing is not blown out top of fluidizer.
To pack column, attach 75 mm funnel to exit end of pre bent
glass tube. Tap tube with pencil or small wooden rod, and add
prepd packing in small amts until exit end is filled to ca 15
mm from end. Move funnel to entrance end of column. Insert
pledget of si lane-treated glass wool in exit end and attach source
AOAC Official Methods of Analysis (1990)
Organophosphates 207
of moderate vac. to this end. Continue to add packing slowly
with tapping until tube is filled to ca 20 mm from entrance
end. Insert pledget of si lane-treated glass wool in entrance end,
compressing it only enough to hold it in place.
Condition column with He carrier gas flowing at 30 mL/
min >15 hr (overnight) at 255° or ca 20° below max. temp.
recommended for liq. phase. Exit end of column should not
be connected to detector during this conditioning.
Connect exit end of column to detector, adjust controls to
conditions given in 979.05A(a), and let instrument come to
equilibrium. Inject 3 julL aliquots std soln C until >3 consec-
utive injections give response ratios agreeing within 2%.
D. Linearity Check
Check gas chromatograph for linearity at least weekly,
whenever new std solns are prepd, and whenever column, new
or used, is newly installed in instrument.
Using digital integration for peak area measurements, det.
appropriate attenuation setting and injection aliquot (2-4 jjlL)
of std soln B to give area count of > 100,000 counts (optimum
electrometer output with acceptable noise level). Use condi-
tions so detd for all samples and stds in series.
Inject triplicate aliquots of detd vol. of std solns A, B, and
C into chromatograph, det. response ratio for each, and av-
erage ratios for each soln. Divide av. ratio for each soln by
corresponding malathion content in mg. Ratio/mg should agree
within 2%. Failure to meet this specification indicates either
weighing error in prepn of a std soln or instrumental difficul-
ties which must be corrected before proceeding with analysis
of samples.
E. Preparation of Sample
(Analyze samples at least in duplicate.)
(a) Liquid formulations and technical materials. — Accu-
rately weigh sample contg ca 200 mg malathion into pre-
weighed 25 mL vol. flask. Pipet in 5 mL internal std soln, dil.
to vol. with CHCI3, and mix well.
(b) Solid formulations containing 10% or more of mala-
thion. — Accurately weigh sample contg ca 1.0 g malathion
and transfer to 200-250 mL (8 oz) bottle. Pipet in 50 mL
CHCI3, stopper tightly, and shake on reciprocating shaker 30
min. Let settle ca 15 min; if not clear, centrf. Layer of solids
will float at interface. Avoid entrainment of particles by ex-
erting pos. pressure from bulb on pipet while it is carefully
inserted into soln for removal of aliquot. Particles in final soln
can clog syringe needle. Transfer 10 mL aliquot clear soln to
25 mL vol. flask, pipet in 5 mL internal std soln, dil. to vol.
with CHCI3, and mix well.
(c) Solid formulations containing less than 10% mala-
thion. — Accurately weigh sample contg ca 400 mg malathion
and transfer to 500 mL (16 oz) bottle. Add exactly 200 mL
CHCI3 and shake 30 min on reciprocal shaker. Let settle, ob-
serving precautions given in (b). Pipet 100 mL aliquot to 500
mL r-b flask and evap. to dryness. Pipet in 5 mL internal std
soln and 20 mL CHC1 3 , swirl to dissolve residue, and mix
well.
F. Determination
Inject duplicate aliquots of appropriate vol. of std soln B as
detd in linearity check, 979. 05D. Response ratios should agree
within 2%; if not, repeat with 2 more injections. Failure to
meet specification with second pair of injections indicates in-
strumental difficulties which must be resolved before proceed-
ing with analysis.
Inject duplicate aliquots of each sample soln of same vol.
as std soln. Average response ratios for each sample. Precision
considerations stated for std soln also apply to sample soln
injection response.
Inject duplicate aliquots std soln B after every 2 sample solns.
Average response ratios of stds immediately before and after
sample solns. Use this av. to calc. malathion content of the 2
sample solns.
Each detn of av. response ratio for std soln B should yield
value within 2% of previously detd value. Failure to meet this
specification indicates instrumental drift which must either be
corrected or compensated for by more frequent measurements
of response of std soln B. In extreme cases, follow each sam-
ple injection with std injection but this would indicate an in-
stability which should be corrected at once.
G. Calculations
For each sample injection, calc. response ratio:
R = area of malathion peak /area of internal std peak
% Malathion = (R/R') x (W'/W) x P x D
where R' and R = av. response ratio for std soln B and sam-
ple soln, resp.; W and W = g malathion std and sample,
resp.; P = % purity of malathion std; and D — diln factor (1
for liqs; (50/10)(25/25) - 5 for solids >10% malathion; and
(200/100X25/25) = 2 for solids <10% malathion).
Ref.: J AOAC 62, 292(1979).
CAS-121-75-5 (malathion)
978.06 Parathion
in Pesticide Formulations
Gas Chromatographic Method
First Action 1978
(Not applicable to dusts and powders)
A. Standard Solutions
(a) Dipentyl phthalate internal std soln. — Dissolve 2.0 ±
0.1 g dipentyl phthalate (Eastman Kodak Co., No. P2473, or
equiv.) in CS 2 and dil. to 500 mL with CS 2 .
(b) Parathion std soln. — Accurately weigh ca 125 mg par-
athion (Monsanto Chemical Co., or equiv.) into 50 mL g-s
erlenmeyer, pipet in 25 mL internal std soln, and mix thoroly.
B. Preparation of Sample
Accurately weigh sample contg ca 125 mg parathion into 50
mL g-s erlenmeyer. Pipet in 25 mL internal std soln and mix
thoroly.
C. Gas Chromatograph
See 977. 04C. Column should have >1200 theoretical plates
for parathion. Vary attenuation and injection vol. (1-2 \xL) so
that peak hts of parathion and dipentyl phthalate are 60-80%
full scale on 1 mv recorder. Retention times for parathion and
dipentyl phthalate are 6-8 and 8-10.5 min, resp.
D. Determination
Proceed as in 977. 04D, except substitute parathion for Me
parathion and dipentyl phthalate for p,p' -DDE.
E. Calculations
Proceed as in 977. 04E, except substitute parathion for Me
parathion and delete F from equation.
Refs.: JAOAC 61, 495(1978); 62, 337(1979).
CAS-56-38-2 (parathion)
208
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
978.07 Parathion
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1978
(Not applicable to dusts and powders)
A. Apparatus
(a) Liquid chromato graph. — See 977.05A(a), except use
eluant flow rate of 1.5 mL/min (ca 800 psi).
(b) Liquid chromatographic column. — See 977.03A(b).
B. Reagents
(a) Choroform.—See 977.05B(a).
(b) Eluant. — Stir 500 mL CHC1 3 on mag. stirrer 3-4 min
under moderate vac. (ca 350 mm fig).
(c) Internal std soln. — Accurately weigh ca 110 mg ben-
zophenone (EM Science, No. BX04I0, or equiv.) into 250 mL
vol. flask, and dissolve and dil. to vol. with CHC1 3 .
(d) Parathion std solns. — (/) Stock soln. — 1500 (xg/mL.
Accurately weigh ca 15 mg anal, grade parathion (Monsanto
Chemical Co., or equiv.) into 50 mL vol. flask, and dissolve
and dil. to vol. with CHC1 3 . (2) Working soln. — (150 (Jig para-
thion + 44 (xg benzophenone)/mL. Pipet 5 mL stock soln and
5 mL internal std soln into 50 mL vol. flask, and dil. to vol.
with CHC1 3 .
C. Preparation of Sample
Accurately weigh sample contg ca 15 mg parathion into 50
mL vol. flask, and dissolve and dil. to vol. with CHC1 3 . Pipet
5 mL sample soln and 5 mL internal std soln into 50 mL vol.
flask, and dil. to vol. with CHCI3.
D. Determination
Proceed as in 977. 05D, except substitute parathion for Me
parathion and benzophenone for acetophenone, and delete F
from equation. Retention times for parathion and benzophen-
one are 4.0-5.5 and 7-9 min, resp.
Refs.: JAOAC 61, 495(1978); 62, 337(1979).
CAS-56-38-2 (parathion)
978.08* Parathion
in Pesticide Formulations
Volumetric Method
First Action 1978
Surplus 1984
(Applicable to dusts and powders only)
See 6.472-6.478, 14th ed.
978.09* Parathion
in Pesticide Formulations
Colorimetric Method
First Action 1978
Surplus 1984
(Applicable to dusts and powders only)
See 6.479-6.483, 14th ed.
977.04 Methyl Parathion
in Pesticide Formulations
Gas Chromatographic Method
First Action 1977
A. Standard Solutions
(a) p,p ! -DDE internal std soln. — Dissolve 5.0 ± 0. 1 g 2, 2-
bis(p~ch]orophenyl)-l,l-dichloroethylene (/?,/> '-DDE, No.
12,389-7, Aldrich Chemical Co., Inc., or equiv.) in CS 2 and
dil. to 1 L with CS 2 .
(b) Methyl parathion std soln. — Accurately weigh ca 125
mg Me parathion (Monsanto Chemical Co., 800 N Lindbergh
Blvd, St. Louis, MO 63167, or equiv.) into 50 mL g-s erlen-
meyer, pipet in 25 mL internal std soln, and mix thoroly.
&. Preparation of Sample
(a) Liquid. — Accurately weigh into 50 mL g-s erlenmeyer
sample contg ca 125 mg Me parathion. Pipet in 25 mL internal
std soln and mix thoroly.
(b) W citable powder, — Accurately weigh into 100-150 mL
(4 oz) round bottle sample contg ca 625 mg Me parathion.
Pipet in 50 mL CHCI 3 -acetone (9 + 1), cap, and shake mech.
30 min. Let settle and pipet 10 mL supernate into 50 mL g-s
erlenmeyer. Place erlenmeyer in 55° H 2 bath and evap. sol v.
under stream of dry air or N. Pipet in 25 mL internal std soln
and mix thoroly.
C. Gas Chromatograph
Use instrument equipped with flame ionization detector and! .2
m x 4 (id) rnm glass column packed with 1.5% SE-30 plus
1.5% OV-210 on 80-100 mesh Gas-Chrom Q.
Prep, column by accurately weighing ca 0.12 g SE-30 and
ca 0.12 g OV-210 into 250 mL beaker. Add 50 mL CHC1 3 -
acetone (3 + 2), cover with watch glass, and heat on steam bath
until stationary phases are dissolved. Speed dissoln of SE-30
by spreading material on walls of beaker with small spatula or
stirring rod. Add enough 80-100 mesh Gas-Chrom Q to yield
1.5% of each phase on solid support. Heat on steam bath, stir-
ring frequently until all sol v. is removed. Air dry 2-3 hr. Pack
in column and condition 24 hr at 245° with N or He at 30 mL/
min. Column should have >:1200 theoretical plates for p,p' -
DDE.
Typical operating conditions: temps (°) — inlet 210, column
180 ± 10, detector 250; N or He carrier gas, 55-75 mL/min;
air and H as specified by manufacturer; attenuation and injec-
tion vol. (1-2 |xL) varied so that peak hts of Me parathion and
p f p' -DDE are 60-80% full scale on 1 mv recorder. Retention
times for Me parathion and p y p' -DDE are 3.5-5.5 and 6-8
min, resp.
D. Determination
Inject aliquots of std soln until peak ht ratio of Me para-
thion:/?,/? '-DDE varies <1% for successive injections. Then
make duplicate injections of sample followed by duplicate in-
jections of std. Peak ht ratios of stds must be within ±1% of
first accepted std values or repeat series of injections. Repeat
for addnl samples.
E. Calculations
Calc. peak ht ratios for both duplicate std injections pre-
ceding and following samples. Average the 4 values (R r ). Calc.
and average peak ht ratios of the 2 samples (/?).
% Me parathion - (R/R f ) X (W /W) X F X P
where W and W = mg sample and std, resp.; F = I for liq.
and 5 for wettable powder samples; and P — % purity of std.
AOAC Official Methods of Analysis (1990)
Organophosphates
209
Ref.: J AOAC 60, 720(1977).
CAS-298-00-0 (methyl parathion)
977.05 Methyl Parathion
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1977
A. Apparatus
(a) Liquid chromato graph. — Waters Model ALC 202/GPC
204 (Waters Associates, Inc.), or equiv., with 254 nm UV
detector and 10 mv recorder. Typical operating conditions: eluant
flow rate, 1.2 mL/m in (ca 700 psi); detector sensitivity, 0.16
A unit full scale; temp., ambient; valve injection vol., 10 (xL.
(b) Liquid chromatographic column. — See 977.03A(b).
(c) Chromatographic tubes. — Glass, 900 x 25 (id) mm, with
coarse porosity frit in bottom (Lurex Scientific, No. 131-1044,
or equiv.).
B. Reagents
(a) Chloroform. — Alcohol-free with <0.01% H 2 (Bur-
dick & Jackson Laboratories, Inc., distd in glass, or equiv.).
(b) Silicic acid-water. — 75% (w/v). Add 25 mL H 2 to 75
g silicic acid (Mallinckrodt Chemical Works, Code 2847, or
equiv.), and shake until lumps disappear.
(c) Water-saturated chloroform. — Shake 700 mL CHC1 3 with
150 mL H 2 2-3 min, and pass thru 900 X 25 mm glass tube
packed with 100 g silicic acid-H 2 0.
(d) Eluant.— Blend 200 mL H 2 0-satd CHC1 3 with 300 mL
CHC1 3 on mag. stirrer 2-3 min under moderate vac. (ca 350
mm Hg).
(e) Internal std soln. — Accurately weigh ca 115 mg ace-
tophenone (Aldrich Chemical Co. Inc., or equiv.) into 250 mL
vol. flask, and dissolve and dil. to vol. with CHC1 3 .
(f) Methyl parathion stdsolns. — (1) Stock soln. — 700 u,g/
mL. Accurately weigh ca 70 mg anal, grade Me parathion
(Monsanto Chemical Co., or equiv.) into 100 mL vol. flask,
and dissolve and dil. to vol. with CHC1 3 . (2) Working soln. —
(70 |xg Me parathion + 46 |xg acetophenone) /mL. Pipet 5 mL
stock soln and 5 mL internal std soln into 50 mL vol. flask,
and dil. to vol. with CHC1 3 .
C. Preparation of Sample
Accurately weigh ca 95 mg tech. Me parathion into 100 mL
vol. flask, or accurately weigh emulsifiable sample contg ca
35 mg Me parathion into 50 mL vol. flask, and dil. to vol.
with CHC1 :> . Pipet 5 mL sample soln and 5 mL internal std
soln into 50 mL vol. flask, and dil. to vol. with CHC1 3 .
D. Determination
Pump sufficient eluant thru column to equilibrate system.
Inject 10 |xL working std soln onto column thru sampling valve,
and adjust operating conditions to give peak hts 60-80% full
scale and retention times of 3.5-5.0 and 5.5-8.0 min for Me
parathion and acetophenone, resp. Repeat injections until ratio
of Me parathion to acetophenone peak hts is within ± 1 % of
previous injection. Without changing conditions, alternately
inject 10 (xL aliquots of working std soln and duplicate 10 |xL
aliquots of sample soln until peak ht ratios for sample soln
vary <1% for successive injections. Average last 2 peak ht
ratios for sample and for std, resp., and calc. % Me parathion.
% Me parathion - (R/R') x (W /W) x (P/F)
where R and R ! = av. peak ht ratios of Me parathion to ace-
tophenone for sample and std, resp.; W and W = mg sample
and std, resp.; P = % purity of std; and F — I for tech. and
2 for emulsifiable samples.
Ref.: JAOAC60, 724(1977).
CAS-298-00-0 (methyl parathion)
980.11 ftflethyl Parathion
or Ethyl Parathion in
Microencapsulated Pesticide Formulations
Gas Chromatographic Method
First Action 1980
Final Action 1982
(Caution: See safety notes on pesticides and acetonitrile.)
A. Principle
Me or Et parathion is released from microcapsules by grind-
ing, and compd is extd into CH 3 CN. Internal std is added and
concn of parathion compd is detd by gas chromatgy.
B. Apparatus
(a) Gas chromato graph. — With flame ionization detector,
such as Perkin-Elmer 900, or equiv., and strip chart recorder,
with full scale reading for I mv and 1 sec full scale response.
Glass column, 1.8 m (6') x 0.25" (od), 2 mm id, packed with
3% OV-17 on 80-100 mesh Supelcoport (Supelco, Inc.). Typ-
ical operating conditions: temps (°): column 200 (Me) or 190
(Et) (isothermal), injection port 225-250 (Me) or 225 (Et),
detector 250; flow rates (mL/min): He 30, H and air optimize
for max. sensitivity; chart speed 0.2" (5 mm)/min.
(b) Syringe. — 10 fxL. Hamilton 702N, or equiv.
(c) Grinder. — (/) Mixer mill. — With tool steel vial and ball
bearing pestles. Spex Mixer Mill No. 8000-1 15, or equiv. (Spex
Industries, 3380 Park Ave, Edison, NJ 08820); or (2) Tissue
grinder. — Corning Glass Works No. 7726-L, large, 40 mL.
(d) Weighing dishes. — Light A1, 60 x 15 (depth) mm; Fisher
Scientific Co., or equiv.
C. Reagents
(a) Ethyl and methyl parathion. — Anal, grade.
(b) Internal stds. — (7) For Me parathion.— R\$(2-m&h-
oxyethyl) phthalate, available from Pfaltz and Bauer, 375 Fair-
field Ave, Stamford, CT 06902, Cat. No. D34510. (2) For Et
parathion. — Dibutyl phthalate (Fisher Scientific Co.).
(c) Std solns. — Accurately weigh ca 0.3 g Et or Me para-
thion and ca 0.1 g (to 0.1 mg) of appropriate internal std, (b)
(7) or (2), into Al dish, and transfer mixt. to 50 mL vol. flask
by washing carefully with CH 3 CN. Dil. to vol.. with CH 3 CN.
Shake several min. to ensure homogeneity. Prep, in duplicate.
D. Determination of Correction Factor
Inject 1 |xL std soln until peaks are reproducible. Calc. cor-
rection factor
C = (777/7) X (W/W) x (P/P r )
where H and 77' = peak ht X attenuation of Me or Et parathion
and of internal std, resp.; W and W' ~ g Me or Et parathion
and of internal std in std soln, resp.; and P and P' — purity
of Me or Et parathion and internal std, resp.
Run the duplicate std solns daily and average the 2 results
to obtain correction factor for that day. Duplicates ordinarily
differ by <0.01.
E. Preparation of Sample
Prep, duplicate sample solns as follows: Thoroly shake sam-
ple container to ensure no sediment remains on bottom and
210
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
remove ca 1 g with medicine dropper (use sep. dropper for
each sample) while stirring. Weigh sample to nearest 0.1 mg
in weighed Al dish and record wt.
Transfer weighed sample to grinder, add ca 30 mL CH 3 CN,
and grind ca 4 min. Accurately weigh ca 0.2 g appropriate
internal std in weighed Al dish and quant, transfer to 100 mL
vol. flask with CH 3 CN. Quant, transfer ground sample to same
vol. flask with CH 3 CN, dil. to vol. with CH 3 CN, and mix
thoroly. Let any sediment settle before withdrawing samples.
F. Determination
Inject 1-2 uX sample soln, using 10 jxL syringe. Identify
peaks on basis of retention times: sol v. elutes almost imme-
diately; Me parathion 2 min and bis(2-methoxyethyl) phthalate
3 min; Et parathion 4 min and dibutyl phthalate 3 min.
Wt % parathion - (/////') x (W x /W s ) x C x 100
where W Y and W s — g internal std and sample weighed, resp.;
and other symbols are defined in 980.1 ID.
Ref.: J AOAC 63, 999(1980).
CAS-298-00-0 (methyl parathion)
CAS-56-38-2 (parathion)
964.05 Phorate in Pesticide Formulations
Infrared Spectroscopic Method
First Action 1964
Final Action 1966
(Applicable to analysis of 5 and 10% granules. Presence of
other pesticides and extractable org. materials such as dis-
persing agents, emulsifiers, and solvs requires testing for in-
terference.)
A. Apparatus
(a) Infrared spectrophotometer. — Capable of measurement
in 7.9-8.6 jxm range; with 0.5 mm cell.
(b) Chromatographic tube. — 15 x 450 mm with stopcock
or Ultramax valve (Fischer & Porter Co., Lab Crest Scientific
Div., Cat. No. 274-019 or 274-100).
B. Reagents
(a) Phorate reference std. — Purified (obtainable from
American Cyanamid Co.).
(b) Phorate std soln. — Accurately weigh by difference from
Smith or Lunge pipet 1.0-1.1 g Phorate Ref. Std into 250 mL
beaker contg 45 mL CH 3 CN.
(c) Cyclohexane . — Practical grade.
(d) Acetonitrile. — Practical grade, bp 82-84°.
C. Preparation of Sample Solution
(Caution: See safety notes on distillation, toxic solvents, and
acetonitrile.)
Accurately weigh 20 ± 0.01 g sample of 5% granular ma-
terial (10 ± 0.01 g for 10%). Place small glass wool plug in
bottom of chromatgc tube, transfer sample to tube, and gently
tap sides with spatula or rod to settle contents. Place 250 mL
beaker under column. Add 50 mL CH 3 CN to column and let
percolate thru at rate of 40-50 drops/min until flow stops.
Place beakers contg std (from 964.05B(b)) and sample solns
in shallow H 2 bath at 30-35° and evap. under gentle stream
of air until odor of CH 3 CN is no longer detectable. (Sample
solns on evapn will change from clear to cloudy and then to
residue of 2 layers.) Treat residue with four 5 mL portions and
one 4 mL portion cyclohexane, quant, transferring cyclohex-
ane layers to 25 mL vol. flask. (Keep cyclohexane-immiscible
layer in beaker during each extn.) Dil. to vol. with cyclo-
hexane.
D. Determination
Using hypodermic syringe, fill 0.5 mm cell with prepd std
soln, and obtain IR spectrum from 7.9 to 8.6 |xm. (With single
beam instrument, adjust to give 75% T at 8.2 jjim with cell
contg std soln in position.) Using same instrument settings,
treat prepd sample solns similarly.
Draw baseline from inflection points 8.10 to 8.48 |mm. Draw
perpendicular from radiation line thru absorption peak, and
measure distance from to baseline (Y) and from to ab-
sorption peak (X) in same units. Calc. A = log (Y/X) for sam-
ple (A) and std (A').
% Phorate = (A /A') x (wt std/wt sample) x % purity of std
Ref.: JAOAC 47, 245(1964).
CAS-298-02-2 (phorate)
980.12 Sulprofos
in Pesticide Formulations
Gas Chromatographic Method
First Action 1980
Final Action 1981
(Caution: See safety notes on pesticides.)
A. Standard Solutions
(a) Tetracosane internal std soln. — Dissolve 2.5 g tetra-
cosane, CH 3 (CH 2 ) 22 CH 3 (Aldrich Chemical Co., No. T875-
2) in toluene and dil. to 1 L.
(b) Sulprofos std soln. — Accurately weigh ca 90 mg anal.
grade sulprofos (Mo bay Corp.) into 50 mL g-s erlenmeyer.
Pipet 25 mL tetracosane soln into flask, and swirl to dissolve.
B. Apparatus
(a) Gas chromato graph. — With flame ionization detector,
recorder, integrator, and provisions for on-column injection.
GC conditions: temps (°) — inlet 200, column oven 185 ± 5,
detector 250; N carrier gas flow (50-75 mL/min) to give re-
tention time of ca 8 min for sulprofos; injection vol. 1.5-2.5
uX; recorder attenuation to give ca 70% full scale deflection
for peaks on 1 mV recorder; integrator adjusted to give opti-
mum slope sensitivity, baseline signal, and area response for
peaks.
(b) Column. — 3 .2 m (4') X 4 mm (id) Pyrex column packed
with 1.5% SE-30/1 .5% OV-210 on Gas-Chrom Q. For prepn
of column packing, see 977. 04C.
C. Preparation of Sample
Accurately weigh tech. sulprofos or sulprofos em u Is if i able
cone, contg ca 90 mg pure material into 50 mL g-s erlen-
meyer, Pipet 25 mL tetracosane soln into flask, and swirl to
dissolve.
D. Determination
Inject aliquots of std soln until response ratios of sulprofos
to tetracosane vary <1% on successive injections. Then make
duplicate injections of sample followed by std injection. Calc.
av. ratio of sulprofos to tetracosane area for each set of du-
plicate injections, and calc. % sulprofos.
% Sulprofos = (/?//?') x (W'/W) x P
where R and R' = av. integrator area ratios for sample and
std, resp.; W = mg tech. material or emulsifiable cone, sam-
ple; W = mg sulprofos anal, std in std soln; and P — purity
(%) of sulprofos anal. std.
AOAC Official Methods of Analysis (1990)
Organophosphates
211
Ref.: JAOAC 63, 120(1980).
CAS-35400-43-2 (sulprofos)
982.07 Temephos
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1982
CIPAC-AOAC Method
(Method is suitable for tech. temephos and formulations with
temephos as only active ingredient.)
A. Principle
Sample is dissolved in ethyl acetate, /?-nitrophenyl /?-nitro-
benzoate is added as internal std, and, after diln with /r-hex-
ane, sample is injected into liq. chromatgc column. LC re-
sponse ratio of insecticide to internal std is compared with
response ratio of std to give content in sample.
B. Apparatus and Reagents
(a) Liquid chromatography — Able to generate >2000 psi
and measure A at 254 nm.
(b) Chromatographic column. — Stainless steel, 300 x 3.9
mm id packed with 10 (xm silica gel (jx-Porasil, Waters As-
sociates, Inc., is suitable).
(c) Ethyl acetate. — Burdick & Jackson Laboratories, Inc.
Dry over molecular sieve, 5 A, 8-12 mesh beads (W.R. Grace
& Co., Davison Chemical Div., 10 E. Baltimore St, PO Box
2117, Baltimore, MD 21203-2117). Filter thru 0.45 p,m Mil-
lipore filter (Millipore Corp., Bedford, MA 01730).
(d) n-Hexane. — Non-spectro, distd in glass (Burdick &
Jackson Laboratories, Inc.). Dry over molecular sieve, 5 A, 8—
12 mesh, and filter thru 0.45 u,m Millipore filter.
(e) p-Nitrophenyl p-nitrobenzoate internal std. — 1,5 g/250
mL ethyl acetate. React p-nitrophenyl p-nitrobenzoate with ex-
cess p-nitrophenyl Na salt (Eastman Kodak Co.) in CH 3 CN.
Alternatively, prep. 1.1% (w/v) dimethyl 4-nitrophthalate in
ethyl acetate.
(f) Reference std soln. — Accurately weigh ca 50, 60, and
70 mg temephos, anal, reagent (American Cyanamid Co.) into
sep. 50 mL vol. flasks. Add by pipet 5 mL internal std soln
and 25 mL dry ethyl acetate to each flask. Shake flasks to
ensure dissolution of std, and dil. to vol. with n-hexane. Des-
ignate solns as A, B, and C. Use soln B as working std soln
for liq. chromatgy; use solns A and C to check linearity of liq.
chromatograph (see Linearity Check) and to guard against
weighing error in prepn of std soln. Supply of soln B can be
replenished from time to time without prepg new supplies of
solns A and C, provided linearity requirement described under
Linearity Check can be met.
(g) LC mobile phase. — Add 100 mL dry ethyl acetate to 1
L vol. flask and dil. to vol. with dry n-hexane.
(h) LC operating conditions. — Column temp, ambient; flow
rate 1.0 mL/min (ca 450 psi); retention times: internal std ca
9.6 min, temephos ca 11.5 min. Pump 50 mL anhyd. MeOH
thru column followed by 100 mL dry ethyl acetate. Pump LC
mobile phase thru column until system is equilibrated (flat
baseline). Inject 5 julL aliquots of std soln B until const, re-
sponse is obtained. If necessary, adjust instrument or injection
vol. (usually 3-6 (jlL) to give 50-60% FSD for internal std
peak. Use same injection vol. and instrument settings for all
samples and stds.
C. Linearity Check
Inject triplicate aliquots of appropriate vol. (as detd above)
of std solns A, B, and C into liq. chromatograph, det. response
ratio for each injection, and av. resulting ratios for each soln.
Divide av. response ratio for each soln by corresponding con-
tent (in mg) and compare resulting response factors. These fac-
tors should agree within 2%.
Liq. chromatograph should be checked for linearity at least
once a week, and same check should be carried out whenever
new std solns are prepd and whenever column, new or used,
is installed in instrument.
D. Sample Preparation
(a) Technical and emulsifiable concentrates. — Accurately
weigh amt sample contg ca 60 mg temephos directly into tared
50 mL vol. flask. For temephos tech., warm and thoroly mix
before sampling. Add by pipet exactly 5 mL internal std soln
and 25 mL dry ethyl acetate. Shake flask to ensure dissolution
and dil. to vol. with n-hexane.
(b) Water-dispersible powders and sand granules. — Ac-
curately weigh amt sample contg ca 60 mg temephos directly
into 2 oz bottles fitted with plastic screw cap. Add by pipet 5
mL internal std soln and 25 mL dry ethyl acetate and shake 1
min. Add 20 mL /?-hexane, mix thoroly, and let particles set-
tle. Filter portion of soln and hold for LC analysis. (In some
cases, centrifugation may be sufficient to remove particles be-
fore LC analysis.)
E. Analysis of Sample Solutions
Inject duplicate aliquots of std soln B. Calc. response ratios
by dividing area (or ht) of temephos peak by that of internal
std peak. Response ratios should agree within 2%. Average
duplicate response ratios obtained with std solns.
Inject duplicate aliquots of each sample soln. Average du-
plicate response ratios for each sample soln. Note: After first
injection of any sample, let instrument run >30 min after
emergence of temephos peak to det. late-eluting peaks due to
impurities. Subsequent injections should be timed so that late-
eluting peaks from sample injections do not interfere with in-
ternal std or temephos peaks of subsequent samples.
Inject duplicate aliquots of std soln B. Average response
ratios of stds immediately before and after sample solns, which
should agree within 2%. Use this av. to calc. temephos content
of sample solns.
F. Calculations
For each injection, response ratio (R) = (area temephos peak/
area internal std peak).
Temephos, wt% = (R/R f ) x (W /W) x P
where/?' and R — av. response ratio for std soln B and sample
soln, resp.; W and W = wt (mg) of temephos std taken (for
std soln B) and sample, resp.; and P = purity of temephos std
(%).
Ref.: JAOAC 65, 580(1982).
CAS-3383-96-8 (temephos)
949.06 TEPP in Pesticide Formulations
Titrimetric Method
Final Action
(Caution: See safety notes on pesticides.)
A. Reagents
(a) Indicator. — 0.1% aq. soln Me red or chlorophenol red.
(b) Amberlite IR-4B(OH) (free base form) resin. — Anal,
grade. Amberlite IR-45, Dowex 3, or equiv., are satisfactory.
212
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
B. Preparation of Resin Column
Screen resin to remove particles <30 mesh. SJurry 30 g
screened resin with H 2 0, and rjour into 100 mL buret contg
small plug of glass wool at bottom. Wash resin column with
150 mL 3% NaOH soln at flow rate of ca 5 mL/min and then
rinse with H 2 until effluent is acid to phthln, adjusting stop-
cock of buret so flow rate is ca 25 mL/min. Wash with aq.
acetone (1 + 3) to displace H 2 0. Column is now ready for
use.
Notes: Because channeling may result if column runs dry,
keep liq. level ca 2.5 cm above resin bed at all times. Because
resin tends to pack in column as it adsorbs acidic material,
expand resin bed after each detn before adding new sample by
back- washing with acetone (1 + 3) as follows: Connect large
funnel to tip of buret with rubber hose, and add the dil . acetone
from funnel until liq. level reaches top of buret; let resin settle,
and then let soln flow from buret until surface is 2.5 cm above
resin bed. Column is now ready to receive next sample.
After 8-10 samples have passed thru column, regenerate
resin by repeating original treatment with 3% NaOH soln, H 2 0,
and acetone (1 + 3). Washing with dil. acetone must be con-
tinued until effluent is colorless.
C. Determination
(a) In purified or technical grades of tetraethy [pyrophos-
phate not mixed with solvent, emulsifying agent, etc. — From
5-10 mL weighing buret, weigh by difference, to nearest mg,
2.5 g sample (1.0 g if tetraethylpyrophosphate content is >50%)
into 50 mL acetone (J 4- 3) in 125 mL separator. Mix by
swirling, and Jet soln stand 15 min at 25 ± 2°. Let soln flow
thru resin column by gravity at ca 25 mL/min, and catch ef-
fluent in 250 mL vol. flask. Wash separator and column with
three 50 mL portions acetone (1 + 3), collecting washings in
same flask. Dil. combined effluent to vol. with H 2 0, mix, and
transfer 100 mL aliquot to 250 mL beaker. Add 50 mL 0.17V
NaOH to beaker, stir well, let stand 30 min at room temp.,
and back-titr. with 0.1W HC1, using pH meter (or indicator,
949.06A(a), if pH meter is not available). Calc. % tetra-
ethylpyrophosphate = net mL 0.17V NaOH X 3.67/wt sample
taken.
(b) In formulations of tetraethylpyrophosphate containing
organic solvent and emulsifying agent. — Proceed as in (a), ex-
cept filter acetone soln thru 25 mm cotton plug in cylindrical
funnel (25 mm diam., 15 mm long) before adding it to column
if oil seps from soln. Pass acetone washings successively thru
separator, cylindrical funnel, and resin column as in (a), (Cot-
ton plug absorbs oil.)
Ref.: Anal. Chem. 21, 808(1949).
CAS- 107-49-3 (tetraethylpyrophosphate)
CARBAMATE, SUBSTITUTED UREA, AND
TR1AZINE PESTICIDES
974.04 Aldicarb
in Pesticide Formulations
Infrared Spectrophotometric Method
First Action 1974
Final Action 1976
{Caution: See safety notes on pesticides.)
A. Apparatus and Reagents
(a) Infrared spectrophotometer. — Perkin-Elmer Model 337,
or equiv. Adjust conditions as required by specific instrument.
(b) Soxhlet extractor. — With 125 mL flask and 25 x 80
mm cellulose thimble.
(c) Aldicarb std soln, — 0.18 g/100 mL. Accurately weigh
(to 0.1 mg) 0.18 ± 0.01 g anal, grade aldicarb (available from
Rhone-Poulenc Ag Co., 2 T.W. Alexander Dr, PO Box 12104,
Research Triangle Park, NC 27709) into 100 mL g-s vol. flask,
add ca 80 mL CH 2 Cl7, mix to dissolve, and dil. to vol. with
CH 2 C1 2 .
B. Determination
Transfer accurately weighed sample contg 0.18 ± 0.01 g
aldicarb to extn thimble, cover with wad of surgical grade cot-
ton, and place thimble in extractor. Add 2-3 Alundum boiling
stones and ca 80 mL CH 2 C1 2 to flask, and ext at rate to provide
5 extns within 60 min. Let cool to room temp., transfer quant,
to 100 mL g-s vol. flask with CH 2 C1 2 , and dil. to vol.
Using matched 0.5 mm NaCl cells, scan sample and std solns
from 5.2 to 6.0 jxm (1900 to 1600 cm" 1 ) against CH 2 C1 2 . Calc.
A of sample and A' of std at 5.75 p,m (1740 cm -1 ), using
corresponding A at 5.4 p-m (1850 cm~ ] ) as/ . (A and A' should
both be ca 0.45.)
C. Determination of Binder Correction
Pipet 50 mL sample soln into .1.00 mL beaker and place in
room temp. H 2 bath in hood. Evap. to dryness, using gentle
stream of clean, dry air. Add 25 mL MeOH, stir well, and
filter thru 30 mL coarse fritted glass gooch. Rinse beaker and
gooch with 25 mL MeOH, applying vac. until all liq. is in
filter flask. Place gooch and contents in original beaker, place
20 mL CH 2 C1 2 in gooch, and swirl to dissolve binder, letting
solv. drip into beaker. Repeat with addnl 20 mL CH 2 C1 2 . Quant.
transfer solv. to 50 mL g-s vol. flask and dil. to vol. with
CH 2 C1 2 . Scan soln as in detn and subtract A of binder soln
(should be <0.005) from that of sample (=AA).
% Aldicarb by wt - (AA/g sample) X (g std /A') X P,
where P is % purity of ref. std.
Ref.: J AOAC 57, 642(1974).
CAS- 116-06-3 (aldicarb)
985.02 Aminocarb Technical
and Pesticide Formulations
Liquid Chromatographic Method
First Action 1985
Final Action 1987
CIPAC-AOAC Method
(Method is suitable for tech. aminocarb and formulations with
aminocarb as only active ingredient.)
A. Principle
Aminocarb is detd by liq. chromatgy, using n-butrophenone
as internal std.
B. Apparatus
(a) Liquid chromato graph. — Able to generate >17.5 MPa
(>2500 psi) and measure A at 246 nm.
(b) Chromatographic column. — 250 X 4.6 mm id packed
with <10 nm C18 bonded silica gel (Partisil-10 ODS-3, What-
man Chemical Separations, Inc.; MicroPak MCH-10, Varian
Instrument Group; Ultrapack-ODS, Beckman Instruments, Inc.,
2350 Camino Ramon, PO Box 5101, San Ramon, CA 94583-
0701; Zorbax Sil, Du Pont Co.; or equiv. is suitable). Oper-
ating conditions: column temp, ambient; mobile phase flow
rate 1.5 mL/min (ca 2000 psi); chart speed 0.5 cm/min; in-
jection vol. 10 julL; A range 0.320 AUFS; retention times: ami-
nocarb ca 2.65 min, internal std ca 3.80 min. Pump LC mobile
phase thru column until system is equilibrated (flat baseline).
Allow ca 6 min after each injection.
AOAC Official Methods of Analysis (1990)
Carbamates
213
(c) Filters. — 0.45 |xm porosity (Gelman Acrodisc-CR, Gel-
man Scientific, Inc., or equiv.).
C. Reagents
(a) n-Butrophenone internal std soln. — 3 g/100 mL tetrahy-
drofuran.
(b) Tetrahydrofuran. — LC grade or distd in glass (Burdick
& Jackson Laboratories, Inc., or equiv.).
(c) Buffer jo/n.— Dissolve 1.36 g KH 2 P0 4 and 2.68 g
Na 2 HP0 4 .7H 2 in 1 L H 2 0.
(d) Water. — LC grade or distd in glass (Burdick & Jackson
Laboratories, Inc., or equiv.).
(e) Mobile phase. — Tetrahydrofuran-buffer soln (60 + 40).
(f) Aminocarb reference std soln. — Accurately weigh ca 250
mg ref. std (Mobay Corp.) into 100 mL vol. flask. Pi pet 5.0
mL internal std soln into flask, dil. to vol. with tetrahydro-
furan, and mix well. Pipet 5.0 mL of this soln into 100 mL
vol. flask, dil. to vol. with mobile phase, and mix well. Filter
portion of soln and hold for LC analysis.
D. Preparation of Sample
Accurately weigh amt sample contg ca 250 mg aminocarb
into 100 mL vol. flask. Pipet 5.0 mL internal std soln into
flask, dil. to vol. with tetrahydrofuran, and shake I min. Pipet
5.0 mL of this soln into 100 mL vol. flask, dil. to vol. with
mobile phase, and mix well. Filter portion of soln and hold
for LC analysis.
E. Determination
Adjust operating parameters to cause aminocarb to elute in
2.6-3. 1 min. Adjust injection size and attenuation to give largest
possible on-scale peaks. Using same injection vol. for samples
and stds, make repetitive injections of std soln and calc. re-
sponse ratios by dividing peak ht of aminocarb by that of in-
ternal std peak. (Note: Peak area measurements are unaccept-
able.) Response ratios must agree within ±1%. Average
duplicate response ratios obtained with std solns.
Inject duplicate aliquots of each sample soln. Average du-
plicate response ratios for each sample soln. Response ratios
must agree within ±1%. If not, repeat detn, starting with std
injections .
Re- inject std soln twice. Average response ratios of stds im-
mediately preceding and following sample injections. These
must agree within ±1%. If not, repeat detn.
F. Calculation
Aminocarb, wt% = (R/R 1 ) x (W /W) x P
where R and R' — av. response ratios for sample and std solns,
resp.; W and W = wt (mg) of aminocarb std and sample solns,
resp.; and P — purity of aminocarb std (%)
Ref.: JAOAC 68, 567(1985).
CAS-2032-59-9 (aminocarb)
988.04 Anilazine
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1988
AOAC-CIPAC Method
(Method is suitable for formulations with anilazine as only ac-
tive ingredient.)
A. Principle
Anilazine is detd by liq. chromatgy using octanophenone as
internal std. Adequate resolution is controlled by monitoring
sepn of bis-compound (major impurity of anilazine) from an-
ilazine and internal std peaks.
B. Apparatus
(a) Liquid chromato graph.— Able to generate >7 MPa
(>1000 psi) and measure A at 250 nm. Operating conditions:
column temp, ambient; mobile phase flow rate ca 1.7 mL/min
(ca 800 psi); chart speed 0.5 cm/min; injection vol. 20 uJL; A
range 0.32 AUFS. Retention times: anilazine ca 2.5 min, bis-
compound ca 4.0 min, octanophenone ca 6.6 min. Pump LC
mobile phase thru column until system is equilibrated (flat
baseline). Allow 1 min after elution of internal std before next
injection.
(b) Chromatographic column. — 250 x 4.6 mm id packed
with <10 |ULm C 18 bonded silica gel capable of resolving bis-
compound from anilazine and internal std peaks (DuPont ODS,
or equiv.)
(c) Chart recorder. — Min. 250 mm span, 10 mV range, 30
cm/h speed.
(d) Bath. — Ultrasonic.
(e) Filters. — 0.45 jmm porosity (Gelman Acrodisc-CR, Gel-
man Scientific, Inc., or equiv.).
C. Reagents
(a) Ac etonitr He .— LC grade or distd in glass (Burdick &
Jackson Laboratories, Inc., or equiv.).
(b) Octanophenone internal std soln. — Dil. 4 mL octano-
phenone (Aldrich Chemical Co., Inc., or equiv.) to 250 mL
with CH 3 CN.
(c) Water. — LC grade or distd in glass (Burdick & Jackson
Laboratories, Inc., or equiv.).
(d) LC mobile phase.— CH 3 CN-H 2 (80 + 20).
(e) Anilazine std soln. — Accurately weigh ca 230 mg ani-
lazine ref. std (Mobay Corp.) into 100 mL vol. flask. Pipet
10 mL internal std soln into flask, dil. to vol. with CH 3 CN,
and mix well. Pipet 5 mL of this soln into 100 mL vol. flask,
dil. to vol. with CH 3 CN, and mix well. Filter portion of final
soln for LC analysis.
D. Preparation of Sample
(a) Formulations excluding flowable. — Accurately weigh
sample contg ca 230 mg anilazine into 100 mL vol. flask. Pi-
pet 10 mL internal std soln into flask, dil. to vol. with CH 3 CN,
and sonicate 1 min. Mix well. Pipet 5 mL of this soln into
100 mL vol. flask, dil. to vol. with CH 3 CN, and mix well.
Filter portion of final soln for LC analysis.
(b) Flowable. — Accurately weigh sample contg ca 230 mg
anilazine into 100 mL vol. flask. Add 5 mL LC grade or distd
in glass H 2 and swirl until sample is thoroughly dispersed.
Pipet 10 mL internal std soln into flask, dil. to vol. with CH 3 CN,
and sonicate 1 min. Mix well. Pipet 5 mL of this soln into
100 mL vol. flask, dil. to vol. with CH 3 CN, and mix well.
Filter portion of final soln for LC analysis.
E. Determination
Inject anilazine std soln and adjust operating parameters so
that anilazine elutes in 2.5-3.0 min. Adjust injection vol. and
attenuation to give largest possible on-scale peaks. Bis-com-
pound must be resolved from anilazine and octanophenone
peaks. If not, change or repack column.
Using same injection vol. for all sample and std injections,
make repetitive injections of ref. std soln and calc. response
ratios by dividing anilazine peak ht by internal std peak ht.
Response ratios must agree within ±1%. Average duplicate
response ratios obtained with std solns.
Inject duplicate aliquots of each sample soln. Average du-
plicate response ratios for each sample soln. Response ratios
214
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
must agree within ±1%. If not, repeat detn, starting with std
injections.
Re- inject ref. std soln twice. Average response ratios of stds
immediately preceding and following sample injection. These
must agree within ±1%. If not, repeat detn.
F. Calculation
Anilazine, wt% = {R/R') x {W'/W) x P
where/? and R' = av. response ratios for sample and std solns,
resp.; W and W = wt (mg) of anilazine in std and sample
solns, resp.; and P — % purity of anilazine std.
Ref.: JAOAC 71, 23(1988).
CAS-101-05-3 (anilazine)
986.09 Bendiocarb
in Technical and Wettabie
Powder Pesticide Formulations
Liquid Chromatographic Method
First Action 1986
(Applicable to tech. bendiocarb and its 20 and 80% wettabie
powder formulations)
A. Principle
Bendiocarb is extd from sample with fixed vol. of CH 3 CN
contg 0.1 % v/v propiophenone internal std. Soln is filtered
and chromatographed on reverse phase column with CH 3 CN-
H 2 (40 + 60) mobile phase. Compd is quantitated by com-
parison of response ratio for bendiocarb /propiophenone in
sample and std.
B. Apparatus and Reagents
(a) Liquid chromato graph. — Const vol. pump, UV detec-
tor, injector. Injection system may be manual or automatic.
Response may be measured by peak ht or peak area. Column:
250 X 4.6 mm id, 6.4 mm od, type 316 stainless steel, slurry-
packed with Partisil 10 ODS 2 (Whatman Ltd). Mobile phase:
Mix 800 mL CH 3 CN with H 2 and dil. to 2 L with H 2 0.
Degas by applying reduced pressure until sol v. just boils.
Maintain this pressure 10 min.
Set UV detector to 254 nm. At 2 mL/min, pump 50 mL
CH 3 CN thru column, followed by 50 mL CH 3 CN-H 2 (75 +
25). Change to mobile phase, pump 50 mL to waste, then con-
nect system to recycle mobile phase.
(b) Internal std soln. — 0. 1% v/v propiophenone in CH 3 CN.
(c) Bendiocarb reference std soln. — Cambridge Animal and
Public Health Ltd, Hauxton, Cambridge CB2 5HU, UK. Weigh
0.49-0.5.1 g bendiocarb into 100 mL g-s conical flask. Add,
by pipet, 25.0 mL internal std soln. Inject 5 |jlL into system.
Adjust flow rate to give bendiocarb peak at 3-5 min and in-
ternal std peak at 1.5 times elution time of bendiocarb.
C. Preparation of Sample
Accurately weigh amt of sample contg ca 0.50 g bendiocarb
into 100 mL g-s conical flask. Use 0.49-0.51 g for tech. ma-
terial, 0.61-0.64 g for 80% formulation, and 2.50-2.55 g for
20% formulation.
Using same pipet as for std, add 25.0 mL internal std soln.
Swirl to dissolve. Filter soln, which contains suspended solids,
thru suitable filter and use filtrate for liq. chromatgy.
D. Determination
It is necessary to establish that LC system has achieved sta-
bility and that it remains stable. After any period of idling,
whether pump is running or not, make at least 3 injections of
std soln. For each injection, measure bendiocarb and internal
std peaks and calc. response ratio, bendiocarb/internal std. Re-
run stds until response ratio achieves acceptable repeatability,
then inject sample soln in duplicate. Follow duplicate sample
injections with std. Average sample responses. Use as std re-
sponse ratio the mean of those which occur on either side of
sample injections.
{R/R') x {W'/W) x p
E. Calculation
Bendiocarb,
where R and R' = av. response ratios for sample and std, resp.;
W and W' = wt (g) of sample and std, resp.; and P = % purity
of std.
Ref.: JAOAC 69, 908(1986).
CAS-2278 1-23-3 (bendiocarb)
984.09 Benomyl in Pesticide Formulations
Liquid Chromatographic Method
First Action 1984
(Method dets benomyl equiv. of benomyl and methyl -2-benz-
imidazole carbamate present.)
A. Principle
Benomyl is extd from inerts with CH 3 CN contg 3% (v/v)
/i-butylisocyanate (BIC). Equilibrium of benomyl spontaneous
decomposition is driven toward benomyl and no significant
degradation occurs. Ext is filtered, chromatographed on re-
verse phase (C 18 ) column, using CH 3 CN-2% HOAc mobile
phase, and quantitated by comparing peak hts of sample exts
and std from UV detector set at 280 or 290 nm. Caution: BIC
is a severe lachrymator!
B. Reagents
(a) Extractant. — 3% (v/v) n-butyl isocyanate (Aldrich
Chemical Co.) in CH,CN (LC grade).
(b) Mobile phase.— CU&N (LC grade)-2% HOAc (80 +
20), or as adjusted to give k' >2 for analyte when delivered
at 1.0 mL/min, and retention time for benomyl of 4-6 min.
(c) Reference std. — Benomyl (E.I. DuPont de Nemours &
Co., Inc.).
C. Apparatus
(a) Liquid chromato graph. — Able to generate >1000 psi
and measure UV absorbance at 290 or 280 nm. Also must be
capable of reproducibly injecting 10 )ulL.
(b) Chromatographic column. — 10 |±m reverse phase C !8
column which produces >:1000 theoretical plates for benomyl
(defined as 5.5 (t/w) 2 , where t — retention time and w = width
at half ht).
(c) Filtration. — 13 mm glass fiber disc (Gel man Sciences)
inserted into 5 mL disposable syringe.
D. Procedure
Accurately weigh stds and samples to contain ca 25 mg ben-
omyl, add 50.0 mL extractant, and shake 30 min. Filter thru
glass fiber pad and inject 10 p,L aliquot. Bracket each 2 sample
injections with std injections and av. std response for calcn of
sample concn.
E. Calculation
% Benomyl
(R/R') x {W'/W) x
where R and R' = peak ht of sample and std, resp.; W and
W' = wt of sample and std; and P — % purity of std.
AOAC Official Methods of Analysis (1 990)
Carbamates
215
Ref.: JAOAC 67, 303(1984).
CAS- 17804-35-2 (benomyl)
976.04 Carbaryl in Pesticide Formulations
Infrared Spectrophotometric Method
First Action 1976
Final Action 1979
(Caution: See safety notes on pesticides, pipets, toxic sol-
vents, and chloroform.)
A. Apparatus
(a) Centrifuge. — Clinical model, 8 place, or equiv.
(b) Hypodermic syringe. — 1 mL, glass barrel with rubber-
tipped plastic plunger (1 mL B-D Glaspak Tuberculin dispos-
able syringe supplied by Becton, Dickinson, and Co., Stanley
St, Rutherford, Nj 07070, is suitable). Disposable syringe may
be used repeatedly. Wash with H 2 and acetone or MeOH,
air-dry, and lubricate rubber plunger tip with silicone stopcock
grease.
(c) Infrared spectrophotometer. — Perkin-Elmer Corp. , Model
337, or equiv. Operator must adapt conditions to instrument.
(d) Rotator. — Tube type, Scientific Equipment Products
(SEPCO), or equiv.
(e) Shaking machine. — Wrist-action shaker (Burrell Corp.,
or equiv.).
(f) Tubes. — Culture tubes, borosilicate glass, 16 x 150 mm
with screw caps and Teflon liners (Corning Glass Works No.
9826, or equiv.).
B. Reagents
(a) Methanol-chloroform soln. — 10% (v/v) MeOH in CHC1 3 .
(b) Carbaryl std solns. — (7) 8 mg/mL. — Transfer 0.12 ±
0.01 g carbaryl (anal, grade, available from Rhone-Poulenc
Ag Co., 2 T.W. Alexander Dr, PO Box 12014, Research Tri-
angle Park, NC 27709), weighed to nearest 0.1 mg, to culture
tube. Pipet 15 mL MeOH-CHCl 3 soln into tube, cap securely,
and rotate or shake mech. 30 min. (2) 2.5 mg/mL. — Transfer
0.25 ±0.01 g carbaryl, weighed to nearest 0.1 mg, to 250 mL
g-s erlenmeyer. Pipet 100 mL CHC1 3 into flask, stopper, and
swirl to dissolve.
C. Preparation of Sample
(a) Carbaryl dust and powder formulations. — Transfer
weighed sample (<2.4 g) contg 0.12 ± 0.01 g carbaryl to
culture tube. Pipet 15 mL MeOH-CHC1 3 soln into tube and
cap securely. Rotate or shake mech. 30 min and centrf. 10
min.
(b) Liquid suspensions. — Following steps must be per-
formed in order described, as any deviation can cause erro-
neous results due to faulty sample transfer and incomplete extn:
Place ca 20 g Na 2 S0 4 in 250 mL g-s erlenmeyer. Pipet 100
mL CHC1 3 into flask. Vigorously shake sample bottle. Draw
appropriate vol. sample into hypodermic syringe without needle.
Use ca 0.5 mL sample for carbaryl 4 lb/gal. and ca 1.0 mL
for carbaryl 2 lb/gal. Wipe outside of syringe with paper towel
and weigh syringe and contents to nearest 0. 1 mg. Add sample
to erlenmeyer by slowly depressing syringe plunger. Do not
let syringe or sample touch sides of flask. Sample must drop
into CHCI3. Reweigh syringe and calc. sample wt by differ-
ence. Stopper flask and shake vigorously 30 min on mech.
shaker.
D. Determination
(a) Carbaryl dust and powder formulations . — Using matched
0.2 mm NaCl cells, scan sample soln against MeOH-CHCl 3
soln from 5.2 to 6.0 |xm (1900-1600 cm -1 ). Repeat scan with
std soln. Measured of carbaryl peak at 5.75 fxm (1740 cm -1 ),
using A at 5.40 \xm (1850 cm -1 ) as point. A - ca 0.4 for
both std and sample.
% Carbaryl by wt - (Ax B' X P)/(A' X B)
where A and A' = absorbance of sample and std, resp., at 5.75
|xm; B and B' - mg sample and mg std/mL, resp.; and P =
% purity of carbaryl std.
(b) Liquid suspensions. — Proceed as in (a), except use
matched 0.5 mm NaCl cells and scan sample soln against CHCI3.
Refs.: JAOAC 50, 566(1967); 56, 576(1973); 59, 753,
1196(1976).
CAS-63-25-2 (carbaryl)
986.10 Carbofuran
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1986
A. Principle
Carbofuran is extd from sample with MeOH contg aceto-
phenone as internal std. Soln is centrfgd and chromatographed
on reverse phase column with MeOH-H 2 (50 + 50) mobile
phase. Compd is quantitated by comparison of response ratio
for carbofuran/acetophenone in sample and std.
B. Apparatus and Reagents
(a) Liquid chromatography — High pressure pump, capable
of 5000 psi up to desired flow rate; sensitivity of 0.2 AUFS;
UV detector with 8—12 uX flow-thru cells, operating at 280
nm; guard column (optional), Brownlee RP-18, No. 140-200
and guard cartridge No. ODS-GU (Rainin Instrument Co., Mack
Rd, Woburn, MA 01801-4628), ambient temp.; column, 150-
300 x 3-5 mm id C18 (typically 250 x 4.1 mm), ca 40°;
injector, Waters Associates Model U6K, Rheodyne Model 7120
or 7125, or Model 728, Alcott Chromatographies, Inc. (One
Micromeritics Dr, Norcross, GA 30093) or equiv.; recorder,
10 in. 10 mV full scale at 0.5 cm /min.
(b) Mobile phase. — Use distd or distd, deionized H 2 and
distd in glass MeOH. Use prefiltered solvs or filter thru sub-
micron filters, 0.5 (xm (Millipore Corp. or Gelman Scientific
Inc.). Measure H 2 and MeOH (50 + 50) and combine; do
not add one to the other to det vol. Alternatively, use 2-pump
liq. chromatgc system with sep. metering for each sol v. Flow
rate 1 mL/min.
(c) Internal std soln. — 0.5 mg acetophenone (5:98%, no.
A 1070-1, Aldrich Chemical Co. Inc.; Pfaltz and Bauer, 375
Fairfield Ave, Stamford, CT 06902; or equiv. )/mL MeOH,
distd in glass.
(d) Carbofuran reference std soln. — 0.5 mg carbofuran (FMC
Corp., Agricultural Chemical Group, Group Quality Assur-
ance, 100 Niagara St, Middleport, NY 14105)/mL MeOH.
Also contg 0.5 mg/mL of 7-hydroxycarbofuran (FMC Corp.).
C. Preparation of Sample
Use balance capable of 0.01 mg resolution, or increase all
wts and vols by factor of 10. Accurately weigh amt of sample
contg ca 8-10 mg carbofuran into 100 mL g-s conical flask.
Add 20 mL internal std soln. Swirl to dissolve. Ext ca 30 min
on reciprocating shaker or 2 min on vortex mixer. Centrf. and
use supernate for liq. chromatgy.
D. Determination
Optimize column as follows: Set column temp, to 40°. Det.
retention time of carbofuran (preferably 12-15 min). Alter
216
Pesticide Formulations
AOAC Official Methods of Analysis (1 990)
mobile phase if necessary. Det. retention time and resolution
of acetophenone relative to carbofuran. Jf acetophenone is not
resolved at baseline from carbofuran, alter mobile phase by
reducing JMeOH. This may result in retention time >15 min
for carbofuran on some columns.
Det. retention time of 7 -hydroxy carbofuran. If it interferes
with carbofuran, further optimization is required: Increase or
decrease column temp., in 5° increments, and alter mobile phase
composition to maintain the resolution and retention times of
carbofuran and acetophenone const. Note retention time and
resolution of 7-hydroxyfuran peak. When it is well resolved
from carbofuran, impurity interferences are minimized and
sample analysis can begin.
Inject 10 \xL sample. Det. response ratio R for peak ht (or
area) of carbofuran /peak ht (or area) of internal std.
Carbofuran, wt % = (R/R') x (W /W) X P
where R' and R - av. response ratio for std and sample solns,
resp.; W and W — wt (mg) of carbofuran std and sample,
resp.; and P — % purity of carbofuran std.
On some high resolution columns, flow of 2 mL/min can
be used to reduce analysis time. If signs of decomposition are
noted, 3-5 drops of H 3 P0 4 /L may be added to mobile phase.
Ref.: JAOAC 69, 915(1986).
CAS- 1563-66-2 (carbofuran)
977.06 Chlorotoluron, Chloroxuron,
or Metoxuron in Pesticide Formulations
Thin Layer Chromatographic Method
First Action 1977
Final Action
CIPAC-AOAC Method
A. Principle
Pesticide is extd from formulations with CH 2 C1 2 , free amines
are removed with acid, and ext is hydrolyzed by alkali to Me 2 NH
which is distd and titrd. Related byproducts, 3-(3-chloro-4-
methylphenyl)- 1 -methylurea (I) , 3-(4-methylphenyl)- 1 , 1 -di-
methylurea (II) (from chlorotoluron), 3-[4-(4-chlorophen-
oxy)phenyl]-l -methylurea (III) and 3-(4-chlorophenyl)-l,l-di-
methylurea (IV) (from chloroxuron), and 3-(3,4-dichlorophenyl)-
1,1-dimethylurea (V), 3-(3-chloro-4-hydroxyphenyl)-l,l-di-
methylurea (VI), and 3-(4-methoxyphenyl)-l,l-dimethylurea
(VII) (from metoxuron), which may interfere, are detd by se-
miquant. TLC. Limit of detection for TLC is 0.1% for each
byproduct. On same TLC plate for chloroxuron, free amine 4-
(4-chlorophenoxy)aniline (VIII) is detd by sep. detection tech-
nic. Other byproducts, l,3-bis(3-chloro-4-methylphenyl) urea
(IX), l,3-bis[4-(chlorophenoxy)-phenyl]urea (X), and l,3-bis(3-
chloro-4-methoxyphenyl) urea (XI), do not interfere with chlo-
rotoluron, chloroxuron, and metoxuron detns, resp.
B. Preparation of Sample
(a) Technical formulation, — Accurately weigh ca 3 g sam-
ple (4 g for chloroxuron) and transfer, using 100 mL CH 2 C1 2 ,
into 250 mL separator, dissolve, and add 50 mL IN HC1.
(b) W enable powder. — Accurately weigh ca 3.5-4.0 g
sample (for 80%) or 6.0-6.5 g (for 50%) into 200 mL beaker.
Add 100 mL CH 2 C1 2 and stir mag. 5 min. Filter thru fritted
glass crucible contg paper and 0.5 g layer of Celite, and rinse
beaker and crucible with portions of CH 2 C1 2 to total vol. of ca
200 mL. Use only slight vac. to prevent crystn of pesticide on
walls of crucible. Transfer quant, to 500 mL separator, and
add 50 mL \N HC1.
C. Determination
Vigorously shake mixt. 1 min and drain lower org, layer
into second separator. Add 25 mL (50 mL for chloroxuron)
\N HC1, shake 30 sec, and drain lower layer into 500 mL
r-b flask. Wash the 2 acid layers successively with same 100
mL portion CH 2 C1 2 (with two 50 mL portions for chloroxuron)
and drain lower layer into the 500 mL r-b flask. Discard acid.
Vac.-evap. CH 2 C1 2 in rotary evaporator to dryness at max.
of 40°. Remove all sol v. to prevent interference in subsequent
titrn. Add 100 mL propylene glycol, 40 g KOH, and some
boiling stones to residue. Immediately connect flask securely
to distn app. (Fig. 977.06) whose joints are lubricated with
thin film of silicone grease. Place end of condenser delivery
tube (>10 mm id) in 400 mL beaker below level of absorbing
soln of 0.2 g H. 3 B0 3 and 1 mL mixed indicator soln (40 mg
methylene blue and 60 mg Me red dissolved in 100 mL al-
cohol) in 150 mL H 2 0. (To enhance end point, use 150 mL
MeOH (2 + 1).)
Gently warm flask until all particles dissolve; then boil 10
min or until propylene glycol distils into condenser. Titr. distd
Me 2 NH continuously with stdzd JW HC1, 936.15. Complete
distn by carefully adding H 2 dropwise from dropping funnel
at rate of 1 drop/sec. Continue titrn until end point persists 2
min (V mL). Perform blank detn (B mL) with each series.
% Pesticide = [(V - B) x N x F/g sample]
- % byproducts (from 977.060)
where F = 21.27 for chlorotoluron, 29.07 for chloroxuron, or
22.87 for metoxuron, and N = normality of stdzd HCI.
D. Determination of Byproducts
(a) For chlorotoluron. — Dissolve 100 mg each of byprod-
ucts I and II (977. 06A) (available from Ciba-Geigy Muench-
wilen Ltd, Analytical Development Agro, CH-4333 Muench-
wilen, Switzerland) together in tetrahydrofuran and dil. to 50
mL in voL flask. Dil. aliquots of 1, 2, 3, 4, and 5 mL to 20
a heating bath or heating mantle
round bottom flask (500 ml)
c distilling column, plain
d dropping funnel (250 m!)
e distilling head
f distilling bridge
g ailihn condenser
h beaker (400 ml)
i magnetic stirrer
ooo\
FIG. 977.06— Distillation apparatus (all dimensions in mm)
AOAC Official Methods of Analysis (1990)
Carbamates 217
mL with tetrahydrofuran, equiv. to 0.2, 0.4, 0.6, 0.8, and
1.0%, resp., of each byproduct.
Dissolve 1.0 g sample in tetrahydrofuran, and dil. to 20 mL
with same sol v.
Spot 5 (JiL each of sample and std solns on 20 x 20 cm
glass plates precoated with 0.25 mm layer of silica gel 60 F-
254 (No. 5715, E. Merck, Frankfurterstrasse 250, Postfach
4119, D6100 Darmstadt, West Germany, or equiv.), and de-
velop by ascending technic in tank, presatd 30 min with de-
veloping solv. CHCl 3 -EtOAc (4 + 1), without filter paper lin-
ings, for ca 70 min (13 cm migration). Expose plate to 254
nm UV light and compare spots of samples with those of stds
to est. concn of byproducts. Approx R { values: chlorotoluron,
0.50; byproduct I, 0.25; byproduct 11, 0.35; and byproduct VI
(does not interfere), 0.82.
(b) For chloroxuron. — Dissolve 100 mg each of byprod-
ucts III, IV, and VIII (available from Ciba-Geigy Muench-
wilen Ltd) together in acetone and dil. to 100 mL in vol. flask.
Dil. aliquots of 1, 3, 5, 8, and 10 mL to 50 mL with acetone,
equiv. to 0.1, 0.3, 0.5, 0.8, and 1% resp., of each byproduct.
Dissolve 1.0 g sample in acetone, and dil. to 50 mL with
same solv. Proceed as in (a), but use CHCl 3 -dioxane (9 + 2)
as developing solv. for ca 80 min (14 cm). Approx R f values:
chloroxuron 0.75; byproduct III, 0.40; byproduct IV, 0.65; and
byproduct X (does not interfere), 0.90.
Det. byproduct VIII on same TLC plate. Place beaker contg
ca 2 g NaN0 2 in empty developing tank and pour ca 3 mL
HO over salt. After 2 min, insert plate into tank 3 min, re-
move, and dry 5 min at room temp, with hair dryer. Spray
with 1% soln of A^-(l-naphthyl)ethylenediamine.2HCl in 0.1N
HC1 and compare violet sample spots with those of stds (R f ,
0.85).
(c) For metoxuron. — Proceed as in (a), except use 100 mg
each of byproducts V, VI, and VII (available from Sandoz
Ltd, AgroDi vision, Development, CH-4002 Basle, Switzer-
land). Approx. R f values: metoxuron, 0.25; byproduct III, 0.34;
byproduct IV, 0.08; byproduct V, 0.13; and byproduct VII
(does not interfere), 0.46.
E. identification
(a) Technical chloroxuron. — Record IR spectrum of 1%
CH 2 C1 2 soln of sample and compare with spectrum of 1 % CH 2 C1 2
soln of authentic ref. std.
(b) 50% W enable powder. — Stir ca 2 g sample and 2 g sil-
ica gel (70-230 mesh) with 100 mL CH 2 C1 2 5 min and per-
colate thru fluted filter. Record IR spectrum of filtrate in NaCl
cell (0.5 mm path length) from 3000 to 650 cm -1 , using blank
solv. as ref. Identity is established if sample spectrum corre-
sponds qual. to that of std.
Refs.: JAOAC 59, 716(1976); 61, 1499(1978); 62, 334(1979).
CAS-1 1 1 1 1-56-1 (chlorotoluron)
CAS- 1982-47-4 (chloroxuron)
CAS-59587-03-0 (metoxuron)
965.1 5 Dithiocarbamates
in Pesticide Formulations
Carbon Disulfide Evolution Method
First Action 1965
Final Action 1966
(Applicable only to cones or formulations contg ferbam,
maneb, nabam, zineb, or ziram and free from interfering
substances)
{Caution: See safety notes on pesticides.)
—IN. Dissolve 112gKOH
filter thru cotton, and add
A. Principie
Dithiocarbamates decompose on heating in acid medium.
Evolved CS 2 is passed thru Pb(OAc) 2 soln traps to remove H 2 S
and S0 2 formed from sample impurities. Washed CS 2 is re-
acted with methanolic KOH, and xanthate formed is titrd with
I soln.
B. Apparatus
Carbon disulfide evolution apparatus. — See Fig. 965.15.
Available from Lurex Scientific, No. JE- 1000-0000.
C. Reagent
Methanolic potassium hydroxide.-
pellets in 500 mL anhyd. MeOH,
addnl 500 mL anhyd. MeOH.
D. Determination
Add 20 mL 10% Pb(OAc) 2 soln to each Pb(OAc) 2 trap and
pipet 50 mL 2N MeOH-KOH soln into MeOH-KOH absorber
(Fig. 965.15). (Absorber must be dry at time of addn and kept
at 25 ± 1°.) Add 50 mL H 2 S0 4 (1 +4) to reaction flask and
heat acid to boiling. Adjust aspiration rate to <1 bubble/sec
thru MeOH-KOH soln, using stopper in reaction flask.
Weigh <5 g sample (contg 0. 1-0.3 g dithiocarbamates) into
small filter paper cone and fold cone to prevent sample loss.
Remove stopper from reaction flask, insert wrapped sample,
and immediately stopper flask. Adjust air flow if necessary
and maintain steady, moderate boil. Do not let acid soln enter
air inlet tube. Some dust formulations react vigorously and
require special care to prevent ejection of hot acid. As reaction
proceeds, adjust system so that rates of boiling and aspiration
are almost in equilibrium, producing only very slow rate of
bubbling thru MeOH-KOH soln. Continue boiling 1.5 hr. Dis-
connect MeOH-KOH absorber and rinse contents into 500 mL
erlenmeyer, using ca 250 mL H 2 0. (To remove absorber con-
37 M MOD. BULBS
WITH 4 INDENTATIONS
EACH
-BMM HEAVY WALL TUBING
— 1 % SPHERICAL JOINT
KOH- MEOH ABSORBER
REACTION FLASK
I2MECK-250ML]
FIG. 965.15 — Carbon disulfide evolution apparatus
218
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
tents, apply slight air pressure to top of absorber and force soln
thru side arm. Rinse with 4 ca 25 mL portions H 2 0, forcing
out rinse H 2 in same manner with air pressure.)
Add 3 drops phthln, and titr. with 30% HOAc until red just
disappears. Immediately titr. with 0.17V I; near end point, add
5 mL starch indicator soln, 922.03A(f), and titr. to faint but
definite color change.
Det. blank (usually 0.1-0.2 mL 0.LV I) by dilg 50 mL
MeOH-KOH soln with 250 mL H 2 0, neutzg with 30% HOAc,
and titrg as above.
Calc. % dithiocarbamate = (Sample titrn — blank)
x (1 normality) x (equiv. wt dithiocarbamate)/
(g sample X 10)
Equiv. wts (V2 MW) of zineb, maneb, ziram, nabam, and
(Va MW) ferbam are 137.87, 132.65, 152.91 ,128.18, and
138.82, resp.
Ref.: JAOAC 48, 562(1965); 52, 385(1969).
CAS-30 1-05-3 (ferbam)
CAS-301-03-1 (maneb)
CAS- 142-59-6 (nabam)
CAS-142-14-3 (zineb)
CAS- 137-30-4 (ziram)
977.07 Fluometuron
in Pesticide Formulations
Gas Chromatographic Method
First Action 1977
Final Action 1978
A. Standard Solutions
(a) Diethyl phthalate internal std soln. — Weigh 1.5 ± 0.1
g tech. diethyl phthalate, dissolve in ca 100 mL alcohol-free
CHC1 3 , dil. to 250.0 mL with CHC1 3 , and mix well. Std should
be >98% pure and contain no impurities eluting at retention
time of fluometuron.
(b) Fluometuron std soln. — Accurately weigh ca 125 mg
tech. fluometuron of known purity (available from Ciba-Geigy
Corp., PO Box 1 1422, Greensboro, NC 27409) into 2 oz round
bottle with Teflon-lined or Poly-Seal screw cap. Pipet in 25
mL diethyl phthalate internal std soln and shake to dissolve.
Pipet in 3 mL trifluoroacetic anhydride and shake mech. 15
min; then place bottle in 55° H 2 bath 30 min. Let cool to
room temp.
B. Preparation of Sample
Accurately weigh sample contg ca 125 mg fluometuron into
2 oz round bottle with Teflon-lined or Poly-Seal screw cap.
Pipet in 25 mL diethyl phthalate internal std soln and shake
well. Pipet in 3 mL trifluoroacetic anhydride and shake mech.
15 min; then place bottle in 55° H 2 bath 30 min. Let cool to
room temp. Let insol. materials settle or centrf. portion of ext
to obtain clear soln.
C. Gas Chromatography
Use instrument equipped with flame ionization detector and
1 .83 m X 2 (id) mm glass column packed with 2% OV-3 (Ap-
plied Science) on 80-100 mesh Gas-Chrom Q. Condition 24
hr at 240° with N or He at ca 40 mL/min. Column should
have si 500 theoretical plates. Use on-column injection to pre-
vent decomposition of derivative.
Typical operating conditions: temps (°) — inlet 150, column
1 15 ± 10, detector 250; N or He carrier gas, 20-22 mL/min;
air and H as specified by manufacturer; attenuation varied so
that peak fits of pesticide and internal std are 60-80% full scale.
Retention times for fluometuron derivative and diethyl phthal-
ate are 3-5 and 8-10 min, resp.
D. Determination
Proceed as in 971. 08C and D, except inject 1 \xL aliquots.
E. Calculations
See 971. 08E.
Refs.: JAOAC 60, 716(1977); 62, 334(1979).
CAS-2 164- 17-2 (fluometuron)
984.10 Methiocarb Technical
and Pesticide Formulations
Liquid Chromatographic Method
First Action 1984
Final Action 1987
CIPAC-AOAC Method
(Method is suitable for tech. methiocarb and formulations with
methiocarb as only active ingredient.)
A. Principle
Methiocarb is detd by liq.
phenone as internal std.
chromatography using aceto-
B. Apparatus
(a) Liquid chromato graph. — Able to generate >1500 psi
and measure A at 266 nm.
(b) Chromatographic column. — 250 X 4.6 mm id packed
with <10 \im C18 bonded silica gel. (Partisil-10 ODS-3,
Whatman Inc., or equiv., is suitable.) LC operating condi-
tions. — Column temp, ambient; mobile phase CH 3 CN-H 2 (60
+ 40); flow rate 2.5 mL/min (ca 1500 psi); chart speed 0.5
cm/min; injection vol. 10 |ulL; A range 0.160 AUFS; retention
times: internal std ca 2.50 min, methiocarb ca 3.70 min. Pump
LC mobile phase thru column until system is equilibrated (flat
baseline). Allow 6 min after each injection and then pump
CH 3 CN 6 min to remove impurities. Pump LC mobile phase
ca 8 min, allowing system to re-equilibrate before next injec-
tion.
(c) Filters. — 0.45 |xm porosity (Gel man Acrodisc-CR, or
equiv . ) .
C. Reagents
(a) Water. — LC grade or distd in glass (Burdick & Jackson
Laboratories, Inc.).
(b) Acetonitrile . — LC grade or distd in glass (Burdick &
Jackson Laboratories, Inc.).
(c) Acetophenone internal std soln. — 10 g/200 mL CH 3 CN.
(d) Tetrahydrofuran. — LC grade or distd in glass (Burdick
& Jackson Laboratories, Inc.).
(e) Methiocarb ref. std soln. — Accurately weigh ca 500 mg
ref. std (Mobay Corp.) into 100 mL vol. flask. Pipet 10 mL
tetrahydrofuran into flask, and swirl to dissolve. Pipet 10.0
mL internal std soln into flask, dil. to vol. with CH 3 CN, and
mix well. Pipet 10.0 mL this soln into 100 mL vol. flask, dil.
to vol. with CH 3 CN, and mix well.
D. Preparation of Sample
Accurately weigh amt sample contg ca 500 mg methiocarb
into 100 mL vol. flask. Pipet 10 mL tetrahydrofuran into flask
and swirl. Pipet 10.0 mL internal std soln into flask, dil. to
vol. with CH 3 CN, and shake 1 min. Pipet 10.0 mL this soln
into 100 mL vol. flask, dil. to vol. with CH 3 CN, and mix well.
Filter portion of soln and hold for LC analysis.
AOAC Official Methods of Analysis (1990)
Carbamates 219
E. Determination
Adjust LC operating parameters to cause methiocarb to elute
in 3.5-4.5 min. Adjust injection size and attenuation to give
>60% FSD peaks for std soln. Make repetitive injections of
std soln and calc. response ratios by dividing peak area (or ht)
of methiocarb by that of internal std. Response ratios must
agree within ±1%. Average duplicate response ratios obtained
with std soln.
Inject duplicate aliquots of each sample soln. Average du-
plicate response ratios for each sample soln. Response ratios
must agree within ±1%. If not, repeat detn, starting with std
injections.
Re-inject std soln twice. Average response ratios of stds im-
mediately preceding and following sample injection. These must
agree within ±1%. If not, repeat detn.
F. Calculation
Methiocarb, wt % = (R/R') x (W /W) x P
where R and R' = av. response ratios for sample and std solns,
resp.; W and W = wt (mg) of methiocarb std and sample,
resp.; and P = purity of methiocarb std (%).
Ref.: JAOAC 67, 492(1984).
CAS-3566-00-5 (methiocarb)
984.11 Metribuzin
in Pesticide Formulations
Gas Chromatographic Method
First Action 1984
Final Action 1986
(Method is suitable for tech. metribuzin and formulations.)
A. Principle
Sample is extd with CH 2 C1 2 contg di-n-butyl phthalate as
internal std and metribuzin is detd by gas chromatgy.
B. Apparatus and Reagents
(a) Gas chromatograph. — Equipped with flame ionization
detector (FID).
(b) Chromatographic column. — 1-2 m X 2 mm id glass
column packed with 3% OV-225 on 60-80 or 80-100 mesh
Gas-Chrom Q, or equiv.
(c) Di-n-butyl phthalate. — Eastman No. 1403 or equiv. that
contains no impurities eluting at retention time of metribuzin.
(d) Reference std metribuzin. — Mobay Corp.
C. Preparation of Standards
(a) Internal std soln. — Weigh 1.6 g di-n-butyl phthalate,
dil. tol L with CH 2 Ci 2 , and mix well.
(b) Metribuzin std soln. — Accurately weigh amt ref. std contg
ca 200 mg ref. std metribuzin into ca 250 mL glass bottle.
Add by pipet 100.0 mL internal std soln. Mix well.
D. Preparation of Sample
Shake liq. flowable formulations vigorously >1 min before
sampling. Accurately weigh sample contg ca 200 mg metri-
buzin into glass bottle (ca 250 mL). Pipet in 100.0 mL internal
std soln. Stopper and mech. shake or ultrasonify 1-5 min. Let
in sol. materials settle and use supernate for injection.
E. Determination
Adjust operating parameters to cause metribuzin to elute in
3-5 min. Maintain all parameters const thruout analysis. Typ-
ical values are as follows: temps (°) — inlet 250, column 210,
detector 250; carrier gas 20-40 mL/min (either He or N); air
and H flows as recommended for FID. Measure peak areas by
electronic integration, or alternatively, peak hts. Retention times
(min) — TV-methyl isomer (impurity in tech. metribuzin) ca 1.5-
2, di-«-butyl phthalate ca 2-3, metribuzin ca 3-5. If internal
std and TV-methyl isomer are not resolved onl m column, sub-
stitute longer column, but do not exceed 2 m.
Make repetitive 2 jjlL injections of metribuzin std soln until
response is stable and ratios of metribuzin peak area (or ht) to
internal std peak area (or ht) for successive injections agree
with ±1% of their mean.
Make duplicate 2 jxL injections of each sample. Metribuzin/
interna] std ratios for 2 sample injections must agree within
±1% of their mean. If not, repeat detn, starting with std in-
jections. After every 4-6 sample injections and after last sam-
ple injection, make 2 injections of calibration soln. Av. me-
tribuzin std soln ratios preceding and following samples must
lie ±1.0% of the mean; otherwise, repeat series of injections.
E. Calculations
Calc. ratios for each injection. Average 2 sample ratios and
4 std ratios (std injections immediately before and after sample
injections).
% Metribuzin = {R/R') x (W /W) x p
where R = av. sample ratio (metribuzin peak/internal std peak);
R' = av. std ratio (metribuzin peak/ internal std peak); W =
mg std; W — mg sample; P = % purity of metribuzin std.
Ref.: IAOAC 67, 840(1984).
CAS-2 1087-64-9 (metribuzin)
982.08 Pirlmicarb
in Pesticide Formulations
Gas Chromatographic Method
First Action 1982
(Caution: See safety notes on pipets, toxic solvents, pesti-
cides, and chloroform.)
A. Principle
Pirimicarb is detd by gas chromatgy, using nonadecane as
internal std and flame ionization detection. Peak areas are
compared with that of std of known purity.
B. Apparatus
(a) Gas chromatograph. — With heated, glass-lined, injec-
tion port and flame ionization detector. Conditions given are
for Hewlett-Packard Model 5710A. Other instruments may re-
quire changing operating parameters to obtain good resolution
and response. Temps (°) — column 210, injection port 240, de-
tector 250; gas flow rates (mL/min) — N carrier gas 40, H 60,
air 240; attenuation 32 x 10; sample size 1.0 fxL; retention
times (min) — pirimicarb 6.8, internal std 8.9. Adjust param-
eters to assure complete sepn of peaks, and peak hts ca 60-
80% full scale on chart at quoted retention times.
(b) Column. — 1.8 m (6 ft) x 0.25 in. (od) x 2 mm (id)
glass column packed with 10% silicone SE-30 on 100-120
mesh Chromosorb W(HP) (Applied Science). Silanize with 30
fxL Silyl 8 (Pierce Chemical Co. and heat to 300° for 16 h
before use.
C. Reagents
(a) Nonadecane internal std soln. — Accurately weigh ca 1
g nonadecane (Aldrich Chemical Co., Cat. No. N2890-6) and
dissolve in 100 mL CHC1 3 . Store in tightly capped bottle to
avoid evapn. Check internal std soln for interfering compo-
nents by injecting 1 |mL into chromatograph.
220
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
(b) Pirimicarb std soln. — Accurately weigh ca 150 mg pi-
rimicarb std of known purity (ICI Americas Inc.) into viaL
Pipet 10.0 mL internal std soln into vial, cap, and shake to
dissolve pirimicarb. Store tightly capped to avoid evapn.
D. Determination
(a) Powder and technical material samples. — Accurately
weigh amt sample contg ca 150 mg pirimicarb into vial. Pipet
10.0 mL internal std soln into vial, cap, and shake to dissolve
pirimicarb. Keep tightly capped to avoid evapn. Allow insol-
uble inerts to settle before use.
(b) Granular formulations. — Grind sample in mortar and
pestle or mech. mill. Accurately weigh amt sample contg ca
150 mg pirimicarb into vial. Add 5.0 mL MeOH and mix to
release pirimicarb. Add 10.0 mL internal std soln, cap, and
shake to dissolve pirimicarb. Store tightly stoppered to avoid
evapn. Allow insoluble inerts to settle before use.
Inject 2 or more aliquots of std soln to set integration pa-
rameters and stabilize instrument. Monitor response factor un-
til results agree within 2%. Inject 4 aliquots of std soln and 2
aliquots of sample soln in succession. Calc. response factor,
R, for each:
R = area pirimicarb peak /area internal std peak
Pirimicarb, % = (R/R r ) X (W'/W) X P
where R and R' - av. response factor for sample and std solns,
resp.; W and W — mg sample and std, resp.; and P ~ purity
(%) of std.
Ref.: JAOAC64, 1315(1981).
CAS-23.1 03-98-2 (pirimicarb)
984.12 Propoxur Technical
and Pesticide Formulations
Liquid Chromatographic Method
First Action 1984
Final Action 1987
CtPAC-AOAC Method
(Method is suitable for tech. propoxur and formulations with
propoxur as only active ingredient.)
A. Principle
Propoxur is detd by liq. chromatography using butyrophen-
one as internal std.
B. Apparatus
(a) Liquid chromato graph. — Able to generate >1500 psi
and measure A at 280 nm.
(b) Chromatographic column. — 250 x 4.6 mm id packed
with <10 (xm C18 bonded silica gel. (Partisil-10 ODS-3,
Whatman Chemical Separations, Inc., or equiv., is suitable.)
LC operating conditions. — Column temp, ambient; mobile
phase CH 3 CN-H 2 (60+40); flow rate 1.5 mL/min (ca 1000
psi); chart speed 0.5 cm/min; injection vol. 20 |ulL; A range
0.160 AUFS; retention times: propoxur ca 3.75 min, internal
std ca 6.50 min. Pump LC mobile phase thru column until
system is equilibrated (flat baseline). Allow 8 min between
injections.
(c) Rotary evaporator . — Equipped with 40-50° H 2 bath.
(d) Soxhlet extractor. — 30 mm id with 25 X 80 mm cel-
lulose extn thimbles.
(e) Filters. — 0.45 u,m porosity (Gelman Scientific, Inc.,
Acrodisc-CR, or equiv.).
C. Reagents
(a) Water. — LC grade or distd in glass (Burdick & Jackson
Laboratories, Inc.).
(b) Acetonitrile . — LC grade or distd in glass (Burdick &
Jackson Laboratories, Inc.).
(c) n-Butyrophenone internal std soln. — 6 g/200 mL CH 3 CN.
(d) Propoxur ref. std soln. — Accurately weigh ca 300 mg
ref. std (Mobay Corp.) into 50 mL vol. flask. Pipet 10.0 mL
internal std into flask, dil. to vol. with CH 3 CN, and mix well.
Pipet 10.0 mL this soln into 100 mL vol. flask, dil. to vol.
with CH 3 CN, and mix well.
D. Preparation of Sample
(a) Technical and formulations excluding bait. — Accu-
rately weigh amt sample contg ca 300 mg propoxur into 50
mL vol. flask. Pipet 10.0 mL internal std into flask, dil. to
vol. with CH 3 CN, and shake 1 min. Pipet 10.0 mL this soln
into 100 mL vol. flask, dil. to vol. with CH 3 CN, and mix well.
Filter portion of soln and hold for LC analysis.
(b) Bait. — Accurately weigh ca 13.5 g sample into extn
thimble. Cover bait with glass wool. Ext 1 h in Soxhlet ex-
tractor contg 75 mL CH 3 CN. Strip ext to oil on rotary evap-
orator (or steam bath). Using 30-35 mL CH 3 CN, transfer all
soluble material to 50 mL vol. flask. Pipet 10.0 mL internal
std into flask, dil. to vol. with CH 3 CN, and mix well. Pipet
10.0 mL this soln into 100 mL vol. flask, dil. to vol. with
CH 3 CN, and mix well. Filter portion of soln and hold for LC
analysis.
E. Determination
Adjust LC operating parameters to cause propoxur to elute
in 3.5-4.5 min. Adjust injection size and attenuation to give
>60% FSD peaks. Make repetitive injections of std soln and
calc. response ratios by dividing peak area (or ht) of propoxur
by that of internal std peak. Response ratios must agree within
±1%. Average duplicate response ratios obtained with std soln.
Inject duplicate aliquots of each sample soln. Average du-
plicate response ratios for each sample soln. Response ratios
must agree within ±1%. If not, repeat detn, starting with std
injections.
Re-inject std soln twice. Average response ratios of stds im-
mediately preceding and following sample injection. These must
agree within ±1%. If not, repeat detn.
F. Calculation
Propoxur, wt % - {R/R') X (W /W) x P
where R and/?' = av. response ratios for sample and std solns,
resp.; W and W — wt (mg) of propoxur std and sample, resp.;
and P = purity of propoxur std (%).
Ref.: JAOAC 67, 497(1984).
CAS- 1 14-26- 1 (propoxur)
981.04 Terbuthylazine in
Pesticide Formulations
Gas Chromatographic Method
First Action 1981
Final Action 1983
CIPAC-AOAC Method
(Caution: See safety notes on pipets and pesticides.)
A. Principle
Terbuthylazine is detd by gas chromatgy using di-«-pentyl
phthalate as internal std. Identity is confirmed simultaneously
by comparing retention times with std.
S. Standard Solutions
(a) Internal std soln. — Weigh 4.0 ± 0.2 g di-n-pentyl
phthalate, dil. to 1 L with CH 2 C1 2 , and mix well. Std should
AOAC Official Methods of Analysis (1990)
Carbamates
221
be >98% pure and contain no impurities eluting at retention
time of terbuthylazine.
(b) Terbuthylazine calibration soln. — Accurately weigh, in
duplicate, ca 250 mg terbuthylazine ref. std of known purity
(available from PAC-Switzerland, Swiss Federal Research
Station, CH-8820 Waedenswil, Switzerland) into 100 mL g-s
flasks. Pipet 50.0 mL internal std soln, (a), into each flask,
stopper, and dissolve terbuthylazine by swirling.
C. Preparation of Sample
Accurately weigh sample contg ca 250 mg terbuthylazine
into 100 mL g-s flask. Pipet in 50.0 mL internal std soln, (a),
stopper, and shake 3 min. Let insol. materials settle and use
supernate for injection.
D. Gas Chromatography
Use instrument equipped with flame ionization detector and
1.8 m X 2 mm id glass column packed with 3% Carbowax K
20M (Applied Science) on 80-100 mesh Gas-Chrom Q. Con-
dition 24 h at 230° with N or He at ca 30 mL/min. Column
must give baseline sepn between peaks of terbuthylazine and
internal std; otherwise, prep, new column.
Operating conditions: temps (°) — inlet 250, column 210, de-
tector 270; N or He carrier gas 35 mL/min; air and H as rec-.
ommended for detector. For monitoring sepn, record chro-
matograms using suitable attenuation. Measure peak areas by
electronic integration. Retention times (min) — terbuthylazine
ca 6, di-n-pentyl phthalate ca 8.
E. Calibration
Alternately inject 2 |xL aliquots of the 2 calibration solns
until calibration factors F — W c /R c of 2 successive chroma-
tograms vary <1%, where W c — mg terbuthylazine std for
calibration solns Q and C 2 , resp.; R c — peak area ratios of
terbuthylazine /internal std. For the next steps, use only cali-
bration soln C t .
F. Determination
Inject 2 uJL calibration soln. Then make duplicate injections
of sample followed by one injection of calibration soln. In-
dividual calibration factors must lie within ±1%, otherwise
repeat series of injections. Repeat for addnl samples.
G. Calculation
Average calibration factors (F) preceding and following
samples. Calc. and average peak area ratios of terbuthylazine/
internal std of the 2 sample injections.
% Terbuthylazine = R s x F av . x P/W,
where /? s = av. peak area ratio of the 2 sample injections; F av
— av. calibration factor; P = % purity of terbuthylazine std;
and W s = mg sample.
H. Identity Test
Identity of terbuthylazine is confirmed if differences of re-
tention times between terbuthylazine and internal std of sample
and calibration solns do not deviate >0.2 min.
Ref.: J AOAC 64, 825(1981).
CAS-5915-41-3 (terbuthylazine)
974.05 Thiocarbamates
in Herbicide Formulations
Gas Chromatographic Method
First Action 1974
Final Action 1988
(Applicable to liq. and granular formulations of EPTC, bu-
tylate, molinate, cycloate, vernolate, and pebulate.)
(Caution: See safety notes on on pesticides.)
A. Apparatus
(a) Gas chromato graph. — With flame ionization detector.
Operating conditions: temps (°) — injection port 225, column
130 (EPTC and butylate), 170 (molinate), 140 (cycloate, ver-
nolate), 150 (pebulate), detector 250; gas flows (mL/min) —
N carrier 30-35, H 25-30, air 200-300 (or as specified by
manufacturer).
(b) Recorder. — 1 mv full scale sensitivity and 1 sec re-
sponse.
(c) Columns. — 6' (1.8 m) x 0.25" od, Pyrex, Al, or stain-
less steel, packed with 3% OV-17 on 60-80 mesh Gas-Chrom
Q, or equiv. (for molinate), and 3% SE-30 or OV-1 on 60-
80 mesh Gas-Chrom Q, or equiv. (for other 5 compds). Con-
dition columns 12 hr at 250° under N flow of 30 mL/min.
B. Preparation of Standards
(a) Internal std .vote.™ -Accurately weigh ca 400 mg each
ref. grade thiocarbamate (EPTC, cycloate, butylate, or pebu-
late; Stauffer Chemical Co., 1200 S 47th St, Richmond, CA
94804; or ICI Americas, Inc.) and transfer to sep. 100 mL
vol. flasks. Dil. to vol. with CS 2 -CHCJ 3 -MeOH (80 + 15 +
5), and mix thoroly.
(b) Std solns. — Accurately weigh ca 100 mg each ref. grade
thiocarbamate into sep. 2 oz (50 mL) polyethylene-lined screw-
cap, conical bottles. Add 25 mL internal std soln indicated
below, and mix thoroly.
Approx.
Approx.
retention
retention
time,
Internal std
time,
Std soln
min
soln added
min
EPTC
2,0
Butylate
2.4
Molinate
4.3
Cycloate
4.8
Cycloate
5.4
Pebulate
2.6
Butylate
2.4
EPTC
2.0
Pebulate
4.0
Cycloate
8.0
Vernolate
3.5
Cycloate
5.5
C. Preparation of Sample
Accurately weigh sample contg ca 100 mg thiocarbamate
into 2 oz (50 mL) polyethylene-lined screw-cap, conical bot-
tle. Add 25 mL appropriate internal std soln, (a), as indicated
in (b), and shake thoroly. Vigorously shake granular formu-
lations 30 min on wrist- action shaker.
0. Determination
Inject 2 |xL clear supernate or soln into chromatograph
preadjusted to appropriate conditions. Make triplicate injec-
tions of sample and appropriate std soln in random order. Det.
peak areas, preferably with digital integrator.
Adjust sensitivity of gas chromatograph so that larger com-
ponent or internal std peak is ca 3 / 4 full scale.
E. Calculations
Wt % compd = (R/R') x (W /W) x P
where R and R' — av. ratios of compd peak area to internal
std peak area for sample and std solns, resp.; W = g sample;
W = g compd std in std soln; P = % purity of compd.
Ref.: JAOAC57, 53(1974).
CAS-759-94-4 (EPTC)
CAS- 11 37-23-2 (cycloate)
CAS-2008-41-5 (butylate)
CAS-22 12-67-1 (molinate)
CAS-1 114-71-2 (pebulate)
CAS- 1929-77-7 (vernolate)
222
Pesticide Formulations
AOAC Official Methods of Analysis (1 990)
966.08 Thiram in Pesticide Formulations
Distillation Method
First Action 1966
Final Action 1977
CIPAC-AOAC Method
(Caution: See safety notes on pesticides.)
A. Principle
Thiram is decomposed by boiling with HOAc and Zn(OAc) 2
to Me 2 NH, CS 2 , and carbonyl sulfide. The gaseous mixt. is
carried by air stream thru CdS0 4 scrubber to remove H 2 S, and
then into absorption system contg MeOH-KOH soln. Mixed
xanthate-monothiocarbamate soln is neutzd and titrd with std
aq. I.
Method is not specific for thiram. Sep. characterization test,
972.29G, must be made.
B. Apparatus
Assembly and operating conditions. — Assemble app. as
shown in Fig. 966.08 with 30 mL CdS0 4 soln in first absorber,
25 mL KOH soln in second absorber, and 5 mL in each bub-
bler. Turn on condenser H 2 and maintain H 2 bath surround-
ing CdS0 4 scrubber at 70-80° thruout test. Keep main KOH
absorber at <25° by immersion in beaker of cold H 2 0. Ab-
sorber must be dry or rinsed with MeOH before adding KOH
soln. Air bleed must reach nearly to bottom of digestion flask.
Make all joints gas-tight, using small amts H 3 P0 4 , petrolatum,
or silicone grease.
Check app. for absorber leaks and efficiency periodically,
using pure Na diethyldithiocarbamate. Recoveries should be
99-101%. Check purity of Na diethyldithiocarbamate by dis-
solving ca 0.5 g, accurately weighed, in 100 mL H 2 and titrg
directly with 0AN I, using ca 2% starch soln as indicator. 1
mL 0AN 1 = 0.02253 g Na diethyldithiocarbamate. % Na di-
ethyldithiocarbamate = 2.253 x mL 0AN I/g sample.
C. Reagents
(a) Acid mixture. — Dissolve 2.5 g ZnO in 100 mL HOAc
d + 1).
(b) Cadmium sulfate soln. — Dissolve 18.5 g 3CdS0 4 .8H 2
in 100 mL H 2 0.
(c) Potassium hydroxide soln. — 2N in MeOH and contg <1
ppm Cu or Fe.
(d) Iodine std soln.— 0.17V. Stdze as in 939.13B.
D. Determination
Accurately weigh and transfer sample contg ca 0.3 g thiram
to digestion flask, using small amt H 2 0, if necessary. Assem-
ble air bleed and dropping funnel, Fig. 966.08, and add 20
mL acid mixt. thru funnel. Connect app. to controlled aspi-
ration (vac. or compressed air) so that ca 3 bubbles /sec pass
thru absorbers. After sample is evenly dispersed, heat and re-
flux 30 min at moderate rate. Turn off cooling H 2 and flush
condenser and first absorber with steam from flask <1 min.
Remove burner and disconnect train. Wash contents of KOH
absorber and bubblers into 600 mL beaker with 300-400 mL
H 2 0, add 1-2 drops phthln, just neutze with HOAc (1+9)
from buret, and add 3 drops excess. With continual stirring,
titr. immediately (preferably within 1 min, as decomposition
of mixed xanthate/monothiocarbamate soln is extremely rapid
under acidic conditions) with 0. IN I (/ mL), using ca 2% starch
soln as indicator. Det. blank in same manner, omitting sample
(b mL). 1 mL 0.1/V I = 0.01202 g thiram.
% Thiram = 1.202 (t - b)/g sample
Refs.: J. Sci. Food Agric. 15, 509(1964). JAOAC 49, 40(1966);
51, 447(1968).
CAS- 137-26-8 (thiram)
971.08 Triazines in Pesticide Formulations
Gas Chromatographic Method
First Action 1971
Final Action 1976
AOAC-CIPAC Method
(See Table 971.08 for applicability to specific compds.)
A. Standard Solutions
(Caution: See safety notes on pipets and pesticides.)
(a) Dieldrin internal std soln. —Std should be >90% pure
and contain no impurities eluting at retention time for pesticide
being detd. (/) For propazine. — Weigh 14.0 ± 0.1 g tech.
dieldrin, dissolve in ca 300 mL CHC1 3 , and dil. to 1 L with
CHC1 3 . (2) For other compounds.— -Weigh 2.00 ± 0.02 g tech.
dieldrin, dissolve in ca 200 mL CHC1 3 , and dil. to 250 mL
with CHCI3.
(b) Aldrin internal std soln.— (For Diazinon®.) Weigh 4.0
±0.1 g tech. aldrin into 600 mL beaker. Slurry with 400 mL
acetone to dissolve, filter thru paper into 1 L vol. flask, wash-
ing with several 100 mL portions acetone, and dil. to vol. Std
should be >90% pure and contain no impurities eluting at re-
tention time of Diazinon.
(c) Dibenzyl succinate internal std soln. — (For chloroben-
zilate and chloropropylate.) Weigh 5.0 ± 0.1 g dibenzyl suc-
cinate, dissolve in ca 300 mL acetone, and dil. to 1 L with
-BIO Joint
20 ml
ShiakJ — H
FIG. 966.08— Absorption system for thiram. Dimensions in cm; M.S. - nonstandard; B10 = J 10/30
AOAC Official Methods of Analysis (1990)
Miscellaneous
223
acetone. Std should be >98% pure and contain no impurities
eluting at retention time for pesticide being detd.
(d) Pesticide std solns. — Accurately weigh 250 mg (125 mg
for Diazinon and 150 mg for simazine) of ref. std of pesticide
being detd (available from Ciba-Geigy Corp., PO Box 11422,
Greensboro, NC 27409) into 4 oz (125 mL) round bottle with
Al-lined screw cap. Pipet in 50 mL internal std soln {see Table
971.08) and shake mech. 30 min.
(e) Dioctyl phthalate internal std soln. — (For simazine.)
Weigh 3.0 ± 0.1 g tech. dioctyl phthalate, dissolve in ca 200
mL DMF, and dil. to 1 L with DMF. (Caution: See safety
notes on dimethylformamide.) Std should be >98% pure and
contain no impurities eluting at retention time of simazine.
B. Preparation of Sample
Accurately weigh amt sample specified in Table 971.08 into
4 oz (125 mL) round bottle with Al-lined screw cap. Pipet in
same vol. internal std used for prepn of std soln, (d), and shake
mech. 30 min. Let insol. materials settle or centrf. portion of
ext to obtain clear soln.
C. Gas Chromatography
Use instrument equipped with flame ionization detector and
4 mm id glass column (length specified in Table 971.08) packed
with 3% Carbowax 20M (Applied Science) on 80-100 mesh
Gas-Chrom Q. (For Diazinon, use 10% silicone DC-200 vis-
cosity 12500.) Condition 24 hr at 240° with N or He at ca 40
mL/min. Column should have >2000 (^1500 for chloroben-
zilate, chloropropylate, propazine, and simazine) theoretical
plates (see 973.126(a)).
Operate at following conditions: temps — as specified in Ta-
ble 971.08; N or He carrier gas, 80-100 mL/min; air and H,
80-100 mL/min; attenuation varied so that peak hts of pes-
ticide and internal std are 60-80% full scale. Retention times
are specified in Table 971.08. (Ametryn and dieldrin peaks
must be resolved. Prep, new column if variation of flow rate
or temp, does not resolve peaks. Resolution may be improved
by increasing column temp.)
D. Determination
Inject 3 juuL aliquots std soln until peak ht ratio of pesti-
cide:internal std varies <I% for successive injections. Then
make duplicate injections of sample followed by duplicate in-
jections of std. Peak ht ratios of stds must be within ±1% of
first accepted std values or repeat series of injections. Repeat
for addnl samples.
E. Calculations
Calc. peak ht ratios for both duplicate std injections pre-
ceding and following samples. Average the 4 values (/?')■ Calc.
and average peak ht ratios of the 2 samples (R)>
% Pesticide - (R/R r ) x (W /W) x P
where W and W = mg sample and std, resp.; and P — %
purity of std.
Refs.: JAOAC 54, 450, 452(1971); 56, 586(1973); 58, 513,
516(1975); 59, 758(1976).
MISCELLANEOUS PESTICIDES
967.06 Amitrole in Pesticide Formulations
Titrimetric Method
First Action 1967
Final Action 1973
(Caution: See safety notes on pi pets and pesticides.)
A. Preparation of Sample Solution
(a) 50% Dry powder formulation. — Transfer 10.00 g sam-
ple to 100 mL g-s vol. flask, using powder funnel. Add 50
Table 971.08 Chemical and Gas Chromatographic Parameters for Triazines and Other Pesticides
Chemical Name
Common or
Trade Name
CA
Registry
No.
Internal
Std Soln
971 .08A
Wt Sample
Length
Column
(m)
Inlet
Temperature (°)
Column Detector
Retention
Pesticide
Times (min)
Internal Std
2-(Ethylamino)-4-(isopropylamino)-6-
(methylthio)-s-triazine
Ametryn
834-12-8
(a)(2)
300 mg 80%
wettable
powder
1.8
240
215±15
240
8-12
9-15
2-Chloro-4-<ethylamino)-6-
(isopropylamino)-s-triazine
Atrazine
1912-24-9
(a)(2)
300 mg 80%
wettable
powder
1.8
240
200±10
240
5-7
9-15
Ethyl-4,4'-dichlorobenzilate
Chlorobenzilate
510-15-6
(c)
500 mg liq.
formulation
1.2
260
230±10
260
5-8
8-10
isopropyl-4,4 f -dichlorobenzilate
Chloropropylate
5836-10-2
(c)
1 g liq.
formulation
1.2
260
230±10
260
4-6
8-10
0,0-Diethyl-0-(2-isopropyl-6-
methyl-4-pyrimidinyl)
phosphorothioate
Diazinon
333-41-5
(b)
Sample
contg 110
mg
1.8
240
190+10
240
5-6
10-12
2,4-Bis(isopropylamino)-6-
methoxy-s-triazine
Prometon
1610-18-0
(a)(2)
1 g liq-
formulation
1.8
240
200 ±20
240
3-5
9-15
2,4-Bis(isopropylamino)-6-
(methylthio)-s-triazine
Prometryn
7287-19-6
(a)(2)
300 mg 80%
wettable
powder
1.8
240
200±10
240
6-8
9-15
2-Chloro-4,6-bis(isopropylamino)-
s-triazine
Propazine
139-40-2
(a)(1)
300 mg 80%
wettable
powder
1.2
250
210±10
240
3-5
7-9
2-Chloro-4,6-bis{ethylamino)-s-
triazine
Simazine
122-34-9
(e)
190 mg 80%
wettable
powder
1.8
250
210±5
250
6-8
10-14
2-(te/?-Butylamino)-4-chloro-6-
(ethylamino)-s-triazine
Terbuthylazine
5915-41-3
2-(terf-Butylamino)-4-(ethyl-
Terbutryn
886-50-0
(a)(2)
300 mg 80%
1.8
240
200 ±20
240
8-10
9-15
amino)-6-(methylthio)-s-triazine
wettable
powder
224
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
mL DMF. Shake 2-3 min to dissolve amitrole. (Undissolved
amitrole is powder and can be differentiated visually from in-
erts which are usually crystals.) Let settle and carefully decant
supernate into 100 mL vol. flask. Repeat extn of residue with
three 15 mL portions DMF, letting settle each time before de-
canting into vol. flask. Dil. combined exts to vol. with DMF
and shake well. Filter 40-50 mL thru fritted glass filter of
medium porosity. Pipet 25 mL into 400 mL beaker contg 50
mL H 2 0.
(b) 90% Dry powder formulation. — Dissolve 1.0000 g
sample in 100 mL H 2 in 400 mL beaker.
(c) Aqueous amitrole, — Pipet 5 mL sample into 400 mL
beaker contg 50 mL H 2 0.
B. Determination
Adjust sample soln or dild aliquot to pH 1.8 with 0.5/V HCL
Stir mech. and titr. with 0.5 mL increments 0.57V NaOH to
pH 3.5-4.0 using pH meter. Add 0.5N NaOH rapidly to pH
6.5 and then dropwise to pH 7.5 (second inflection point). Plot
pH against mL 0.5N NaOH and det. first inflection point (oc-
curs at pH 2.5-2.9).
% Amitrole by wt = (B - C) x 0.5 x 8.408/F
where C ~ mL 0.57V NaOH required to titr. to first inflection
point; B = mL 0.57V NaOH required to titr. to pH 7.5; and F
^2.5 for 50% dry powder formulation, (a), g sample for 90%
dry powder formulation, (b), and 5.0 x sp gr sample for aq.
amitrole, (c).
lb Amitrole in aq. amitrole/U.S. gal.
- % amitrole X sp gr X 8.32/100
Ref.: J AOAC 50, 568(1967).
CAS-61-82-5 (amitrole)
983.11 Brodifacoum (Technical)
and Pesticide Formulations
Liquid Chromatographic Method
First Action 1983
Final Action 1989
(Not applicable to wax bait formulations)
A. Principle
Weighed sample of tech. brodifacoum, brodifacoum cone,
or bait ext is dissolved in tripheny I benzene internal std soln
and detd by reverse phase liq. chromatgy and UV detection.
B. Apparatus
(a) Liquid chromato graph. — Flow rate 1 mL/min; loop in-
jection 10 (xL; mobile soJv. MeOH-H 2 0-HOAc (94.2 + 5.0
+ 0.8), filtered and degassed; UV detection at 254 nm with
range to give peak hts ca 60-80% full scale. Retention times
(min) — brodifacoum 6.2, internal std 11.7.
(b) Column. — 25 cm x 4.6 mm Zorbax ODS 5 fim reverse
phase column (E.I. DuPont de Nemours & Co., Instrument
Products Div.).
(c) Centrifuge. — Equipped with 15 mL capped tubes.
(d) Macerator. — With 400 mL stainless steel cup /impeller
assembly, such as Sorvall Omnimixer (DuPont Instruments
Inc.).
(e) Rotary evaporator. — Fitted with vac. and cold H 2
supplies.
(f) Filter. —Spread 10 g Celite 545 on 9 cm No. 5 filter
paper wetted with MeOH in buchner. Press filter and pre wash
with 30 mL MeOH.
C. Reagents
(a) 1 ,3 ,5 -Tripheny Ibenzene internal std soln. — Accurately
weigh ca 100 mg pure 1,3,5-triphenylbenzene into 500 mL
vol. flask, dissolve in 200 mL CH 2 C1 2 , and dil. to vol. with
MeOH.
(b) Brodifacoum std soln. — Accurately weigh ca 100 mg
brodifacoum std of known purity (ICI Americas Inc.) into 100
mL vol. flask. Dissolve in 40 mL CH 2 C1 2 . Dil. to vol. with
MeOH. Transfer 10.0 mL each of brodifacoum std soln and
interna] std soln to 50 mL vol. flask and dil. to vol. with dilg
soln.
(c) Diluting soln.— CH 2 Cl 2 -MeOH (2 + 3).
(d) Extracting soln. — CH 2 Cl 2 -formic acid (50 4- 1).
Store reagents in tightly capped dark bottles to avoid evapn
and decomposition. Check internal std soln for interfering
components by injecting 10 \xL into liq. chromatograph.
D. Determination
(a) Technical material. — Accurately weigh ca 100 mg sam-
ple into 100 mL vol. flask. Dissolve in 40 mL CH 2 C1 2 . Dil.
to vol. with MeOH. Transfer 10.0 mL each of sample soln
and internal std soln to 50 mL vol. flask. Dil. to vol. with dilg
soln.
(b) Powder concentrate. — Accurately weigh amt sample
contg ca 5 mg brodifacoum into 250 mL capped conical flask.
Add 100 mL extg soln and shake 1 min. Filter thru Celite,
using two 30 mL washes of extg soln. Evap. filtrate at 60°
under vac. Dissolve residue in 20.0 mL dilg soln and 5.0 mL
internal std soln.
(c) Liquid concentrate. — Accurately weigh amt sample contg
ca 5 mg brodifacoum into 25 mL vol. flask. Add 5.0 mL in-
ternal std soln, and dil. to vol. with dilg soln.
(d) Pelleted bait. — Grind amt sample contg ca 2 mg bro-
difacoum in anal. mill. Transfer to tared macerator cup and
accurately weigh. Add 250 mL extg soln and homogenize 10
min. Filter thru Celite using three 50 mL washings of extg
soln. Rotary-evap. filtrate at 60° under vac. Dissolve residue
in 8.0 mL dilg soln and 2 mL internal std soln. Centrf. sample
to remove remaining solids.
Inject 2 or more aliquots of std soln into liq. chromatograph
to set integration parameters and stabilize instrument. Monitor
response factor until results agree within 2%. Inject 4 aliquots
of std soln and 2 aliquots of sample soln in succession. Calc.
response factor, R, for each:
R = area brodifacoum peak/area internal std peak
Peak hts can be used in place of peak areas for tech. material
and bait formulations, but not for liq. formulations.
Brodifacoum, % = (R/R r ) x (W /W) x P x F
where R and/?' = av. response factor for sample and std solns,
resp.; W and W = mg sample and std, resp.; P = purity (%)
of std; F = scaling factor - 1 for technical material, V20 for
powder and liq. cones, l /so for pelleted bait.
Ref.: J AOAC 66, 993(1983).
CAS-56073-10-0 (brodifacoum)
AOAC Official Methods of Analysis (1990)
Miscellaneous
225
988.02 Cyhexatin Technical and
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1988
CIPACAOAC Method
A. Principle
Sample is extd with n-decylbenzene internal std soln contg
HO Ac, MeOH, and H 2 0. Cyhexatin is detd by liq. chromatgy
using peak ht for quantitation.
B. Apparatus
(a) Liquid chromato graph. — With peak ht integrator or re-
corder, 10 |ulL sample loop, and detector at 214 nm. Operating
conditions: column ambient; flow rate 2.0 mL/min; A range
1.0 AUFS; injection vol. 10 jxL; retention times, cyhexatin ca
7 min and internal std ca 10 min. Adjust parameters to give
peak ht for cyhexatin ca 75% full scale.
(b) Chromatographic column. — ODS bonded silica, 10 |xm
particle size, stainless steel, 25 cm X 4.6 mm id (E. Merck,
available from Curtin Matheson Scientific, Inc., or VWR Sci-
entific), or equiv.
C. Reagents
(a) Methanol. — LC grade,
(b) Acetic acid. — Glacial.
(c) HCl soln. — \M.
(d) Sodium chloride. — Analytical reagent grade.
(e) Mobile phase.— MeOH-H 2 0-HCl-NaCl (93 + 7 +
0.0001M + 0.005M). In 1 L g-s flask, combine 1 mL 1M
HCl, 69 mL H 2 0, and 0.29 g NaCl. Add 930 mL MeOH, mix,
and degas.
(f) n-Decylbenzene. — Eastman Laboratory Chemicals No.
9195 (Eastman Kodak Co.), or equiv.
(g) Cyhexatin reference std. — Available from Dow Chem-
ical Co.
D. Preparation of Standards
(a) n-Decylbenzene internal std soln. — Weigh 1.0 g n-de-
cylbenzene into 1 L vol. flask. Add 49 mL H 2 and 1 mL
HO Ac. Dil. to vol. with MeOH and sonicate until dissolved.
(b) Cyhexatin std soln. — Accurately weigh ca 110 mg pure
cyhexatin ref. std into 150 mL g-s flask. Add by pipet 100
mL internal std soln, shake well, sonicate for 10 min, and cool
to ambient temp.
E. Preparation of Sample
(a) Technical material. — Accurately weigh ca 120 mg sam-
ple into 150 mL g-s flask. Add by pipet 100 mL internal std
soln, shake well, and sonicate for 10 min. Cool to ambient
temp, and centrifuge at 2000 rpm.
(b) W enable powder. — Accurately weigh sample contg 1 10
mg cyhexatin into 150 mL g-s flask. Proceed as for technical
material, beginning, "Add by pipet. . ."
(c) Suspension concentrate. — Accurately weigh sample contg
ca 120 mg cyhexatin into 150 mL g-s flask. Add 10 mL H 2
and swirl until completely homogeneous. Proceed as for tech-
nical material, beginning. "Add by pipet. . ."
F. Determination
Inject 10 |jlL std solns until response ratio (cyhexatin peak
ht/internaJ std peak ht) varies <2%. Make 2 sample injections
followed by 1 std injection. Average peak ht ratios of stds
immediately preceding and following sample injections, and
average peak ht ratios of the 2 samples. Calc. cyhexatin as
follows:
Cyhexatin,
(R/R') x (W'/W) x p
where R and R' = av. peak ht ratios for sample and std, resp.;
W = g cyhexatin in std soln; W = g sample extd for analysis;
and P = % purity of std.
Ref.: JAOAC71, 26(1988).
CAS- 13 121 -70-5 (cyhexatin)
969.08 Diquat in Pesticide Formulations
Spectrophotometric Method
First Action 1969
Final Action 1972
AOAC-CIPAC Method
A. Reagents
(a) Acetate buffer soln. — pH 4.05. Dissolve 10.88 g
NaOAc.3H 2 in H 2 0, add 19 mL HOAc, dil. to 2 L with
H 2 0, and mix.
(b) Diquat std solns. — (/) Stock soln. — 0.2 mg diquat/mL.
Prep, stock soln by dissolving 0. 1968 g pure diquat dibromide
monohydrate (C ]2 H 12 N 2 Br 2 .H 2 0, MW 362.1; 50.87% cation;
ICI Americas, Inc.) in buffer soln, dil. to 500 mL with buffer
soln, and mix. (2) Working soln.— 0.02 mg diquat/mL. Dil.
10.0 mL stock soln to 100 mL with buffer soln. Prep, dild
stds fresh as required.
B. Determination
Using buret, transfer 10.0, 20.0, and 30.0 mL std diquat
soln, contg 0.2, 0.4, and 0.6 mg diquat, resp., to three 100
mL vol. flasks, dil. each soln to vol. with buffer soln, and
mix. Measure A of stds at 310 nm in 1 cm silica cell, with
buffer soln as ref. , and draw std curve relating A to mg diquat.
Accurately weigh portion (w g) of well mixed sample contg
ca 0.5 g diquat, transfer to 250 mL vol. flask, dil. to vol. with
buffer soln, and mix (Soln J). Transfer 10.0 mL Soln J to 200
mL vol. flask, dil. to vol. with buffer soln, and mix (Soln 2).
Transfer 5.0 mL Soln 2 to 100 mL vol. flask, dil. to vol. with
buffer soln, and mix (Soln 3).
Measure A of Soln J at 310 nm in 1 cm silica cell, with
buffer soln as ref. Read diquat content of Soln 3 (y mg) di-
rectly from std curve or calc. diquat content by interpolation.
% Diquat, w/w = 100 y/w
Rets.: Analyst 92, 375(1967). JAOAC51, 1304, 1306(1968).
CAS-2764-72-9 (diquat)
970.07 Dodine in Pesticide Formulations
Titrirnetric Method
First Action 1970
Final Action 1971
(Caution: See safety notes on acetic acid, perchloric acid, and
pesticides.)
A. Reagents
(a) Perchloric acid.—0.05N. Dissolve 4.2 mL 72% HC10 4
in HOAc and dil. to 1 L with HOAc. Stdze as follows: Ac-
curately weigh 0.200 g KHC 8 H 4 4 into 250 mL erlenmeyer.
Dissolve in 20 mL HOAc by gently heating flask on hot plate.
Add 80 mL Ac 2 and 8 drops metanil yellow indicator, (b).
Place erlenmeyer contg bar on mag. stirrer and titr. with HC10 4
226
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
to first definite red (magenta). Titr. reagent blank and correct
sample titer.
Normality = 0.200/(0.20422 X net mL HC10 4 )
(b) Metanil yellow. — 0.20%. Dissolve 0.200 g metanil yel-
low powder in 100 mL MeOH.
(c) Potassium acid phthalate. — NIST SRM KHC 8 H 4 4 .
B. Determination
Accurately weigh sample contg ca 0.600 g dodine into 250
mL erlenmeyer. Add 10 mL HO Ac followed by 90 mL Ac 2 0.
Mix by swirling 5 min. Filter slurry with vac. thru large, me-
dium porosity fritted glass buchner into 250 mL vac. flask.
Wash erlenmeyer and residue in funnel with two 10 mL por-
tions HOAc-Ac 2 (10 + 90). Place vac. flask contg bar on
mag. stirrer, add 8 drops metanil yellow indicator, and titr.
with stdzd ca 0.05JV H00 4 to first definite red (magenta). Titr.
reagent blank and correct sample titer.
% Dodecylguanidine acetate
= (net mL HC10 4 x normality x 28.75)/g sample
Ref.: JAOAC52, 1292(1969).
CAS-2439-10-3 (dodine)
898.01 Formaldehyde in
Pesticide Formulations
Hydrogen Peroxide Method
Final Action
(Applicable only to solns)
A. Reagents
(a) Sulfuric acid std soln. — IN. Prep, and stdze as in 890.01.
(b) Sodium hydroxide std soln. — IN. Stdze against (a), us-
ing litmus or bromothymol blue indicator. 1 mL = 30.03 mg
HCHO.
(c) Hydrogen peroxide soln. — Com., contg ca 3% H 2 2 . if
acid, neutze with NaOH, (b), using litmus or bromothymol
blue indicator.
(d) Litmus indicator. — Soln. of purified litmus of such concn
that 3 drops gives distinct blue color to 50 mL H 2 0.
(e) Bromothymol blue indicator. — Dissolve 1 g bromothy-
mol blue in 500 mL alcohol, 50% by vol.
B. Determination
Pipet 50 mL IN NaOH soln into 500 mL erlenmeyer and
add 50 mL H 2 2 , (c). Add weighed amt sample (ca 3 g) from
weighing pipet, letting point of pipet reach nearly to liq. in
flask. Place funnel in neck of flask and heat on steam bath 5
min, shaking occasionally. Remove from bath, wash funnel
with H 2 0, cool to room temp., and titr. excess NaOH with
std acid, using bromothymol blue or litmus. (Cool flask before
titrn to obtain sharp end point with litmus.) From mL IN NaOH
used and wt sample, calc. % HCHO according to following
equation
NaOH + HCHO + H 2 2 = HCOONa + 2H 2
If HCHO soln contains appreciable free acid, titr. sep. por-
tion and calc. acidity as % HCOOH. Correct for this acidity
in calcg % HCHO.
Refs.: Ber. 31, 2979(1898). J. Am. Chem. Soc. 27,
1183(1905). USDA Bur. Chem. Bull. 99, p. 30;
132, p. 49; 137, p. 47.
CAS-50-00-0 (formaldehyde)
897.01 Formaldehyde in
Pesticide Formulations
Cyanide Method
Final Action
(Applicable only to dil. solns)
Treat 15 mL 0. IN AgN0 3 , 941.18A-C, with 6 drops HN0 3
(1 + 1) in 50 mL vol. flask, add 10 mL KCN soln (3.1 g in
500 mL H 2 0), dil. to vol., shake well, filter thru dry filter,
and titr. 25 mL filtrate with 0.17V NH 4 SCN, 942.26, as in
915.01B. Acidify another 15 mL portion 0.1 N AgN0 3 with
6 drops HN0 3 (1 + 1) and treat with 10 mL of the KCN soln
to which has been added measured amt of sample (wt calcd
from sp gr) contg <25 mg HCHO. Dil. to 50 mL, filter, and
titr. 25 mL aliquot with the 0AN NH 4 SCN as before. Differ-
ence between mL NH 4 SCN used in these 2 titrns X 2 — mL
0. IN NH4SCN corresponding to KCN used by the HCHO. Calc.
% HCHO present. 1 mL 0.17V NH 4 SCN - 3.003 mg HCHO.
Refs.: Z. Anal. Chem. 36, 18(1897). USDA Bur. Chem. Bull.
132, p. 49.
931.03
Formaldehyde
in Seed Disinfectants
Final Action
(Applicable to detn of HCHO absorbed in inert carrier, e.g.,
bentonite, talc, charcoal, sawdust)
Weigh ca 5 g sample contg 0.3-0.5 g HCHO in weighing
bottle and transfer to 800 mL Kjeldahl flask. Add 25 mL H 2
and 12 mL H 2 S0 4 (1 +4). Steam distil rapidly, passing vapors
thru condenser with delivery end dipping into 25 mL H 2 in
500 mL vol. flask. Collect ca 450 mL distillate, keeping vol.
in distg flask nearly const with aid of small flame. After distn,
wash delivery tube, and dil. distillate to vol. with H 2 0.
Into each of two 200 mL vol. flasks measure 20 mL 0.1 AT
AgN0 3 . To each flask add 12 drops HN0 3 (1 + 1) and 30 mL
H 2 0. To one flask add slowly, with const shaking, 30 mL
KCN soln (3.1 g in 1 L H 2 0). Dil. to vol., shake well, and
filter thru dry filter. To 100 mL filtrate add 3 mL HNO ? and
5 mL ferric indicator, 929.04A(e), and titr. with 0. IN KSCN.
Pipet 25 mL HCHO distillate into small beaker contg 30 mL
of the KCN soln, mix well, and add slowly, with const shak-
ing, to second flask contg the acidified AgN0 3 soln. Dil. to
vol. with H 2 0, filter, acidify 100 mL filtrate with 3 mL HN0 3 ,
and titr. with the KSCN soln, using FeNH 4 (S0 4 ) 2 indicator.
Difference between mL KSCN soln used in these 2 titrns X
2 = mL 0. IN KSCN equiv. to HCHO. Calc. % HCHO pres-
ent. 1 mL 0.17V KSCN - 3.003 mg HCHO.
Refs.: Ind. Eng. Chem. Anal. Ed. 3, 357(1931). JAOAC 25,
80, 668(1942).
CAS-50-00-0 (formaldehyde)
986.08 Oxythioquinox
in Pesticide Formulations
Liquid Chromatographic Method
First Action 1986
Final Action 1989
AOAC-CIPAC Method
(Method is suitable for tech. oxythioquinox and formulations
with oxythioquinox as only active ingredient.)
AOAC Official Methods of Analysis (1990)
Miscellaneous
227
A. Principle
Sample with 1 -phenyl- 1-pentanone internal std is extd with
CH 3 CN, and oxythioquinox is detd by reverse phase liq. chro-
matgy.
B. Apparatus and Reagents
(a) Liquid chromato graph. — Able to generate >10 MPa
(>1430 psi) and measure A at 280 nm. Operating conditions:
column temp, ambient; flow rate 2 mL/min (ca 5 MPa); chart
speed 0.5 cm/min; injection vol. 10 |ulL; A range 0.320 AUFS;
retention times: 1 -phenyl- 1-pentanone ca 3.1 min, oxythio-
quinox ca 5.4 min. Pump LC mobile phase thru column until
system is equilibrated (flat baseline). Allow each injection ca
7 min run time, then pump CH 3 CN ca 4 min to remove im-
purities. Pump LC mobile phase ca 4 min, allowing system to
re-equilibrate before next injection.
(b) Chromatographic column. — 250 x 4.6 mm id packed
with <10 u>m CI 8 bonded silica gel.
(c) Acetonitrile . — LC grade or distd in glass (Burdick &
Jackson Laboratories, Inc., or equiv.).
(d) Chloroform. — Spectrophtric grade or equiv.
(e) Filters. — 0.45 (xm porosity (Gel man Acrodisc-CR, or
equiv.).
(f) 1 -Phenyl- 1-pentanone (n-valerophenone) internal std
soln. — \ g/100 mL CHC1 3 .
(g) Reference std oxythioquinox. — Mobay Corp.
(h) Water. — LC grade or distd in glass (Burdick & Jackson
Laboratories, Inc., or equiv.).
(i) LC mobile phase.— CH 3 CN-H 2 (80 + 20).
C. Preparation of Standard
Accurately weigh ca 100 mg ref. std into 100 mL vol. flask.
Pipet 10 mL internal std soln into flask and swirl to mix. Add
ca 50 mL CH 3 CN, sonicate 4 min, dil. to vol. with CH 3 CN,
and mix well. Filter portion of soln for LC analysis.
D. Preparation of Sample
Accurately weigh amt of sample contg ca 100 mg oxythio-
quinox into 100 mL vol. flask. Pipet 10 mL internal std soln
into flask, and swirl to mix. Add ca 50 mL CH 3 CN, sonicate
4 min, dil. to vol. with CH 3 CN, and mix well. Filter portion
of soln for LC analysis.
E. Determination
Adjust operating parameters to elute oxythioquinox in 5.0-
5.9 min. Adjust injection size and attenuation to give largest
possible on-scale peaks. Make repetitive injections of ref. std
soln and calc. response ratios (R) = oxythioquinox peak area
(or ht)/internal std peak area (or ht). Response ratios must
agree ±1%. Average duplicate response ratios obtained with
ref. std soln.
Inject duplicate aliquots of each sample soln. Average re-
sponse ratios for each sample soln. Response ratios must agree
±1%. If not, repeat detn, starting with std injections.
Re- inject ref. std soln twice. Average response ratios of stds
immediately preceding and following sample injections. These
must agree ±1%. If not, repeat detn.
F. Calculations
Oxythioquinox, wt% - (/?//?') x (W /W) x P
where R and R' = av. response ratios for sample and std solns,
resp.; W W — wt (mg) of oxythioquinox std and sample, resp.;
P — % purity of std oxythioquinox.
Ref.: JAOAC 69, 490(1986).
CAS-2439-01-2 (oxythioquinox)
969.09 Paraquat
in Pesticide Formulations
Spectrophotometric Method
First Action 1969
Final Action 1971
A. Reagents
(a) Sodium dithionite. — 1% soln in 0. IN NaOH. (Sodium
dithionite, Na 2 S 2 04.2H 2 0, is also called sodium hydrosulfite
and sodium hyposulfite.) Do not keep soln >3 hr; solid is un-
stable in presence of moisture. Store solid in small air-tight
bottles in vac. desiccator.
(b) Paraquat std soln. — 0.25 mg paraquat/mL. Dry anal.
std (ICI Americas, Inc.) to const wt at 100-120° before weigh-
ing (paraquat salts are hygroscopic). Dissolve 0.1728 g pa-
raquat dichloride (72.40% cation) in H 2 0, dil. to 500 mL with
H 2 0, and mix. Prep, soln fresh as required.
(c) Extracting soln. — Dissolve 11 g Na 2 SO 4 .10H 2 O in 500
mL H 2 0, add 500 mL alcohol, and mix.
B. Preparation of Standard Curve
Pipet 50 mL std soln into 250 mL vol. flask, dil. to vol.
with H 2 0, and mix. Pipet 5, 10, 15, and 20 mL aliquots of
this dild std soln into sep. 100 mL vol. flasks. (When dild to
vol. these solns contain 2.5, 5.0, 7.5, and 10.0 |JLg paraquat/
mL, resp.) Proceed as in 969. 09D. Plot A against fig para-
quat/mL at final diln.
C. Preparation of Sample
(Caution: Open aerosol can behind safety shield.)
(a) Formulations not containing oil base. — Accurately weigh
portion well mixed sample contg ca 0.25 g paraquat. Transfer
to 500 mL vol. flask, dil. to vol. with H 2 0, and mix well (Soln
1). Pipet 10 mL Soln 1 into 100 mL vol. flask, dil. to vol.
with H 2 0, and mix well (Soln 2). Pipet 10 mL Soln 2 into 100
mL vol. flask and proceed as in 969.090.
(b) Aerosol formulations containing oil base. — Weigh
aerosol can to nearest 0.1 g (C). Clamp can with bottom up
and puncture smallest possible hole with punch and hammer.
After hiss of escaping propellent is no longer heard, cut bottom
7 / 8 open with hand can opener. Push nearly detached lid into
can. Immerse can 15 min in 50-70° H 2 bath or in hot tap
H 2 running into 1 L beaker.
Add 50 mL extg soln, (c), and 50 mL pentane to 250 mL
separator. Remove can from H 2 bath, dry well (especially
inside cap and around valve), and weigh (D). Place pipet with
capacity to deliver ca 20 mg paraquat in can, and weigh both
(E). Withdraw liq., transfer contents to separator, replace pi-
pet in can, and weigh (F). (Disregard material left in and on
pipet.) Empty can, rinse completely with acetone, air dry, and
weigh (G).
Stopper separator and shake 30 sec, venting frequently. Let
layers sep, and drain lower layer into 200 mL vol. flask. Add
25 mL extg soln to separator, repeat extn, and drain lower
layer into same vol. flask. Dil. to vol. with extg soln and mix
well. Pipet 5 mL into 100 mL vol. flask and proceed as in
969.09D
D. Determination
(Complete analysis of one soln before adding
dithionite to next soln.)
Add 10 mL Na dithionite soln to one 100 mL vol. flask and
dil. to vol. with H 2 0. Mix by inverting end-over-end 3 times
at such speed that air bubble travels from one end to other; do
not shake flask vigorously, as this tends to cause fading of
228
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
color due to oxidn. Immediately measure A of soln at 600 nm,
using reagent blank (no paraquat) to set the 100% T or for ref.
side for dual beam instruments. Similarly, treat each flask in
turn, completing color measurement without delay before add-
ing dithionite to next soln.
% Paraquat - (|d,g/mL from std curve) x 5/g sample
% Paraquat (in aerosol formulations)
= [(|xg/mL from std curve) x (D - G) X 0.4]/
[(C - G) X (E - F)]
Refs.: Analyst 92, 375(1967). JAOAC51, 1304, 1306(1968);
55, 857(1972).
CAS-4685-14-7 (paraquat)
960.14 Quaternary Ammonium Compounds
First Action 1960
Final Action 1961
A. Potentiometric Titration Method
Transfer sample contg 30-35 mg CI to 600 mL beaker, dil.
to 200 mL with H 2 0, and add 5 mL HN0 3 (1 + 1). Add just
enough acetone to dissolve ppt that forms and titr. with 0. IN
AgN0 3 , using app. for potentiometric titrn. Calc. % CI (1 mL
O.liV AgN0 3 = 3.545 mg CI) and equiv. % quaternary NH 4
salt.
Adsorption Indicator Method
B. Reagents
(a) Bromothymol blue indicator. — Dissolve 1 g indicator in
500 mL50% alcohol.
(b) Dichlorofluorescein soln. — 0.1%. Dissolve 100 mg in-
dicator in 100 mL 70% alcohol.
C. Determination
Transfer sample contg 30-140 mg CI (usually ca 1 g qua-
ternary NH 4 salt) into 300 mL erlenmeyer, dil. to 75 mL with
H 2 0, and add 25 mL isopropanol. Neutze if necessary with
HO Ac (1 + 9), using 1 drop bromothymol blue (pH 4-6).
Add 10 drops dichlorofluorescein, and titr. with 0.1/V AgN0 3 ,
avoiding direct sunlight. Ppt becomes red at end point and may
flocculate just before end point. Calc. % CI and equiv. % qua-
ternary NH 4 salt.
Ref.: JAOAC43, 352(1960).
(e) Wash soln.— To 300 mL H 2 add I mL H 2 S0 4 (1 +4),
1 g (c), 10 mL (d), and 12 g Na 2 S0 4 , and pass S0 2 into soln
10 min.
B. Preparation of Sample
Weigh sample preferably contg ca 0.03 g thiocyanate N into
250 mL g-s erlenmeyer. (If SCN content is very low, do not
unduly increase amt sample without correspondingly increas-
ing ami mixed sulfide soln used; 20-25 g fly spray is usually
enough.) Add 35 mL mixed sulfide soln and shake vigorously
at room temp. 10 min, during which time reaction is nearly
completed. Heat to 70° on steam bath, carefully releasing pres-
sure resulting from heating, shake 15 min at 70°, and cool.
Removal of petroleum oil. — Transfer mixt. to separator with
ca 200 mL H 2 0. Add 50 mL pet ether, shake, and drain aq.
layer into 600 mL beaker. Wash pet ether layer with two 10
mL portions H 2 0, adding washings to main soln. (If emulsions
form during washing, break by acidifying with H 2 S0 4 (1 +
4).) Drain aq. layer and wash pet ether layer with H 2 as above.
Discard pet ether layer.
C. Determination of Thiocyanate Nitrogen
Dil. combined aq. soln to ca 300 mL and neutze with H 2 S0 4
(1 + 4), using litmus paper as outside indicator. Add 2 mL
H 2 S0 4 (1 + 4), quickly bring mixt. to bp, and boil 8 min to
remove H 2 S. Cool. If fatty acids or oils are present, transfer
to separator, ext with pet ether, and return aq. phase to original
beaker. Filter thru small buchner and transfer filtrate to beaker.
Neutze to litmus paper with 10% KOH soln and add 1 mL
H 2 S0 4 (1 +4). Add 1 g Na bisulfite and stir until dissolved.
Add excess (ca 15 mL) CuS0 4 soln and pass S0 2 into soln 10
min.
Let pptd CuSCN settle 2 hr, and filter with suction thru 56
mm buchner coated with layer of asbestos {Caution: See safety
notes on asbestos.), upon which is placed No. 42 Whatman
paper, or equiv., second layer of asbestos, layer of diatomite,
and finally third layer of asbestos. If filtrate is not clear, centrf.
soln at 2000 rpm 10-15 min, and pour thru filter again. Wash
filter and ppt once or twice with wash soln, continue suction
until filter pad is dry, and transfer to 800 mL Kjeldahl flask.
(Filter pad may be folded in filter paper together with bits of
moist filter paper used to wipe out buchner, and whole placed
in Kjeldahl flask.) Add few glass beads, 35 mL H 2 S0 4 , 10 g
K 2 S0 4 , and ca 0.7 g HgO or 0.65 g Hg. (Caution: See safety
notes on sulfuric acid and mercury.) Digest until colorless; then
15 min more. Det. N as in 955. 04C, second par. Perform blank
analysis on paper, filter pad, and reagents.
Ref.: JAOAC 34, 677(1951).
951.02 Organic Thiocyanate
in Livestock or Fiy Sprays
Kjeldahl Method
Final Action
(Caution: See safety notes on pesticides.)
A. Reagents
(a) Strong potassium poly sulfide soln. — Dissolve 180gKOH
in 120 mL H 2 0. Sat. 100 mL of this soln with H 2 S (ca 42 g)
(Caution: See safety notes on hydrogen sulfide.) while cool-
ing. Add remaining 100 mL KOH soln and 80 g S. Shake until
dissolved.
(b) Mixed sulfide soln.— To 100 mL (a) add 50 g
Na 2 S.9H 2 0, 30 g KOH, and 200 mL H 2 0.
(c) Sodium bisulfite. — Na 2 S 2 5 or NaHS0 3 .
(d) Copper sulfate soln. — 20% aq. soln CuS0 4 .5H 2 0.
985.08 Triadimefon Technical
and Pesticide Formulations
Liquid Chromatographic Method
First Action 1985
Final Action 1987
CIPAC-AOAC Method
(Method is suitable for tech. triadimefon and formulations with
triadimefon as only active ingredient.)
A. Principle
Triadimefon is detd by liq. chromatgy, using 4-chlorophenyl
sulfoxide as internal std.
B. Apparatus
(a) Liquid chromato graph.— Able to generate >7 MPa
(>1000 psi) and measure A at 475 nm.
AOAC Official Methods of Analysis (1990)
Miscellaneous
229
(b) Chromatographic column. —250 x 4.6 mm id packed
with <10 juim silica gel capable of resolving 4-chIorophenol
from triadimefon and internal std peaks (Du Pont Zorbax-Sil
or equiv.). LC operating conditions . — Column temp, ambient;
mobile phase flow rate 1 .5 mL/min (ca 500 psi); chart speed
0.5 cm/min; injection vol. 20 jxL; A range 0.320 AUFS; re-
tention times: 4-chlorophenyl sulfoxide ca 4.0 min, triadime-
fon ca 5,9 min. Pump LC mobile phase until system is equil-
ibrated 15 min (flat baseline). Allow 6.5 min between injections.
(c) Mechanical shaker.
(d) Filters. — 0.45 (xm porosity (Gel man Acrodisc-CR, Gel-
man Scientific, Inc., or equiv.).
C. Reagents
(a) Butyl chloride. — LC grade or distd in glass (Burdick and
Jackson Laboratories, Inc.).
(b) Ethanol.—Anhyd., 200 proof.
(c) Mobile phase. —Butyl chloride-EtOH (100 + 1). Pipet
10 mL anhyd. EtOH into I L butyl chloride, mix, and degas.
(d) 4-Chlorophenol stock soln. — Weigh ca 20 mg 4-chlo-
rophenol into 100 mL vol. flask and dil. to vol. with mobile
phase.
(e) 4-Chlorophenyl sulfoxide internal std soln. — About 275
mg/250 mL mobile phase; sonicate to dissolve.
(f) Triadimefon reference std soln. — Accurately weigh ca
200 mg ref. std (Mobay Corp.) into 100 mL vol. flask. Pipet
10 mL cholorophenol stock soln into flask. Pipet 20 mL in-
ternal std soln into flask, dil. to vol. with mobile phase, and
mix well. Filter portion of soln for LC analysis.
D. Preparation of Sample
Accurately weigh amt sample contg ca 200 mg triadimefon
into 100 mL vol. flask. Pipet 20.0 mL internal std into flask.
Add ca 50 mL mobile phase and shake 20 min. Dil. to vol.
with mobile phase and mix. Filter portion of soln for LC anal-
ysis.
FiG. 985.08— LC chromatogram of 4-chlorophenol (A), 4-chlo-
rophenylsulfoxide (B), and triadimefon (C)
E. Determination
Inject triadimefon std soln and adjust operating parameters
to cause triadimefon to elute in 5.5-6.0 min. Adjust injection
size and attentuation to give largest possible on-scale peaks.
Chlorophenol in std injection must be resolved from triadi-
mefon and internal std peaks (Fig. 985.08). If not, change sil-
ica columns.
Using same injection vol. for all sample and std injections,
make repetive injections of std and calc. response ratios by
dividing peak area (or ht) of triadimefon by that of internal std
peak. Response ratios must agree within ±1%. Average du-
plicate response ratios obtained with std solns.
Inject duplicate aliquots of each sample soln. Average du-
plicate response ratios for each sample soln. Response ratios
must agree within ±1%. If not, repeat detn, starting with std
injections.
Reinject std soln twice. Average response ratios of stds im-
mediately preceding and following sample injection. These must
agree within ±1%. If not, repeat detn.
F. Calculation
Triadimefon, wt % - (R/R') x (W /W) x P
where R and /?' = av. response ratios for sample and std solns,
resp.; W f and W — wt (mg) of triadimefon std and sample
solns, resp.; and P purity of triadimefon std (%).
Ref.; J AOAC 68, 586(1985).
CAS-43 1 2 1 -43-3 (triadimefon)
960.15 Warfarin
in Rodenticide Formulations
Spectrophotometric Method
First Action 1960
Final Action 1961
(Applicable to baits contg ca 0.025% and to cones contg >0.5%
warfarin. Not applicable to pelleted baits or baits consisting of
cracked corn treated with ale. warfarin soln and aq. sugar soln,
and then dried.)
A. Reagents
(a) Sodium pyrophosphate soln. — -1%. Dissolve 5 g Na 4
•P 2 O 7 -10H 2 O in 500 mL H 2 0.
(b) Petroleum ether, purified. — Ext 200 mL pet ether with
three 20 mL portions }% Na 4 P 2 7 soln.
(c) Warfarin std soln. — 10 |xg/mL. Dissolve 100 mg pure
warfarin (Biocenotics, Inc. , 4880 Hudson Rd, Osseo, MI 49266)
in 100 mL 1% Na 4 P 2 ? soln. Dil. 10 mL to 100 mL with 1%
Na 4 P 2 7 soln, and dil. 10 mL of second soln to 100 mL with
1% Na 4 P 2 7 soln.
B. Determination
Weigh 10 g sample (0.025%), 0.600 g (0.5%), or equiv. wt
of higher concn, into 125 mL g-s flask or 100 mL centrf. tube
and add 50 mL Et ether from pipet. Stopper tightly and shake
mech. ca 30 min. Transfer 5 or 10 mL to centrf. tube (or centrf.
directly), stopper, and centrf. 5 min at high speed or until clear.
Take precautions to avoid evapn of ether.
230
Pesticide Formulations
AOAC Official Methods of Analysis (1990)
Pipet 10 mL 1% Na 4 P 2 7 soln into g-s 16 x 150 mm test
tube and add 2 mL centrfd ether ext from pipet. Stopper and
shake vigorously 2 min. Centrf. at high speed until aq. layer
is clear. Draw off ether layer, including any emulsion that re-
mains, using fine-tip glass tube attached to aspirator. Add ca
2 mL Et ether, shake vigorously, centrf. , and completely draw
off ether layer. Repeat ether extn, and then ext twice with puri-
fied pet ether in same manner.
Prep, blank soln similarly, using 2 mL ether instead of 2
mL ether ext.
Det. A of aq. soln in 1 cm silica cell at 308 nm against blank
soln in Beckman spectrophtr, model DU (replaced by models
24/25), or equiv. Det. A' (ca 0.46) of the std warfarin soln
against 1% Na 4 P 2 7 soln.
% Warfarin = (A/A f ) x (JO" 5 g std/mL)
x [100/(g sample x (2/50)(l/10))]
= (A/A f ) x (0.250/g sample)
Ref.: JAOAC 43, 365(1960).
CAS-81-81-2 (warfarin)
968.05* Sulfoxide Pesticide Formulations
Spectrophotometry Method
First Action 1968
Surplus 1974
5^6.296-6.302, 11th ed.
Common and Chemical Names of Pesticides in this chapter
Common Name
Chemical Name
Alachlor
Aldicarb
Aldrin
Allethrin
d-fra/is-Allethrin
Aminocarb
Amitrole
Anilazine
Azinphos-methyl
Bendiocarb
Benfluralin
Benomyl
7-BHC
Brodifacoum
Bromoxynil
Butachlor
Butylate
Captan
Carbaryl
Carbofuran
Chloramben
Chloramine-T
Chlordane
Chlordimeform
Chlorotoluron
Chloroxuron
Chiorpyrifos
Cycloate
Cyhexatin
Cypermethrin
2,4-D
Dalapon
DCPA
DDT
DDVP
Diazinon
Dicamba
Dichlobenil
Dichlorvos
Dicofol
Dieldrin
Diflubenzuron
Diquat
2-Chloro-W-(2,6-diethylphenyl)-/V-(methoxymethyl)acetamide
2-Methyl-2-(methylthio) propionaldehyde 0-(methylcarbamoyl)oxime
1, 2,3,4, 10,10-Hexachloro-1,4,4a,5,8,8a-hexahydro-exo-1,4-enc/o-5,8-dimethanonaphthalene, not less than 95%
o7-3-Allyl-2~methyl-4-oxocyclopent-2-enyl dt-cis /trans chrysanthemate
d/-2-Allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of d-trans chrysanthemum monocarboxylic acid
4-(Dimethylamino)-3-methylphenol methylcarbamate (ester)
3-Amino-1 ,2,4-triazole
4,6-Dichloro-/V-(2-chlorophenyl)-1,3,5-triazin-2-amine
0,0-Dimethyl S-[(4-oxo-1 ) 2,3-benzotriazin-3(4H)-yl)methy|]phosphorodithioate
2,2-Dimethyl-1 ,3-benzodioxol-4-yl methylcarbamate
A/-Butyl-N-ethyl-«,a,a-trifluoro-2,6-dinitro-p-toluidine
Methyl 1 -(butylcarbamoyl)-2-benzimidazolecarbamate
1,2,3,4,5,6-Hexachlorocyclohexane, gamma isomer
3-[3-(4'-Bromo-(1 ,1 '-biphenyl)-4-yl)-1 ,2,3,4-tetrahydro-1 -napthalenyl]-4-hydroxy-2H-1-benzopyran-2-one
3,5-Dibromo-4-hydroxybenzonitrile
2-Chloro-A/-(2,6-diethylphenyl)/V-(butoxymethyl)-acetamide
S-Ethyl diisobutylthiocarbamate
W-[(Trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide
1-Naphthyl /V-methylcarbamate
2,3-Dihydro-2,2-dimethy!-7-benzofuranol methylcarbamate
3-Amino-2,5-dichlorobenzoic acid
W-Chloro-4-methylbenzenesulfonamide sodium salt
1,2,4,5,6,7,8,8-Octachlor-2,3,3a,4,7.7a-hexahydro-4,7-methanoindane
W-(4-Chloro-2-methylphenyl)-N,A/-dimethylmethanimidamide
A/'-(3-Chloro-4-methylpheny!)-/V'A/-dimethyl urea
3-[p-(p-Chlorophenoxy) phenyl]- 1 ,1-dimethylurea
0,O-Diethyl 0-(3,5,6-trichloro-2-pyridyl)phosphorothioate
S-Ethylcyclohexylethylthiocarbamate
Tricyclohexylhydroxystannane
3-(2,2-Dichloroethenyl)-2,2-dimethylcyc!opropanecarboxylic acid cyano(3-phenoxyphenyl)methyl ester
2,4-Dichlorophenoxyacetic acid
2 f 2-Dichloropropionic acid
Dimethyl tetrachloroterephthalate
1 ,1 '-{2,2,2-Trichloroethylidene)bis[4-chlorobenzene]
2-2-Dichlorovinyl dimethyl phosphate
0,0-Diethyl 0-(2-isopropy!-4-methyl-6-pryimidinyi) phosphorothiote
2-Methoxy-3,6-dichlorobenzoic acid
2,6-Dichiorobenzonitrile
2-2-Dichlorovinyl dimethyl phosphate
4-Chloro-alpha-(4-chSorophenyl)-alpha-(trichloromethyl)benzenemethanol
3,4,5 J 6,9,9-Hexachloro-1a J 2,2a ) 3 J 6,6a ! 7,7a-octahydro-2 J 7:3,6-dimethanonaphth{2,3-b)oxirene
1-(4-Chlorophenyl) 3-(2,6 difluorobenzoyl) urea
1,1'-Ethylene-2,2'-bipyridylium ion OR 6,7-Dihydrodipyridol (1,2-a:2',1'-c) pyrazdiium ion
{Continued)
AOAC Official Methods of Analysis (1990)
Miscellaneous
231
Common and Chemical Names of Pesticides in this chapter (Continued)
Common Name
Chemical Name
Disulfoton
Dodine
Endosulfan
Endrin
EPTC
Ethion
Fenitrothion
Fensulfothion
Fentin
Ferbam
Fluazifop-butyl
Fluometuron
Folpet
Formothion
Glyphosate
Heptachlor
Isofenphos
Lindane
Malathion
Maneb
MCPA
MCPP
Methazole
Methiocarb
Methyl parathion
Metolachlor
Metoxuron
Metribuzin
Molinate
Nabam
Nicotine
A/-Octyl bicycloheptene
dicarboximide
Oxythioquinox
Paraquat
Parathion
PCNB
Pebulate
Permethrin
P ho rate
Picloram
Piperonyl butoxide
Pirimicarb
Propachlor
Propoxur
Pyrethrins
Rotenone
Sulfoxide
Sulprofos
2,4,5-T
TCA
Temephos
TEPP
Terbuthylazine
Tetrad if on
Thiram
Toxaphene
Triadimefon
Trifluralin
Vernolate
Warfarin
Zineb
O-O-Diethyl S-[2-(ethylthio)ethyl] phosphorodithioate
n-Dodecylguanidine acetate
6,7,8,9,1 0,1 0-Hexachloro-1, 5, 5a,6,9,9a-hexahyro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide
Hexachloroepoxyoctahydro-endo,endo-dimethanonaphthalene
S-Ethyl dipropylthiocarbamate
0,0,0,0-Tetraethyl S,S- methylene bisphosphorodithioate
Phosphorothioic acid 0,0dimethyl 0(3-methyl-4-nitrophenyl) ester
Phosphorothioic acid 0,0-diethyl 0-[4-(methylsulfinyl)phenyl] ester
Triphenyltin
Ferric dimethyldithiocarbamate
Butyl 2-{4-{5-trifluoromethyl-2-pyridinyloxy) phenoxy} propanoate
1 , 1 -Dimethyl-3-(a,a,a-trifluoro-/77-tolyl) urea
A/-(Trichloromethylthio)phthalimide
S-(2-(Formylmethylamino)-2-oxoethyl) O-O-dimethyl phosphorodithioate
Isopropylamine salt of A/-{phosphonomethyl) glycine
1,4,5, 6,7,8, 8-Heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene
2-[[Ethoxy[(1-methylethyl)amino]phosphinothioyl]oxy]benzoic acid 1-methylethyl ester
Gamma isomer of 1 , 2,3,4,5, 6-hexachlorocyclohexane
0,0~Dimethyl S-{1 ,2-dicarbethoxyethyl) phosphorodithioate
Manganese ethylenebisdithiocarbamate
4-Chloro-2-methyl phenoxyacetic acid
2-(4-Chloro-2-methylphenoxy) propanoic acid
2-(3,4-Dichlorophenyl)-4-methyl-1,2,4-oxadiazolidine-3,5-dione
3,5-Dimethyl-4-(methylthio)phenol methylcarbamate
O-O-Diimethyl O-p-nitrophenyl phosphorothioate
2-Chloro-A/-(2-ethyl-6-methylphenyl)-A/-(2-methoxy- 1-methylethyl) acetamide
A/'-{3-Chloro-4-methoxyphenyl)-/V,A/-dimethylurea
4-Amino-6-(1 ,1 -dimethyethyl)-3-(methythio)-1 ,2,4-triazin-5(4H)-one
S-Ethyl hexahydro-1H-azepine-1-carbothioate
Disodium ethylene-1 ,2-bisdithiocarbamate
3-{1 -Methyl-2-pyrrolidyl) pyridine
Same as common name OR A/-(2-Ethylhexyl)-5-norborene-2,3-dicarboximide
6-Methyl-1,3-dithiolo[4,5-b]quinoxalin-2-one
1 ,1 '-Dimethyl-4,4'-bipyridinium ion
0,0-Diethyl O-p-nitrophenyl phosphorothioate
Pentachloronitrobenzene
S-Propyl butylethylthiocarbamate
3-(2,2-Dichloroethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxy phenyl) methyl ester
0,0-Diethyl S-[(ethylthio)methyl] phosphorodithioate
4-Amino-3,5,6-trichloropicolinic acid
a-[2-(2-Butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-propylto!uene
2-(Dimethylamino)-5,6-dimethyl-4-pyrimidinyl dimethylcarbamate
2-Chloro-A/-(1-methylethyl)-/V-phenylacetamide
2-(1-Methylethoxy) phenol methycarbamate
Standardized mixture of pyrethrins I and II (Mixed esther of pyrethrolone)
1,2,12,12a-Tetrahydro-8,9-dimethoxy-2-(1-methyletheny!)-(1)benzopyrano(3,4-b)furo(2,3-h)(1)benzopyran-6(6aH)-one
1 ,2-Methylenedioxy-4-(2-(octylsulfidnyl)propyl) benzene
O-Ethyl 0-[4-(methylthio)phenyl] S-propyl phosphorodithioate
2,4,5-Trichlorophenoxyacetic acid
Trichloroacetic acid
0,0, 0',0'-Tetramethyl O,0'-thiodi-p-phenylene phosphorothioate
Tetraethyl diphosphate
4-tert-Butylamino-2-chloro-6-ethylamino-s-triazine
4-Chlorophenyl 2,4,5-trichlorophenyl sulfone
Bis(dimethylthiocarbamoyl) disulfide
Chlorinated camphene (67-69% chlorine)
1 -(4-Chlorophenoxy)-3,3-dimethy!-1 -(1 H-1 ,2,4-triazol-1 -yl)-2-butanone
a^.a-Trifluoro^^-dinitro-A/^-dipropyl-p-toluidine
S-Propyldipropylthiocarbamate
3-(«-Acetonylbenzyl)-4-hydroxycoumarin
Zinc ethylenebisdithiocarbamate
Sources: The Merck Index (1983) 10th ed., Merck & Co., Inc., Rahway, NJ; The Agrochemicals Handbook (1987) 2nd ed. t The Royal Society of Chemistry,
Nottingham, UK; Farm Chemicals Handbook (1988) 74th ed., Meister Publishing Co., Wiiloughby, OH.
8. Hazardous Substances
975.05 Cadmium and Lead in Earthenware
See 973.32 and 973.33.
911.01 Carbonate and Hydroxide
in Soda Lye
Titrimetric Method
Final Action
Weigh ca 10 g sample from weighing bottle, dissolve in
C0 2 -free H 2 0, and dil. to definite vol. Titr. aliquot with 0.5jV
HCI, 936. ISA and B, using 0.5% aq. Me orange soln,
930.11A(e); note total alky found. Transfer equal aliquot to
vol. flask and add enough 10% BaC1 2 soln to ppt all carbonate,
avoiding any unnecessary excess. Dil. to vol. with C0 2 -free
H 2 0, stopper, shake, and let stand. When liq. clears, pipet off
one-half and titr. with the 0.5/V HCI, using phthln; mL 0.5N
acid required for this titrn X 2 = mL 0.5jV acid equiv. to
NaOH present in original aliquot. Difference between this fig-
ure and mL 0.5N HCI required for total alky = mL 0.5N acid
equiv. to Na 2 C0 3 present in aliquot. Calc. % Na 2 C0 3 and
NaOH.
Ref.: Sutton "Systematic Handbook of Volumetric Analysis,"
10th ed., p. 61(1911).
CAS-5968-1 1-6 (sodium carbonate)
CAS-1 3 10-73-2 (sodium hydroxide)
975.06 Denaturants (Volatile)
in Alcoholic Products
Gas Chromatographic Method
First Action 1975
A. Apparatus and Reagents
(a) Gas chromato graph and integrator. — With flame ioni-
zation detector (F&M Model 400, 402, Hewlett-Packard 7600
series, or equiv.). Column 1.2 m (4') x 2 mm id glass packed
with 100-120 mesh Chromosorb 102; column temp. 160° (iso-
thermal), detector and inlet 200°; He flow rate 50 mL/min;
relative retention times: EtOH 1.00 (<100 sec), n-PrOH 2.06,
and tetrahydrofuran 3.04. Integrator: Hewlett-Packard 3370A
(new model 3370B), or equiv.
(b) Stdsolns. — 6% (v/v). Dil. 6.00 mL of each denaturant
of interest to 100 mL with anhyd. alcohol in sep. vol. flasks.
Approx. slopes and retention times relative to n-PrOH are given
in Table 975.06.
B. Determination
Pipet 25 mL of each expected denaturant std soln into sep.
flasks and add 1.00 mL n-PrOH as internal std. Cap imme-
diately with rubber stoppers, shake 3 min, and let stand 10
min at room temp. Inject 0.3 fiL portions from 1 jxL micro-
syringe. Det. peak areas and calc. slope for each compd as:
5 X = {PAJPA-M6.0O
where PA X and PA { — peak areas of compd X in std soln and
of n-PrOH internal std, resp., and 6.00 = % compd X in std
soln. Slopes and retention times should approximate those of
Table 975.06.
% Compd X in sample = (PA/PA,) - (1/5 X )
where PA — peak area of compd X in sample.
Ref.: JAOAC 57, 148(1974).
973.10 Fluorides in Hazardous Substances
Potentiometric Method
First Action 1973
Final Action 1975
A. Apparatus
(a) pH meter. — With expanded mv scale (digital Model 110,
Corning Scientific Instruments, 63 North St, Medfield, MA
02052, or equiv.), fluoride ion-selective electrode (Model 94-
09, Orion Research Inc., or equiv.), and single junction ref.
electrode, plastic sleeve-type (Model 90-01, Orion Research
Inc., or equiv.).
(b) Magnetic stirrer. — With Teflon-coated stirring bar. Use
asbestos or foam mat to insulate sample from motor heat.
(c) Beakers. — 4.5 oz (135 mL), polypropylene, or equiv.
(d) Graph paper. — Linear or semi-antilog, vol. corrected
No. 90-00-90 Gran's plot paper (Orion Research Inc., or equiv.).
B. Reagents
(a) Buffer soln.— pH 6.0. Add 77.0 g NH 2 OAc and 0.452
g NH 4 citrate to 1 L H 2 0. Adjust to pH 6.0 with HO Ac.
(b) Fluoride std soln. — I mg F/mL. Prep. 2.2108 g NaF
(reagent grade, dried 4 hr at 105°)/L buffer soln. Store in leak-
proof plastic bottles. Compare with 1 mg F/mL soln prepd
from USP Ref. Std; equiv. reading of ±1 mv is satisfactory.
C. Determination
(Stir all solns constantly at same rate thruout titrns. Let elec-
trodes equilibrate ^2 min before addn of F std soln and 30
sec after each addn of F std soln.)
(a) Blank. — Record mv values (E f ) of 100 mL buffer soln
after addn of 4 mL std F soln from 10 mL buret and after each
addnl mL up to 10 mL. (Preliminary mv values will not fall
on linear range of response curve.) Vol. std soln added - V .
(b) Samples. — Est. molarity of samples from direct read-
ing. Dil. samples, if necessary, to ca 0.00 1M F. Transfer 50
mL sample soln to beaker and add 50 mL buffer soln. Record
initial mv reading, using expanded scale (E ). If initial reading
Table 975.06
Compound
Approximate Slopes and Retention Times Rel-
ative to n-Propyl Alcohol (RT) for Denaturants
Slope
RT
Acetone CAS-67-64-1
Benzene CAS-71-43-2
n-Butyl alcohol CAS-71-36-3
sec-Butyl alcohol CAS-78-92-2
Chloroform CAS-67-66-3
Ethyl acetate CAS-1 41 -78-6
Ethylene glycol monoethyl ether CAS-629-14-1
Ethylene glycol monomethyl ether CAS-1 09-86-4
Isopropanol CAS-67-63-0
Methanol CAS-67-56-1
Methyl isobutyl ketone CAS-1 08-1 0-1
Toluene CAS-1 08-88-3
0.207
0.694
0.646
2.309
0.269
2.283
0.246
1.621
0.058
1.543
0.192
1.640
0.187
3.868
0.151
2.071
0.210
0.727
0.130
0.266
0.275
5.436
0.454
5.302
232
AOAC Official Methods of Analysis (1990)
Hazardous Substances 233
is < — 50 mv, soln is too coned. Dil. sample to avoid asymp-
totic slope. Record mv values (E) after each mL F std soln is
added up to 10 mL. Rinse electrodes with H 2 between sam-
ples. Vol. std soln added = V.
D. Calculations
(a) Linear graph paper. — For each addn of F std soln and
corresponding E value, calc. for blank:
Z' = antilog [log(V + V) - 0.017(£')]
where V is original vol. soln to which F std soln was added
(100 mL) and E' is treated algebraically (+ or — as read). Plot
Z' against mL (mg) F std soln added and extrapolate to inter-
section of mL (mg) F axis to obtain mL (mg) F in blank, V e \
In graph, assign horizontal axis to mL (mg) F, with at center
and mL (mg) F increasing in both directions to left and right.
Assign Z values to vertical axis. Plot actual readings of mL
(mg) F on right portion of horizontal axis so that extrapolation
will fall on left portion of axis.
Similarly, for original readings and each addn of F std soln
and corresponding E value, calc. for sample:
Z - antilog [Jog(V + V) - 0.017(E)]
where V is original vol. soln to which F std soln was added
(100 mL). Plot Z against mL (mg) F std soln on same graph
as blank and extrapolate to intersection of mL (mg) F axis to
obtain mL (mg) F in sample, V c .
(b) Semi-antilog paper. — Plot E directly for both blank and
sample, descending 5 mv for each major line crossing vertical
axis. At top of vertical axis place most neg. E reading which
still allows extrapolation of V c on left portion of mL (mg) side
of horizontal axis. Obtain V e and V c ' by extrapolation to left
side of mL (mg) F.
% F - (V c - V c ') x (B x 100)/[W x C x 1000 (mg/g)]
where B = vol. of diln, W — mL or g sample, and C = aliquot
(50 mL max.) buffered to 100 mL.
Ref.: J AOAC 56, 798(1973).
CAS-7782-41-4 (fluorine)
ment model PC300), or equiv.). Sand bath may be used in-
stead of Al block.
(d) Boiling chips. — Unglazed boiling chips, 1 .5 mm diam.,
Pb-free.
B. Determination of Solids
Thoroly mix samples manually for 10 min or mech. for 5
min. Accurately weigh 0.3-0.4 g into weighed Al dish, 63
mm diam. Add 3-5 mL hexane or pet ether to oil-based paints
or H 2 to latex paints and swirl to disperse. Warm on hot plate
while swirling until sol v. has evapd and film is formed. Heat
in oven 4 hr at 105°, cool, and weigh.
% Solids = g dried sample X 100/g sample
C. Determination of Lead
Introduce ca 0.6 g (0.3 mL) thoroly mixed sample near bot-
tom of 16 X 150 mm test tube with syringe and weigh ac-
curately. Add 5 ± 0.2 mL HN0 3 and 2 boiling chips to each,
including blanks. Place in block or bath at 90-100° so that liq.
surface is slightly above heated surface. (Use hood.) After ini-
tial fuming has subsided, increase temp, until vapors are con-
densing in top 1-2 cm of tube (bath temp., 160-170°) and
maintain at this temp. 3 hr. Cool to 50—60°, transfer to 25 mL
vol. flask, including chips and any ppt, and rinse with four
4 mL portions H 2 0, transferring as much residue as possible.
Dil. to vol. with H 2 and let settle 0.5-1 hr. Floating residue
may be removed by aspiration thru disposable pipet.
Aspirate solns and stds into AA spectrophotometer, avoid-
ing introduction of ppt. If A of sample is greater than highest
std, dil. sample and re-aspirate. Det. jxg Pb/mL from std curve.
% Pb in paint solids
= (|xg Pb/mL) X F X 10~ 2 /(g sample x % solids in sample)
F (diln factor) - l/[(l/25) (b/c) (die) . . .]
where 25 — vol. original sample digest, h = aliquot of original
25 mL dild to c mL; d = aliquot of c (mL) dild to e mL; etc.
For dry paint films, % solids in sample = 100.
Ref.: JAOAC 57, 614(1974).
CAS-7439-92-1 (lead)
974.02 Lead in Paint
Atomic Absorption Spectrophotometric Method
First Action 1974
Final Action 1976
A. Reagents and Apparatus
(a) Lead std solns. — (1) Stock soln. — 1 mg Pb/mL 1%
HN0 3 . Dissolve 159.9 mg Pb(N0 3 ) 2 in HNO3 (1 + 99) and
dil. to 100 mL with HNO3 (1+99). (2) Intermediate soln.—
300 |xg/mL dil. HNO3. Dil. 15 mL stock soln to 50 mL with
0.5 mL HNO3 and H 2 0. (3) Working solns. — To each of seven
100 mL vol. flasks contg 1 mL HN0 3 , add resp. 0, 1, 2, 3,
4, 5, and 6 mL intermediate soln and dil. to vol. with H 2
(0, 3, 6, 9, 12, 15, and 18 [ig Pb/mL).
(b) Atomic absorption spectrophotometer. — With Pb hol-
low cathode lamp and 4" single slot or 3 slot Boling burner
head, capable of detecting 0.5 |xg Pb/mL, such as Perkin-
Elmer Model 403. Operating conditions: 283.3 nm, 0.7 nm
band width slit, recorder response (if used) 0.25-1 sec time
constant, air-C 2 H 2 flame, with gas flows adjusted according to
directions of manufacturer.
(c) Heater for digestion. — Drill 7.5 cm Al block to hold
>16 test tubes, 16 x 150 mm. Place on hot plate capable of
maintaining medium at 160-170° (Corning PC 35 (replace-
971.03 Methanol in Hazardous Substances
Gas Chromatographic Method
First Action 1971
Final Action 1973
(Applicable in presence of acetone, BuOAc, EtOH, isopropanol,
hexane, MeEt ketone, CH 2 C1 2 , Me Cellosolve, paraffin, toluene,
and H 2 0. This includes many paint removers, fuels, liq. sanders,
antifreezes, and paint products.)
A. Apparatus and Reagents
(a) Gas chromatograph. — With flame ionization detector
and oven capable of temp, changes >5°/min near 160° or pref-
erably temp, programmer. Column. — 1.8 m (6') x 4 mm id
packed with 120-150 mesh Porapak R (Waters Associates, Inc.);
condition 2 hr at 235°. Conditions: Temps (°): injection ca 200,
column ca 160, detector ca 210; N flow ca 25 mL/min; set
electrometer so that 8 \xL std soln provides at least half scale
peak. Adjust column temp, and N flow so that MeOH retention
time is ca 5-7 min.
(b) Methanol std soln.— 0.4% (v/v). Dil. 4.00 mL MeOH
to 100 mL with dioxane; dil. 10.0 mL of this soln to 100 mL
with dioxane. Rinse pipet into flask before dilg to vol. with
dioxane. Prep, fresh daily.
234
Hazardous Substances
AOAC Official Methods of Analysis (1990)
B. Preparation of Sample
(a) For asphalt-base tar compounds and viscous adhe-
sives. — Refrigerate unopened sample container >3 hr (longer
for larger containers) at 1-10°, open container, and mix well;
close container and refrigerate 30 min more. Transfer 1.5-3
g sample to tared, 250 mL, wide-mouth g-s erlenmeyer (tared
with stopper in place). Let sample reach room temp, in stop-
pered erlenmeyer and weigh. Refrigerate 30 min and quickly
add 100.0 mL dioxane. Stopper and shake mech. 1 hr. Re-
frigerate 30 min and filter thru rapid paper (S&S sharkskin,
or equiv.). Filter as quickly as possible, covering funnel with
watch glass and placing funnel against neck of narrow-mouth
g-s receiver. Proceed as in 971. 03C, dilg with dioxane, if
necessary.
(b) For other less viscous products. — Prep, soln with pi-
pets and vol. flasks to contain ca 0.4% (v/v) MeOH, dilg with
dioxane. Avoid excessive shaking of semiviscous products and
do not fill pipet above mark. (Use safety pipet filler to draw
liq. to mark and hold until transfer.) Wash pipet with dioxane
and add washings to soln.
If MeOH concn is unknown, prep. 2% soln. Prep, addnl
dilns as needed.
C. Determination
Inject portion std soln with 10 |ulL syringe. Note vol. AtR MA
(retention time relative to MeOH) ca 0.5, inject portion sample
soln. Note vol. At R MA ca 2 (from second injection), repeat
injection of std soln. At R MA ca 0.5 (from third injection),
repeat injection of sample soln. After MeOH from fourth in-
jection elutes, increase column temp, to 235° as rapidly as pos-
sible for time ca 4 X R MA until all dioxane (R MA ca 5) is re-
moved from column. Cool column to 160° and repeat sequence
for subsequent sample. Modify injection time if necessary to
sep. MeOH from other peaks. (Note: Injection sequence is used
only to save time; it need not be used if desired.)
D. Calculation
Det. retention areas for each MeOH peak by multiplying
peak ht by retention distance. Average retention areas for sam-
ple (RA) and for std (RA'). Presence of solv. in column changes
retention times, requiring use of retention areas in calcn.
% MeOH (w/v) in sample soln
- F x (RA/RA f ) x (V/V) x C x 0.79
where F = diln factor, C — % (v/v) std soln, V and V =
vol. sample and std soln injected, resp., and 0.79 = density
of MeOH.
Refs.: JAOAC 54, 558(1971); 55, 242(1972).
CAS-67-56-1 (methanol)
986.01 M-Nitrosodibutylamine
in Latex Infant Pacifiers
Gas Chromatographic Method
First Action 1986
A. Principle
Volatile AMNitrosamines are extd from cut-up latex pacifier
nipples with CH 2 Cl 2 . Ext is coned and subjected to high temp.
purge and trap, and A'-nitrosamines are eluted from trap and
detd by gas chromatgy with thermal energy analysis.
B. Reagents
Use all glass-distd solvs (Burdick & Jackson Laboratories,
Inc., or equiv.).
(a) N-Nitrosamine stock std solns. — (J) External stock std
soln. — 10 juLg/mL each of NDMA (JV-nitrosodi methyl amine),
NDEA (N-nitrosodiethylamine), NDPA (N-nitrosodipropylam-
ine), NDBA (iV-nitrosodibutylamine), NP.IP (JV-nitrosopiperi-
dine), NPYR (/V-nitrosopyrrolidine), and NMOR (/V-nitroso-
morpholine) in alcohol. (2) Internal stock std soln. — 10 jxg
NDPA/mL alcohol.
Caution: Volatile A'-nitros amines are extremely hazardous
compds. Carry out all manipulations involving handling neat
liqs or solns in adequately ventilated and filtered fume hood
or glove box.
(b) Mineral oil. — White, lightwt Saybolt viscosity 125/135
(No. 6358, Mallinckrodt Chemical Works).
(c) Nitrosation inhibitor. — 10 mg a-tocopherol/mL mineral
oil.
(d) Keeper solns. — (1) For K-D evaporation. — 80 mg min-
eral oil/mL CH 2 C1 2 . (2) For N evaporation. — 20 mg mineral
oil/mL isooctane.
C. Apparatus
(a) ThermoSorb/N !M cartridges. — Use as received for quant.
trapping of volatile N-nitrosamines (Thermedics, Inc., Div. of
Thermo Electron Corp., 470 Wild wood St, PO Box 2999,
Woburn, MA 01801).
(b) Variable temperature oil bath. — Thermostatically con-
trolled, capable of operating at 150 ± 3° and of moving ver-
tically with aid of laboratory jack (The Lab Apparatus Co.,
PO Box 42070, Cleveland, OH 44142).
(c) Soxhlet extraction apparatus. — (Kimble Glass Inc.). Al-
lihn condenser with 34/45 ¥ joint. Extn tube with 34/45 ¥
upper joint and 24/40 ¥ lower joint. Extn thimble, 25 x 85
mm boro silicate glass fitted with coarse porosity frit.
(d) Kuderna-Danish evaporative concentrator. — (Kontes
Glass Co.). 3-ball Snyder column with 24/40 I joints, 250
mL flask with 24/40 I joint and 19/22 ¥ lower joint, and 4
mL graduated concentrator tube with 19/22 ¥ joint.
(e) Gas chromatography — Hewlett-Packard Model 5710A,
or equiv., equipped with 6 ft x 4 mm id glass column packed
with 10% Carbowax 20M/2% KOH on 80-100 mesh Chro-
mosorb WAW (No. 1-1805, Supelco). Condition column
overnight at 215°. Operate at temp, program mode from 150
to 190° at 4°/min. Injection port temp. 250°. Carrier gas pre-
purified Ar at flow rate 40 mL/min. Interface GC app. to ther-
mal energy analyzer, (f), via Vs in. od stainless steel tube con-
nected to Swagelok fittings and operate at 170°.
(f) Thermal energy analyzer .—Model 502, Thermo Elec-
tron Corp., 115 Second Ave, Waltham, MA 02154, or equiv.
Operate pyrolysis chamber at 500° in GC mode. O flow to
ozonator, 10 mL/min. Keep cold trap at -150° using liq. N/
2-methylbutane slush bath. Pressure of reaction chamber, ca
0.9 torr. Record TEA detector response on Hewlett-Packard
3380 integrator.
(g) Purge and trap apparatus. — Fig. 986. 01 A contains fol-
lowing parts: (I) Ar gas cylinder and gauge (Air Products Spe-
cialty Gas, Tamaqua, PA 18252); (2) metering valve; (3) purge
gas manifold, 4-position; (4) Nalgene needle valve type CPE
(No. 6400-0125, Nalge Co., 75 Panorama Creek Dr, PO Box
20365, Rochester, NY 14602); (5) 18/7 g-g outer joints with
pinch clamps (No. 772398, The Wheaton Agency, A Div. of
Wheaton Industries, 1000 N Tenth St, Millville, NJ 08332);
(6) impingers, 50 mL graduated glass tubes with 24/40 J clear-
seal, grease-free joints 18/7 g-g ball joints, and 1 mm id noz-
zle ca 5 mm above bottom of impinger (No. 753463, Wheaton
Scientific); (7) variable scale flow-check, calibrated for purge
rate in mL Ar/min (No. 7083, Alltech Associates, Inc.). Bub-
ble meter for measuring gas flow rates for GC may be sub-
stituted.
AOAC Official Methods of Analysis (1990)
Hazardous Substances 235
Fig. 986.01 A — Diagram of purge and trap apparatus equipped
with 4 impinger tubes
Note: Do not use any rubber tubing, gaskets, O-rings, or
other items made of rubber in any part of this method.
D. Description and Use of Purge and Trap Apparatus
App. shown in Fig. 986. 01 A is designed for high temp,
purging and trapping of 7 volatile yV-nitrosamines from coned
sample ext/mineral oil mixt. on 4 samples simultaneously.
Cylinder contg prepurified Ar gas equipped with high pressure
regulator is used to supply 20 psig to flow-metering valve which
regulates final purge flow thru samples. Gas stream is diverted
into tubular stainless steel manifold, 250 X 20 mm od, contg
4 exit tubes spaced 50 mm apart and measuring 40 x 10 mm
od. Each of these tubes is coupled using 3 / 8 in. Tygon tubing
to Nalgene needle valves which serve dual purposes: as shut-
off valve when less than 4 samples are analyzed; and for mak-
ing minor adjustments in purge rate due to slight differences
in flow characteristics of impinger and cartridges. An 18/7 g-
g outer spherical joint is attached to Nalgene valve to permit
quick gas-tight connection to 18/7 g-g ball joint on impinger
inlet, using appropriate pinch clamp. As shown in Fig. 986. 01B,
impingers are assembled by inserting glass nozzle (1 mm id
ThermoSorb/N
^sample
Fig. 986.01 B— Close-up diagram of impinger tube fitted with
ThermoSorb/N cartridge
orifice) into sample mixt. and coupling 24/40 ¥ grease-free
male and female joints together to form leak-free seal. Once
sealed, Ar gas is allowed to purge thru sample mixt. , thru out-
let tube of impinger (see Fig. 986. 01B). Tygon tubing is used
to connect impinger outlet tube to inlet side marked "AIR IN"
of cartridge, which is std male Luer connector. Purged volatile
N-nitrosamines are then collected on sorbent contained in car-
tridge with Ar effluent exiting from female Luer connector.
Flow rate of Ar is measured directly from cartridge with vari-
able scale flow meter which has been previously calibrated for
flow rate of Ar gas (mL/min). Bubble meter can be substituted
for variable scale flow meter. Temp, of sample mixt. during
purge is controlled by immersing impinger up to sample vol.
mark (ca 25 mL line) in thermostatically controlled oil bath
capable of operating isothermal ly up to 150°. Gas manifold,
as well as each impinger, is secured by clamps to support grid;
therefore, oil bath is moved vertically in and out of position
for high temp, purge.
E. Extraction and Cleanup of Pacifier Samples
Accurately weigh 5 g from each sample into 250 mL r-b
flask and add 100 mL CH 2 C1 2 . Oil. internal stock std soln to
50 ng/mL with CH 2 C1 2 and spike contents of flask with 2 mL
dild std. Seal flask and let contents stand overnight (16—21 h)
at ambient temp.
Transfer ext and rubber pieces to glass extn thimble fitted
with coarse porosity glass frit in Sox h Jet extn app. Rinse 250
mL r-b flask with 25 mL CH 2 C1 2 and transfer rinse to Soxhlet
app. Ext rubber pieces for 1 h in app. at rate of 8 cycles/h.
Let cool and transfer Ch 2 Cl 2 ext to 250 mL K-D evaporator.
Rinse extn flask with two 10 mL portions of CH 2 C1 2 and com-
bine rinses with 125 mL ext. Add 1 mL keeper soln / and 2
or 3 boiling chips (Boileezers, Fisher Scientific Co.) to ext.
Evap. ext in K-D unit using 3-ball Snyder column on 55° water
bath until vol. is reduced to 3-4 mL.
Let K-D unit cool to room temp., allowing excess sol v. in
Snyder column to rinse down walls of unit into 4 mL K-D
tube (total = 3-4 mL). Remove 250 mL reservoir and 3-ball
Snyder column, reduce vol. of ext to 2 mL in same K-D tube
under gentle stream of N (ca 50 mL/min), and transfer 2 mL
ext using disposable Pasteur pipet with two 1 mL mineral oil
rinses to 50 mL purge and trap app. contg 20 mL mineral oil
and 1 mL of 10 mg/mL of a-tocopherol in mineral oil as ni-
trosation inhibitor.
Assemble purge and trap app. and connect cartridges to exit
tubes with Tygon connector. Adjust Ar flow rate to 400 mL/
min thru cartridge ±5% (i.e., 380-420 mL Ar/min). Note:
Check flow rate intermittently during purging, especially within
first 15 min because of initial increase in temp, of sample.
Immerse purge tubes (up to sample line) or to ca 25 mL mark
in 150 ± 3° oil bath for 1.5 h. Remove cartridge and tightly
cap. (Note: This is good stopping point; cartridge can be eluted
on following day if necessary.)
Elute cartridge using 10 or 20 mL glass Luer-Lok syringe
connected to female Luer adapter (air exit side) with 20 mL
acetone-CH 2 CI 2 (1 + 1, v/v). Collect eluate in 30 mL culture
tube. (Note: 30 mL tube(s) should be scored with file or piece
of tape placed at 5 mL vol. mark.)
Evap. ext to ca 5 mL and then transfer with three 1 ml
rinses of CH 2 C1 2 to 10 mL graduated tube. Add 0.5 mL keeper
soln 2. Evap. sample (vol. = 8.5 mL) to 2 mL under gentle
stream of N. (Note: If 2 mL sample cannot be analyzed same
day as evapd, it is advantageous to refrigerate sample at larger
vol., i.e., 4-5 mL, and evap. next day before analysis by GC-
TEA.)
Analyze 2 mL sample by injecting 8 fxL aliquot into GC-
TEA.
236
Hazardous Substances
AOAC Official Methods of Analysis (1 990)
F. Quantitation
Use internal std technic. Dil. external stock std soln with
CH 2 C1 2 to 50, 100, and 200 ng/mL to be used as working stds
for analysis. Inject 8 |jlL into GC-TEA to det. responses (peak
hts) of NDPA and other nitrosamines for use in internal stdzn
calcn. Inject 8 |xL of each 2 mL sample ext into GC-TEA.
Det. responses (peak hts) of NDPA and any other Af-nitrosa-
mines detected for use in internal stdzn calcn. Calc. results as
follows:
ppb TV-Nitrosamine X = [(PH X ) X (F x ) x (100 ng NDPA)]/
[(PH NDPA ) x (F NDPA ) X (g sample)]
where PH X = peak ht in mm of yV-nitrosamine X in sample;
F x = ng TV-nitrosamine X/mL in external std soln divided by
peak ht in mm of 7V-nitrosamine X in external std soln; 100
ng NDPA = total ng NDPA (internal std) added to sample;
PH NDPA = peak ht in mm of NDPA (internal std) in sample;
Fndpa = ng NDPA/mL in external std soln divided by peak
ht in mm of NDPA in external std soln; g sample = g rubber
sample analyzed.
Ref.: J AOAC 69, 504(1986).
930.11 Phenol In Hazardous Substances
Colorimetric Method
Final Action
Method i
(Applicable to com. cresols, saponified cresol solns, coal tar
dips, and disinfectants, and to kerosene solns of phenols in
absence of salicylates or f$-naphthol)
A. Reagents
(Caution: See safety notes on nitric acid, formaldehyde, and
mercury.)
(a) Dilute nitric acid. — Aerate HN0 3 until colorless and dil.
1 vol. with 4 vols H 2 0.
(b) Millon reagent. — To 2 mL Hg in 200 mL erlenmeyer
under hood, add 20 mL HN0 3 . After first violent reaction,
shake as needed to disperse Hg and maintain action. After ca
10 min, when action practically ceases even in presence of
undissolved Hg, add 35 mL H 2 0, and if basic salt seps, add
enough dil. HN0 3 to dissolve it. Add 10% NaOH soln drop-
wise with thoro mixing until curdy ppt that forms after adding
each drop no longer redissolves but disperses as permanent
turbidity. Add 5 mL dil. HN0 3 and mix well. Prep, fresh daily.
Millon reagent is dangerously poisonous and should not be
transferred with ordinary pipet and mouth suction unless pro-
tective trap is used.
(c) Phenol std soln. — Dissolve weighed amt pure phenol
(congealing point >40°) in enough H 2 to make 5:1% soln.
On day it is to be used, dil. to make 0.025% aq. soln (final
std).
(d) Formaldehyde soln. — Dil. 2 mL 37% HCHO soln to
100 mL with H 2 0.
(e) Methyl orange indicator. — 0.5% aq. soln.
B. Apparatus
(a) Nessler cylinders. — 50 mL tall-form, matched.
(b) Test tubes. — Approx. 180 x 20 mm, with rubber stop-
pers, marked at 25 mL.
(c) Water bath for heating test tubes. — Beaker contg disk
of wire gauze raised ca 2.5 cm from bottom may be used.
C. Preparation of Sampie
(a) Commercial cresol. — Weigh by difference ca 2.5 g
sample into 250 mL vol. flask, dissolve in 10 mL 10% NaOH
soln, and dil. to vol. with H 2 0.
(b) Saponified cresol solns, coal tar dips and disinfectants,
kerosene solns of phenols, etc. — Weigh by difference ca 5 g
sample (or use 5 mL and calc. wt from density) into 250 mL
vol. flask and dil. to vol. with H 2 0. With products consisting
largely of kerosene, bring H 2 level to mark and take aliquots
from aq. portion only.
D. Determination
Transfer 5 mL aliquot prepd soln to 200 mL vol. flask and
promptly dil. to ca 50 mL. Add 1 drop Me orange, (e), and
then dil. HN0 3 until soln is practically neut. Dil. to vol. and
shake well.
Place 5 mL dild soln in each of 2 marked test tubes; in each
of 2 addnl test tubes place 5 mL std phenol soln. Flow 5 mL
Millon reagent down side of each tube, mix, and place tubes
in boiling H 2 bath; continue boiling exactly 30 min, cool
immediately and thoroly by immersion in bath of cold H 2
^10 min, and add 5 mL dil. HN0 3 to each tube.
Mix well and add 3 mL HCHO soln to one of each pair of
tubes. Dil. all tubes to 25 mL mark with H 2 0, stopper, shake
well, and let stand overnight. (Tubes contg HCHO fade to yel-
low; others show orange or red color.)
Pipet 20 mL from each of the 2 phenol tubes to 100 mL
vol. flasks; add 5 mL dil. HN0 3 to each, dil. to vol., and mix.
(Red flask contains "phenol std," yellow flask "phenol blank.")
Transfer these solns to burets. Pipet 10 mL of each sample
soln into Nessler tubes. (The orange or red constitutes the "un-
known" and the yellow the "sample blank." Mark each Ness-
ler tube distinctly to avoid confusion.) To "sample blank" tube
add measured amt of "phenol std" and add same vol. "phenol
blank" to "unknown." Agitate thoroly (aided by insertion of
rubber stoppers, if necessary), and compare colors. When tubes
are brought to match , each mL phenol std used = 1 % phenol
if sample weighing exactly 5 g was used, or 2% if exactly 2.5
g was used.
Note. — Take following precautions: Pair of phenol tubes pro-
vides enough final solns to assay several unknowns, but all the
latter must have accompanied phenol solns thruout entire pro-
cess with identical reagents and treatment. If end point is in-
advertently overrun it is possible to work back to it, but since
mistakes may be made in this operation it is better to repeat
comparison on fresh portions from original tubes. Too much
delay in matching tubes must be avoided after titrn is started,
otherwise excess HCHO present in blanks may have time after
mixing to affect intensity of red color.
Refs.: USDA Bull. 1308, p. 17. JAOAC 13, 160(1930).
Method II
E. Determination
(Applicable to detn of phenol in presence of salicylates)
Weigh by difference 10 g sample into separator (or use 10
mL and calc. wt from density of sample). Add 50 mL kerosene
and ext with three 100 mL portions H 2 0. Filter aq. exts thru
wet filter into 500 mL vol. flask, dil. to vol. with H 2 0, and
proceed as in 930. 11D.
When tubes are brought to match, each mL phenol std used
= 1% phenol if sample weighing exactly 10 g was used.
Ref.: Ind. Eng. Chem. Anal. Ed. 1, 232(1929).
CAS-108-95-2 (phenol)
9. HHetais and Other Elements at Trace Levels in Foods
Stephen G. Capar, Associate Chapter Editor
Food and Drug Administration
MULTIELEMENT METHODS
986.15 Arsenic, Cadmium, Lead,
Selenium, and Zinc in Food
Multielement Method
First Action 1986
Final Action 1988
A. Principle
Sample is digested with HN0 3 in closed system. Cd and Pb
are detd by anodic stripping voltammetry (ASV). As, Se, and
Zn are detd by atomic absorption spectrophotometry (A AS)
after generation of metal hydrides (for As and Se).
(Caution: See safety notes on pipets, nitric acid, perchloric
acid, sodium hydroxide, and arsenic trioxide.)
B. Apparatus
(a) Polarograph. — With anodic stripping accessories. Typ-
ical operating parameters for Model 174 with hanging drop Hg
electrode are: Scan rate, 5 mv/sec; scan direction, +; scan
range, 1.5 v; initial potential, —0.7 v; modulation amplitude,
25 mv; operation mode, differential pulse; display direction,
"-"; drop time, 0.5 sec; low pass filter, off; selector, off;
pushbutton, initial; output offset, off; and current range, 5-10
|mamp, or as needed.
Other instruments and electrodes such as wax impregnated
graphite may be used according to manufacturer's directions.
(b) Atomic absorption spectrophotometer. — Perkin-Elmer
Corp. Model 403, or equiv., with Zn, As, and Se hollow cath-
ode lamps or As and Se electrodeless discharge lamps, 3 slot,
10 cm Boiling burner head, air-C 2 H 2 and H-N-entrained air
flames, and deuterium arc background corrector.
(c) Decomposition vessel. — 70 m'L. See 974. 14A.
(d) Hydride generator. — See Fig. 986. 15A. Constructed from
following: (V) Flat bottom flask. — Borosilicate glass, 50 mL
(Corning No. 5160, or equiv.). (2) Stopper fittings. — Two-
hole (1 thru center) No. 9 rubber stopper, fitted with gas outlet
tube of 100 mm X Vs" (3 mm) id polyethylene tubing thru
center hole. Place bottom of gas outlet tube thru cut off bottom
1" segment of 5 /s" polyethylene test tube with hole in bottom
so that 3 mm of tube protrudes thru test tube. Insert thru sec-
ond hole 75 mm x Vs" (3 mm) id polyethylene tubing as N
inlet tube. Seal bottom end of tube with burner and then punch
several holes at sealed end with 21 gage needle. Alternatively,
prep, similarly 500 mm x Vie" 0-5 mm) id polyethylene tub-
ing and hold in place in stopper with hole-thru septum. Con-
nect other end of tubing to AA spectrophotometer with 500
mm Tygon tubing by cutting auxiliary line at ca 75 mm from
mixing chamber and attaching tubing. (3) Generator mount. —
(Optional) 64 mm x 0.5" id pipe secured to laboratory ring
stand by means of clamp holder. Insert extension clamp into
pipe and attach another clamp to back of clamp to hold clamp
in place and to serve as handle; clamp is now free to rotate ca
180°. Attach rubber stopper of hydride generator to extension
clamp with stiff wire and position just at level of clamp jaws.
In operation, place flask of generator between jaws of exten-
sion clamp, insert stopper firmly into neck of flask, then tighten
clamp jaws around neck of flask. Unit can be rapidly and uni-
formly inverted by rotating handle on extention clamp, thus
allowing sample and Na borohydride to mix rapidly and re-
producibly.
(e) Pipets. — 50 and 100 \xL Eppendorf micropipets, or equiv.
C. Reagents
(Use double distd H 2 0. Rinse all glassware with HN0 3 (1 +
1) followed by thoro H 2 rinse. Decontaminate digestion ves-
sels by digesting with reagents to be used in digestion. Rinse
thoroly with the H 2 0. Decontamination is necessary to reduce
blanks, especially for Pb, to acceptable level.)
(a) Acids. — (J) Nitric acid. — Redistd. (2) Perchloric acid. —
70%, double vac. distd (G. Fredrick Smith Chemical Co., or
equiv.). (3) Hydrochloric acid. — 8M. Oil. 66 mL HCl to 100
mL with H 2 0.
(b) Nitrate soln. — Equimolar soln of KN0 3 and NaN0 3 .
Dissolve 54.3 g KN0 3 and 45.7 g NaN0 3 (available as Su-
prapur®, Nos. 5065 and 6546, resp., EM Science) in H 2 in
200 mL vol. flask, dil. to vol., and mix. To further purify,
add 1-2 drops NH 4 OH to 25 mL aliquot and ext with 2 mL
10 |JLg dithizone/mL CC1 4 until lower solv. layer is colorless.
(c) Magnesium solns. — (/) Magnesium chloride soln. — 37.5
mg/mL. Dissolve total of 3.75 g MgO, USP, by adding small
amts at time to 100 mL 8M HCl. (2) Magnesium nitrate soln. —
75 mg/mL. Mix 3.75 g MgO, USP, with ca 30 mL H 2 0,
slowly add HN0 3 to dissolve (ca 10 mL), cool, and dil. to 50
mL with H 2 0.
(d) Sodium borohydride soln. — 4.0 g NaBH 4 /100 mL 4%
NaOH.
(e) Potassium iodide soln. — Dissolve 20 g KI in H 2 and
dil. to 100 mL. Prep, just before use.
(f) Metal powders. —Purity: 99.99 + % Cd, Pb, Zn; 99.99%
Se. Alfa Products, Morton Thiokol, Inc., 152 Andover St,
Dan vers, MA 01923.
(g) Cadmium std solns. — (I) Stock soln. — 1 mg/mL. Dis-
solve 1.000 g Cd powder in 20 mL HN0 3 (1 + 1) in I L vol.
flask, and dil. to vol. with H 2 0. (2) Working soln. — 2 fig/
mL. Pipet 10 mL stock soln into 100 mL vol. flask, and dil.
to vol. with H 2 0. Pipet 2 mL dild soln into 100 mL vol. flask
and dil. to vol. with H 2 0.
(h) Lead std solns. — (/) Stock soln. — 1 mg/mL. Dissolve
1.000 g Pb powder in 20 mL HN0 3 (1 + 1) in 1 L vol. flask,
and dil. to vol. with H 2 0. (2) Working soln. — 5 (xg/mL. Pipet
1 mL stock soln into 200 mL vol. flask and dil. to vol. with
H 2 0.
(i) Zinc std solns. — (/) Stock soln. — 1 mg/mL. Dissolve
1.000 g Zn powder in 20 mL HCl (I + 1) in 1 L vol. flask,
and dil. to vol. with H 2 0. (2) Working solns. — 0.2, 0.5, 1.0,
and 1.5 jxg/mL. Pipet 1 mL stock soln into 100 mL vol. flask
and dil. to vol. with H 2 0. Pipet 2, 5, 10, and 15 mL dild soln
into sep. 100 mL vol. flasks, each contg 1 mL HCI0 4 , and
dil. to vol. with H 2 0.
(j) Arsenic std solns. — (/) Stock soln. — Dissolve 1.320 g
As 2 3 in min. vol. 20% NaOH in 1 L vol. flask, acidify with
HCl (1 + 1), and dil. to vol. with H 2 0. (2) Working solns. —
1, 2, 3, 4, and 5 |xg/mL. Pipet 10 mL stock soln into 100
237
238 Metals and Other Elements
AOAC Official Methods of Analysis (1990)
FIG. 986.1 5A— Hydride generator: 1, polyethylene tubing; 2,
rubber stopper; 3, flame sealed polyethylene tubing with holes
punched at one end; 4, reagent cup; 5, sodium borohydride
solution; 6, sample solution; 7, nitrogen inlet from ''auxiliary"
line of AAS
mL vol. flask, and dil. to vol. with H 2 0. Pipet 1, 2, 3, 4, and
5 mL dild soln into sep. 100 mL vol. flasks, and dil. to vol.
with H 2 0.
(k) Selenium std solns. — (1) Stock soln. — 1 mg/mL. Dis-
solve 1.000 g Se powder in min. vol. HND 3 in 200 mL beaker
and evap. to dryness. Add 2 mL H 2 and evap. to dryness.
Repeat addn of H 2 and evapn to dryness twice. Dissolve in
min. vol. HC1 (1 + 9) in 1 L vol. flask, and dil. to vol. with
HC1 (1 + 9). (2) Working solns. — 1, 2, 3, 4, and 5 |xg/mL.
Pipet 10 mL stock soln into 100 mL vol. flask and dil. to vol.
with H 2 0. Pipet 1, 2, 3, 4, and 5 mL dild soln into sep. 100
mL vol. flasks and dil. to vol. with H 2 0.
D. Closed System Digestion
(Do not exceed manufacturer's specifications of 0.3 g solids
with 70 mL vessel. Proceed cautiously with new or untried
uses. Let such samples stand with HN0 3 overnight or heat on
hot plate cautiously until any vigorous reaction subsides. Then
proceed with closed vessel digestion. Open vessel in hood since
N oxides are released.)
Weigh 0.3 g sample (dry basis) into decontaminated decom-
position vessel, add 5 mL HN0 3 , close vessel with lid, and
heat in 150° oven 2 hr. Cool in hood, remove vessel from
jacket, and transfer contents to 10 mL vol. flask. Add 4 mL
H 2 to vessel, cover with lid, and while holding lid tightly
against rim, invert several times, and add rinse to flask. Dil.
to vol. with LLO and mix.
E. Anodic Stripping Voltammetry
(For Cd and Pb)
Pipet aliquot of digested sample soln into decontaminated
50 mL Vycor crucible and add 2 mL nitrate soln, (b). Conduct
reagent blank simultaneously. Heat on hot plate at low heat to
dryness; then increase heat to max. (ca 375°). Nitrate salts will
melt and digest org. matter in 15-20 min. Place crucibles in
450° furnace to oxidize any remaining carbonaceous matter (10-
20 min). Digestion is complete when melt is clear. Let cool,
add 1 mL HNO ? (1 + 1) to solidified melt, and heat on hot
plate to dryness to expel carbonates and nitrites and to control
acidity. Dissolve in 5.0 mL HN0 3 (0.5 mL/L), warming on
hot plate to speed soln. Transfer to polarographic cell with 5.0
mL H 2 0. Bubble O-free N thru soln 5 min; then direct N over
soln.
Set dial for Hg drops at 4 jxm divisions. Stir soln with mag.
stirrer at const and reproducible rate so Hg drop is not dis-
turbed. Slide selector switch to "Ext. Cell" and measure time
for 120 sec with stopwatch. Turn off stirrer and let stand 30
sec. Press "Scan" button to obtain peaks corresponding to Cd
and Pb at ca —0.57 and -0.43 v, resp., against satd calomel
electrode.
Add known vols of each std to sample soln in cell from
Eppendorf pipet. Amts added should be ca 1.x, 2x, etc. of
amt metal present initially in cell, and each addn should not
change original vol. significantly. After each addn, bubble N
thru soln briefly and perform deposition and stripping opera-
tions exactly as for original soln. Plot jxg metal added on x-
axis against peak ht on y-axis. Extrapolate linear line to x axis
to obtain jxg metal in cell.
(jig metal/g sample
= \{M - M')/g sample]
X (10/mL aliquot taken)
where M and M' ~ |uLg metal from std curve for sample and
blank, resp.
F. Atomic Absorption Spectrophotometry
(For As, Se, and Zn)
(a) Arsenic. — Pipet aliquot digested sample soln into de-
contaminated 50 mL round, flat-bottom borosilicate flask, and
add 1 mL Mg(N0 3 ) 2 soln, (e)(2). Heat on hot plate at low heat
to dryness; then increase heat to max. (ca 375°). Place flask
in 450° furnace to oxidize any carbonaceous matter and to de-
compose excess Mg(N0 3 ) 2 (S:30 min). Cool, dissolve residue
in 2.0 mL 8M HC1, add 0.1 mL 20% Kl to reduce As +3 to
As t3 , and let stand >2 min. Conduct reagent blank with sam-
ple.
Prep, stds as follows: To six 50 mL flasks (same type as
used for sample) add 2.0 mL MgCI 2 soln, (c)(7), and to 5
flasks add 50 u,L aliquots of respective working std solns so
that series will contain 0, 0.05, 0.1, 0.15, 0.20, and 0.25 |xg
As. (Other amts may be used depending on sensitivity of sys-
tem.) Add 0.1 mL 20% Kl to each flask, mix, and let stand
^2 min.
Connect generator to instrument as shown in Fig. 986. 15B
and adjust pressures and flows as in Table 986.15. Operate
instrument according to manufacturer's instructions, with lamp
in place and recorder set for 20 mm/min.
Add 2.0 mL 4% NaBH 4 soln to reagent dispenser of gen-
erator, and insert rubber stopper tightly into neck of flask contg
sample or std. With single rapid, smooth motion, invert flask,
letting soln mix with sample or std. (This operation must be
performed reproducibly.) Sharp, narrow A peak will appear
immediately. When recorder pen returns to baseline, remove
stopper from flask, and rinse reagent dispenser with H 2 from
squeeze bottle; then suck out H 2 0. Proceed with next sample
or std. When series is complete, rinse glassware thoroly.
Plot calibration curve of |xg As against .A, and obtain \xg As
in sample aliquot from this curve. Correct for reagent blank.
(b) Selenium. — Proceed as in (a), using Se lamp and stds,
but omit addn of Kl soln. Kl will reduce Se to elemental state
and cause loss of signal. Instead, cover flask with small watch
glass and place on steam bath 10 min, and cool to room temp.
(c) Zinc. — Pipet 1 mL aliquot digested sample soln into de~
AOAC Official Methods of Analysis (1990)
Multielement Methods
239
FIG. 986.1 5B — Hydride generator and mount connected to auxiliary line of spectrophotometer. Test tube acid trap connected be-
tween generator and instrument is not included in method
Table 986.15 Flow Rates and Pressures for Arsenic and
Selenium Determinations
Gas
Tank,
psi
AA Control
Box, psi
Perkin-Elmer Model 403
Flowmeter, divisions
20
40
10
30
20 (4 L/min)
25 (10 L/min)
contaminated 25 mL erlenmeyer, and add 0. 1 mL HC10 4 . Heat
on hot plate to white fumes of HC10 4 . Sample should be com-
pletely digested as indicated by clear, practically colorless soln.
If sample chars, add 0.5 mL portions HN0 3 and again heat to
white fumes. Finally, heat just to dryness but do not bake.
Cool, and dissolve residue in 3.0 mL HC10 4 (1 + 99).
Operate instrument in accordance with manufacturer's in-
structions, using air-C 2 H 2 flame, and measure A of sample and
stds, (i)(2). Dil. sample soln with HC10 4 (1 + 99), if soln is
too coned. Plot calibration curve of fig Zn against A, and ob-
tain fxg Zn in sample aliquot from this curve. Correct for re-
agent blank.
Ref.: J AOAC 63, 485(1980).
CAS-7440-38-2 (arsenic)
CAS-7440-43-9 (cadmium)
CAS-7439-92-1 (lead)
CAS-7782-49-2 (selenium)
CAS-7440-66-6 (zinc)
982.23 Cadmium and Lead in Food
Anodic Stripping Voitam metric Method
First Action 1982
Final Action 1988
(Not applicable to fats and oils)
(Caution: See safety notes on pipets and nitric acid.)
A Principle
Sample is dry-ashed with K 2 S0 4 and HNO ? at ca 500°. Pb
and Cd are detd by anodic stripping voltammetry (ASV). Estd
quantitation limits, based on 10 g sample, are 0.005 ppm Cd
and 0.010 ppm Pb.
B. Apparatus
(Thoroly soak all glassware and plasticware in 20% (v/v) HN0 3
for >24 h and rinse with distd, deionized H 2 0.)
(a) Voltammetric analyzer. — Capable of ASV and equipped
with necessary accessories, i.e., cells, electrodes, recorders,
Hg capillaries, micrometer or similar device for adjusting drop
size, stirring motor, etc. (EG&G Princeton Applied Research
Corp., PO Box 2565, Princeton, NJ 08540, Models 174A,
315A, and 303, or equiv.., for differential pulse anodic strip-
ping voltammetry (DPASV) at hanging Hg drop electrode; En-
vironmental Sciences Associates, 45 Wiggins Ave, Bedford,
MA 01730, Model 3010A, or equiv., for linear sweep anodic
stripping voltammetry (LSASV)).
(b) Ashing vessels. — 150-250 mL quartz, Vycor, or Pyrex
beakers equipped with suitable glass covers (Fisher Scientific
Co., No. 2-609A, or equiv.). Quartz is preferred. Vycor or
Pyrex may be used if quartz beakers are not available. Note:
For best results, quartz beakers should be fire-polished to re-
tard etching.
(c) Drying oven. — Controllable within range 50-150° with
<5° variation.
(d) Furnace. — Controllable within range of 100-1000° with
<5° variation. Check calibration of oven temp, control to en-
sure accurate temps. Furnace must be operated in suitable fume
hood.
(e) Controllable hot plate. —Corning Glass Works, Corn-
ing, NY, PC-35 (replacement Model PC 300), or equiv.
(f) Micropipets. — 10 thru 100 \xL (Eppendorf, or equiv.).
C. Reagents
Note: Use only distd, deionized FLO.
(a) Nitric acid. — J.T. Baker Inc. No. 9598, or equiv.
(b) Potassium sulfate ashing soln. — 10 g/100 mL. Dis-
solve 50.0 g K,S0 4 (J.T. Baker Inc. No. 3278, or equiv.) in
400 mL H 2 contg 10 mL HN0 3 . Dil. to 500 mL with H 2 C
(c) Nitrogen. — Prepurified, FLO-pumped.
(d) Electrolyte soln. — 1.7M in HOAc, 1.25M in Na ace-
240
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
tate trihydrate, and 0.1M in tartaric acid. Dissolve 170.0 g
NaOAc.3H 2 (ACS) in 300 mL H 2 0. Add 97 mL glacial HOAc
and 15 g tartaric acid (ACS). Dil. to 1 L with H 2 0. pH should
be 4.7 ±0.1.
(e) Cadmium std soln. — 1.0 mg/mL. Dissolve 1.000 g Cd
(99.99%) in 10 mL HN0 3 in 1 L vol, flask. Dil. to vol. with
H 2 0.
(f) Lead std soln.—\.0 mg/mL. Dissolve 1.000 g Pb
(99.99%) in 10 mL HN0 3 in 1 L vol. flask, Dil. to vol. with
H 2 0.
(g) Working std solns. — Prep, either sep. or mixed working
std soln for Cd and Pb in the range 0.1-10 |xg/mL from std
solns (e) and (f) by dissolving appropriate aliquots in 1% (v/
v) HN0 3 .
Note: Electrolyte soln (d) and K 2 S0 4 soln (b) may require
further cleanup for sufficiently low reagent blanks. For stated
quantitation limits, analyte concns in final cell solns (electro-
lyte and sample solns) of reagent blank should not be >0.5
ng Cd/mL and >1 ng Pb/mL. Controlled potential electrol-
ysis is recommended means of cleaning reagents.
D. Preparation of Sample
Note: Laboratory contamination control is important. Take
all precautions possible to avoid contamination of samples, re-
agents, and equipment. Prep, at least 3 control reagent blanks
which include any addnl H 2 and HN0 3 used for sample ash-
ing. Carry control reagent blanks thru entire method.
Weigh 5.0—10.0 g homogenized sample into ashing vessel
(b). Use 5.0 g for dry materials such as cereals. Add 5.0 mL
K 2 S0 4 ashing soln (b) and mix thoroly, using glass stirring
rod. If needed, add H 2 to ensure sample and ash aid are well
mixed. Cover ashing vessel with glass cover and dry in 110-
120° oven (c) until thoroly dry (usually 2-3 h or, if desired,
overnight). Place vessel in cold furnace (d) and set tempera-
ture at 500-550°. Caution: Do not heat >500° if using Pyrex
beakers, and avoid excessive overshooting of temp. Maintain
set temp. >4 h (may be ashed overnight). Remove vessel from
furnace, and cool. Ash should be white and essentially carbon-
free.
Wash down sides of vessel with min. amt. H 2 and add 2.0
mL HN0 3 . Use glass stirring rod to break up solid particles.
Dry thoroly on hot plate (e) at low setting. If samples such as
sugars and cereals splatter on hot plate during HN0 3 treat-
ment, dry under IR lamp instead. Increase hot plate setting to
medium for several minutes to ensure dryness. Return vessel
to 500° furnace 30 min. Cool; if necessary, repeat HN0 3 treat-
ment using 1 mL increments of HNO3, until white, C-free ash
is obtained.
Add 1.0 mL HNO3 and 5 mL H 2 to vessel, swirl to dis-
solve, and let stand 5 min. If residue remains undissolved,
warm gently on 80-90° hot plate not >5 min. Minimize pos-
sible Sn(II) formation by heating dil. acid soln as little as pos-
sible. Small amt of white, siliceous-like ppt may remain un-
dissolved. Cool, and quant, transfer sample to 50 mL vol. flask
with aid of H 2 0. Dil. to vol. with H 2 and mix well. Let stand
to allow any ppt present to settle. Do not filter. Use clear su-
pernate to det analytes by either DPASV or LSASV below.
E. Differential Pulse Anodic Stripping Voltammetry
Transfer 5.0 mL aliquot of sample soln to electrolysis cell
containing Teflon-coated stirring bar and add 5,0 mL electro-
lyte soln (d) to cell. (Aliquot vol. may be varied as long as
1:1 ratio is maintained between sample soln and electrolyte.)
pH of cell soln should be 4.3 ± 0.3. Room temp, should be
constant (±l°/2 h) and between 20 and 30°. Purge soln 5 min
with N (c). Adjust gas inlet to let N flow gently above and
across soln surface. If hanging Hg drop electrode is used, add
fresh drop of Hg to capillary tip with micrometer or similar
device to ensure reproducibility of drop. Turn on stirrer motor
and electrolyze soln at —0.8 V vs satd calomel electrode (SCE)
or Ag/AgCl electrode. Deposition time may vary with instru-
ment (see manufacturer's instructions). When using PAR 174
polarographic analyzer, 1-2 min is sufficient, depending on
level of analytes of interest in cell soln. Stop stirring and let
soln equilibrate 30 s. Linerarly increase applied voltage an-
odically. Follow manufacturer's instructions for rate of scan,
e.g., 2-6 mV/s. Measure wave ht at peak potentials for Cd
at -0.62 ± 0.05 V and for Pb at -0.45 ± 0.05 V vs SCE or
Ag/AgCl. For widely varying concns of Cd and Pb, change
current sensitivity to appropriate range by momentarily stop-
ping stripping scan at end of Cd peak, switching to appropriate
sensitivity setting for Pb, and then continuing scan before Pb
peak begins.
Quantitate total amts of Pb and Cd in cell soln by using
method of std addns in cell as follows: Record voltammogram
from known vol. of cell soln. From working std soln (g), add
known amts of Pb and Cd, using appropriate micropipets (f )
and being certain to add amt of each element sufficient to gen-
erate peak hts ca twice those given by sample cell soln. Repeat
with 2 more similar addns of working std soln to cell soln.
For each analyte, plot |xg added on Jt-axis vs peak ht in \xA
current on v-axis. Extrapolate linear plot to x-axis intercept to
det. total amt of analyte in sample aliquot. If available, use
computer program based on method of least squares to calc.
regression line and det. amt of analyte in sample aliquot. Sim-
ilarly, det. amt of each analyte in reagent blank aliquots, using
same vol. of aliquots for reagent blank as for sample.
Calc. ppm analyte in sample as follows:
ppm (|xg/g) = [(B - Q/A] x (50/W0
where A — mL sample soln taken for analysis; B — \xg analyte
in sample soln aliquot; C - av. \xg analyte in reagent blank
soln aliquots; and W = total g sample.
F. Determination by Linear Sweep Anodic Stripping
Voltammetry
Transfer 2.0 mL aliquot of sample soln to electrolysis cell
and add 3.0 mL electrolyte (d). pH of cell soln should be 4.3
±0.3. Deposit elements of interest onto composite Hg graph-
ite electrode (CMGE) at -0.9 V vs Ag/AgCl ref. electrode
for 30 min. Bubble N through cell soln during entire deposi-
tion period. Linearly increase applied voltage anodic ally at 60
mV/s from -0.9to-0.2Vvs Ag/AgCl ref. electrode. Mea-
sure peak current (julA) for each analyte.
Run reagent blank in same manner using same size aliquot
as for sample and det. peak current (pA) for each analyte. For
each analyte, make std addn to cell soln and measure peak
current (pA). Calc. conversion factor, u-g/uA, for each an-
alyte as |xg of addn divided by diff . between peak current be-
fore and after addn of analyte std. Verify conversion factors
periodically. Multiply sample peak current (fxA) by conversion
factor to det. |xg of each analyte in sample soln aliquot. Calc.
ppm, using equation in 982.23E.
G. Interference
Tl may interfere with Pb detn, but its occurrence in food is
unlikely. If Tl interference is suspected, treat as follows: Transfer
5.0 mL aliquot of sample soln to electrolysis cell and make
basic with 3.0 mL NaOH. Det. elements of interest in this soln
by ASV in the usual manner. Plating potential is —1,0 V vs
SCE or similar ref. electrode. Strip deposited elements by an-
odically scanning from -1.0 to -0.3 V vs SCE. In this man-
ner, Cd and Pb peaks shift to -0.78 ± 0.05 V and -0.73 ±
0.05 V vs SCE, resp. Tl peak remains at -0.47 V vs SCE.
Ref.: JAOAC 65, 970, 978(1982); 70, 295(1987).
AOAC Official Methods of Analysis (1990)
Multielement Methods
241
CAS-7440-43-9 (cadmium)
CAS-7439-92-1 (lead)
Leachable Cadmium and Lead from Foodware
The following 2 methods are applicable to detn of Cd and Pb
from foodware contact surfaces used to cook, serve, or store
food. 973.32 is applicable to ceramic, glass, enameled, or other
ware used to serve or store food at room temp, or below. 984.19
is applicable to ceramic, glass, enameled, or other ware in
which food is cooked before serving. Decision of which method
to use should be based on how ware being tested will be used
in practice.
984.19 Cadmium and Lead
in Cookware
Hot Leach Atomic Absorption Method
First Action 1984
Final Action 1986
WHO-AOAC Method
A. Principle
Enameled and ceramic cookware contg 4% acetic acid is
heated by elec. hot plate (or by internal heating elements, if
present, that are not exposed to the leach solution) to produce
slow boil or simmering of sol v. soln for 2 h. Pb and Cd in
extg solv. are detd by AAS.
B. Apparatus
See 973.32A plus the following:
(a) Hot plate.— Thermolyne Model HP-A1915B (Barn-
stead /Thermolyne Corp., 2555 Kerper Blvd, Dubuque, IA
52001), or equiv.
(b) Variable transformer. — Cat No. 09-521-100 (Fisher
Scientific Co.), or equiv.
C. Reagents
See 973.32B. Use only deionized distd H 2 0.
Plus (e) Acetic acid. — Glacial.
D. Cleaning of Laboratory Glassware
After normal cleaning, soak all glass and plastic ware used
to prep., transfer, or store anal, solns in HN0 3 -H 2 (4 -f 6)
^24 h; thoroly rinse with H 2 before use.
E. Preparation of Standard
See 973.32B(c) and (d).
F. Extraction
Samples of ware must be free of grease or other material
which could influence test. Gently wash sample with detergent
soln, using pad of absorbent cotton. Rinse ware thoroly with
H 2 and let drain dry.
Det. total vol. required to fill ware to overflowing or to cover
rest, if one is present.
Fill ware to 2 / 3 total vol. with H 2 0; cover with self-cover or
clean sheet of opaque borosilicate glass to prevent evapn of
soln. When leach soln is to be analyzed for Cd, ensure that
light is excluded from test surface. Heat on hot plate adjusted
by variable transformer to produce simmer or slow boil of
leaching soln, or use internal heating element, if present. Also
use variable transformer to prevent excessively rapid boiling
in ware containing heating elements. If contained heating ele-
ment is not able to produce temp, high enough to boil soln,
then highest temp, reached is test temp.
When boiling or highest temp, has been reached, add suf-
ficient glacial acetic acid to make soln 4% acetic acid, cover,
and continue heating 2 h.
At end of 2 h, re-establish initial vol. of solv. with 4% acetic
acid. Dip-stick (glass rod marked for depth of soln required)
is useful for replacing losses. Stir thoroly and remove test sam-
ple at once.
G. Determination
See 973.32D.
Ref.: JAOAC66, 610(1983).
CAS-7740-43-9 (cadmium)
CAS-7439-92-1 (lead)
973.32 Cadmium and Lead
in Earthenware
Atomic Absorption Spectrophotometric Method
First Action 1973
Final Action 1977
AOAC-ASTM Method
A. Apparatus
Atomic absorption spectrophotometer . — Equipped with 4"
single slot or Boling-type burner head and operated as follows:
Pb hollow cathode lamp, 283.3 or 217.0 nm; Cd hollow cath-
ode lamp, 228.8 nm; flame, air-C 2 H 2 . (Caution: See safety
notes on AAS.) App. should have sensitivity of ca 0.5 |xg Pb/
mL and 0.25 p,g Cd/mL for 1% absorption. Use operating
conditions specified by manufacturer.
B. Reagents
(Use glassware of chemically resistant borosilicate glass.)
(a) Acetic acid.— 4%. Mix HOAc and H 2 (1 +24). Ana-
lyze each new batch of reagent for Pb and Cd.
(b) Detergent wash. — Add 15 g alk. detergent (e.g., Cal-
gonite, Calgon Corp., PO Box 1346, Pittsburgh, PA 15230,
or equiv.) to 1 gal. (3.8 L) lukewarm tap H 2 0.
(c) Lead std solns. — (/) Stock soln. — 1000 |xg/mL. Dis-
solve 1.5985 g Pb(N0 3 ) 2 in 4% HOAc and dil. to 1 L with
same soln. (2) Working solns.— Dil. 0.0, 1.0, 3.0, 5.0, 10.0,
and 15.0 mL stock soln to 1 L with 4% HOAc (0, 1, 3, 5,
10, and 15 jig/mL).
(d) Cadmium std solns. — (J) Stock soln. — 1000 |xg/mL.
Dissolve 0.9273 g anhyd. CdS0 4 in 250 mL HCl (1 + 37),
and dil. to 500 mL with HCl (1+37). (2) Intermediate soln. —
10 [Ag/mL. Dil. 10 mL stock soln to 1 L with 4% HOAc. (3)
Working solns.— Dil. 0.0, 3.0, 5.0, 10.0, 15.0, and 20.0 mL
intermediate soln to 100 mL with 4% HOAc (0.0, 0.3, 0.5,
1.0, 1.5, and 2.0 fxg/mL).
C. Extraction
Take, at random, 6 identical units of product and cleanse
each with detergent wash. Rinse with tap H 2 followed by
distd H 2 0, and dry. Fill each unit with 4% HOAc from grad-
uate to within 6-7 mm of overflowing. (Measure distance along
surface of test unit, not vertical distance.) Record vol. acid
required for each unit in sample. Cover each unit with fully
opaque glass plate (so that extn is carried out in total darkness)
to prevent evapn of soln, avoiding contact between cover and
surface of leaching soln (if opaque glass is not available, cover
glass with Al foil or other material to prevent exposure to light).
Let stand 24 hr at room temp. (22 ± 2°).
If test unit is extremely shallow or has scalloped brim, evapn
losses should be anticipated. In those cases, record head space
after filling. After 24 hr leaching period, adjust soln vol. to
same recorded headspace, using 4% HOAc.
242
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
D. Determination
(a) Lead. — Set instrument for max. signal at 283.3 or 217.0
nm, using Pb hollow cathode lamp and air and C 2 H 2 flow rates
recommended by manufacturer. Stir sample soln and decant
portion into clean flask. Det. A of sample and Pb working std
solns. Flush burner with H 2 and check point between read-
ings. Det. Pb from std curve of A against jxg Pb/mL or cal-
ibrate DCR unit in concn mode with Pb working solns, and
read and record sample concn directly. Bracket sample soln
with next higher and lower working solns.
Dil. samples contg >20 jxg Pb/mL with 4% HOAc. Cone.
samples contg <1 jxg Pb/mL by accurately transferring min.
of 50.0 mL of soln to 250 mL beaker, evapg almost to dryness
on steam bath (do not use hotplate); then add 1 mL HO and
evap. to dryness. Dissolve residue in 4% HOAc by adding
exactly 0.1 vol. of soln taken for concn (i.e., for 50.0 mL
soln, add exactly 5.0 mL 4% HOAc), cover with watch glass,
and swirl to complete dissoln. Det. Pb as above, except sub-
stitute "3.0 mL" and "3 |xg/mL" for "20.0 mL" and "20 jutg/
mL", resp., in 973.32B(c)(2).
(b) Cadmium. — Proceed as for Pb, setting instrument for
max. signal at 228.8 nm, using Cd hollow cathode lamp. Dil.
samples contg >2 (xg Cd/mL with 4% HOAc. Cone, samples
contg <0.1 jxg/mL as in (a)
Report type of units tested and for each, vol. acid used and
Pb and Cd leached in jjig/mL.
Refs.: JAOAC 56, 869(1973); 59, 158(1976); 62, 380(1979);
64, 396(1981); 71, 92(1988). ASTM C 738-72.
CAS-7440-43-9 (cadmium)
CAS-7439-92-1 (lead)
979.16* Cadmium and Lead
in Earthenware
Rapid Screening Method
First Action 1979
Surplus 1982
(Detects 0.3 jxg Pb and 0.05 p.g Cd/mL 4% HOAc)
See 25.028-25.030, 14th ed.
971.20 Copper and Nickei in Tea
Atomic Absorption Spectrophotometric Method
First Action 1971
Final Action 1976
(Caution: See safety notes on AAS, wet oxidation, nitric acid,
and perchloric acid.)
A. Principle
Samples are wet ashed and after diln are detd by A A at
232.0 nm (Ni) and 324.7 nm (Cu). Matrix of std solns is
matched to that of sample to avoid interference from Na and
K.
B. Apparatus
Atomic absorption spectrophotometer. — Capable of mea-
suring content or change of content of 0.05 (ucg Ni or Cu/mL
in aq. soln.
C. Preparation of Standard Solutions
(a) Copper std soln. — 1000 jxg/mL. Dissolve 1.000 g
99.99% Cu in 20 mL HN0 3 , cool, and dil. to 1 L with H 2 0.
(b) Nickel std soln.— 1000 |xg/mL. Dissolve 1 .000 g 99.99%
Ni in 20 mL HNQ 3 , cool, and dil. to 1 L with H 2 0.
(c) Matrix std solns. — Prep, solns contg 0, 0.2, 0.4, 0.8,
1.6, 2.0, 4.0, 8.0, and 10 |xg Ni and Cu/mL and major metal
matrix components: (/) For 3 g sample tea. — To contain 180
\ig Ca, 100 (xg Mg, and 40 jig Al/mL with final concn of 8%
(v/v) HC10 4 . (2) For 6 g sample instant tea. — To contain
7000 [xg K, 70 jxg Na, 700 juLg Mg, and 130 jxg Ca/mL with
final HNO3 concn of (1 + 9).
D. Preparation of Calibration Curve
Let instrument stabilize. Optimize conditions for Cu or Ni
according to manufacturer's instructions.
Aspirate 10 (xg/mL std enough times to establish that A
reading is not drifting. Record 6 readings and calc. std devia-
tion (a) — (x — y) x 0.40, where x and y are max. and min.
readings, resp., and 0.40 is factor to convert range of 6 values
to o\
Beginning with soln contg Cu, aspirate each matrix std
soln and record A. If value for 10 [xg/mL soln differs from
av. of the 6 values used to calc. a by >0.01 x (av. of the 6
values), repeat measurements. If these detns indicate drift, det.
cause (e.g., deposits in burner or clogged capillary), correct
it, and repeat calibration. Repeat for Ni solns. Plot A against
|xg metal /mL.
E. Determination
Select sample wt to give soln contg >0.05 but <10 juug Ni/
mL, usually 3 g for teas and 6 g for instant teas.
(a) Wet ashing. — Accurately weigh sample into 400 mL
beaker, add 100 mL HN0 3 , and swirl. Cover, and Jet react 10
min; then place on hot plate. Evap. to near dryness and cool.
Add 50 mL HN0 3 , and for tea, add 10 mL HCIO4. Continue
evapn to obtain clear soln.
Transfer to 50 mL vol. flask and dil. to vol. with H 2 0. (In-
sol. KCIO4 which settles to bottom of flask does not interfere.)
Prep, reagent blank contg same amts of acids taken from
same lots, evapd as above.
(b) Photometry. — Aspirate sample and blank solns, and
record A. Measure A of matrix std soln contg 10 |ULg/mL. If
this value differs from value of av. of the 6 values used to
calc. o by >2cr, repeat measurement. If these values indicate
drift, det. cause, correct it, and repeat calibration and sample
and blank readings.
(c) Calculations . — Correct readings of sample soln for blank.
Convert corrected A to fjig/mL from calibration curve.
ppm Ni (or Cu) = (C x V)/W
where C = \xg metal/mL from curve, V = final vol. sample
soln (50), and W = g sample.
Refs.: JAOAC 53, 531(1970); 54, 658(1971).
CAS-7440-50-8 (copper)
CAS-7440-02-0 (nickel)
SINGLE ELEMENT METHODS
964.16
Antimony in Food
Spectrophotometric Method
First Action 1964
Final Action 1976
American Conference of Governmental Industrial
Hygienists-AOAC Method
A. Principle
Pentavalent Sb in aq. HC1 soln reacts with Rhodamine B to
form colored complex extractable with org. solvs. Intensity of
extd color is measured spectrophtric at 565 nm.
AOAC Official Methods of Analysis (1990)
Arsenic
243
B. Reagents
(H 2 for aq. reagents should be double distd; final distn from
glass.)
(a) Hydrochloric acid soln. — 6N. Dil. coned acid with H 2
(i + d:
(b) Dilute phosphoric acid.—3N. Dil. 70 mL H 3 P0 4 (85%)
to 1 L with H 2 0.
(c) Rhodamine B soln. — 0.02% in H 2 0.
(d) Antimony std solns. — (I) Stock soln. — 100 |mg Sb/mL.
Dissolve 0.1000 g pure Sb in 25 mL H 2 S0 4 with heat; cool,
and cautiously dil. to 1 L with H 2 0. (2) Working soln. — 1
(xg/mL. Dil. 2.0 mL stock soln to 200 mL with H 2 0.
(Cool reagents (a), (b), (c), ca 100 mL benzene, and eight
125 mL separators with Teflon stopcocks in refrigerator before
use; maintain temp, of 5-10° during extn and color develop-
ment. Work in subdued light.)
C. Preparation of Sample
Digest sample as in 963.21 C. Oxidizing conditions must be
maintained.
D. Determination
(Caution: See safety notes on wet oxidation, perchloric acid,
and sulfuric acid.)
Transfer digest or aliquot to 125 mL g-s erlenmeyer, add
enough H 2 S0 4 to make total of 5 mL H 2 S0 4 , and evap. to
fumes of S0 3 . Cool flask, add 10 drops 70% HC10 4 , and again
evap. to white fumes. Cool digest in ice bath >30 min; then
slowly add 5 mL precooled 6N HO by pipet. Let stand in ice
bath 15 min; then add 8 mL precooled 3 N H 3 P0 4 . (Until color
is extd into benzene, perform subsequent operations as quickly
as possible. Color is stable in benzene several hr.) Immedi-
ately add 5 mL precooled Rhodamine B soln, stopper, and
shake vigorously. Transfer to precooled 125 mL separator. Pi-
pet 10 mL precooled benzene into separator, shake vigorously
1 min, and discard aq. layer. Transfer benzene layer (red if
Sb is present) into test tube and let H 2 settle. Rinse 1 cm
cell with ext, fill cell, and read at 565 nm against benzene
blank taken thru entire detn. Refer readings to std curve.
E. Preparation of Standard Curve
Pipet 0, 2, 4, 6, 8, and 10 mL Sb working std soln into 125
mL g-s erlenmeyers, add 5 mL H 2 S0 4 to each, and proceed
as in detn. Plot A against |xg Sb.
Ref.: Manual of Analytical Methods ACGIH, May 1963.
J AOAC 47, 191, 630(1964).
CAS-7440-36-0 (antimony)
trioxide and toxic dusts.) in 25 mL 20% NaOH soln and dil.
to 1 L. (2) Intermediate soln. — 10 u,g/mL. Dil. 10 mL stock
soln to 1 L. (J) Working soln. — I fig/mL. Dil. 100 mL in-
termediate soln to 1 L.
(e) Hydrazine sulfate soln. — 1.5% N 2 H 4 .H 2 S0 4 in H 2 0.
(f) Potassium iodide soln. — 15%. Keep in dark. Discard
when soln turns yellow.
(g) Stannous chloride soln. — Dissolve 40 g As-free
SnCl 2 .2H 2 in HC1 and dil. to 100 mL with HC1.
(h) Dilute hydrochloric acid soln. — Dil. 144 mL HC1 to
200 mL with H 2 0.
(i) Lead acetate soln. — 10% Pb(OAc) 2 .3H 2 in H 2 0.
(j) Zinc metal. — 30 mesh.
(k) Sea sand. — To clean sand ("30 mesh") before use and
between detns, mount piece of 3 mm id glass tubing thru rub-
ber stopper in suction flask. Fit piece of rubber or Tygon tub-
ing over top to take bottom of sulfide absorption tube easily
and to maintain it upright. Add, in turn, with suction, aqua
regia, H 2 0, HN0 3 , and H 2 to remove all traces of acid (>5
washings). Wet sand with Pb(OAc) 2 soln and remove excess
with suction.
(1) Silver diethyldithiocarbamate . — Chill 200 mL 0.1M
AgN0 3 soln (3.4 g/200 mL) and 200 mL 0.1M Na diethyl-
dithiocarbamate soln (4.5 g/200 mL) to 10° or lower. Add
carbamate soln to AgN0 3 soln slowly with stirring. Filter thru
buchner, wash with chilled H 2 0, and dry under reduced pres-
sure at room temp. Dissolve salt in pyridine (reagent grade)
with stirring, chill, and add cold H 2 slowly until completely
pptd. Filter thru buchner, and wash with H 2 to remove all
pyridine. Dry pale yellow crystals under reduced pressure (mp
185-187°; recovery 85-90%). Store in amber bottle in refrig-
erator. (Second recrystn may be necessary to obtain correct
mp.)
(m) Silver diethyldithiocarbamate soln. — Dissolve 0.5000
g salt, (1), in colorless pyridine in 100 mL vol. flask, and dil.
to vol. with pyridine. Mix, and store in amber bottle. Reagent
is stable several months at room temp.
B. Generators and Absorption Tubes
See Fig. 963.21. Use 2 oz (60 mL) wide-mouth bottles of
uniform capacity and design as generators, and fit each by means
of perforated stopper with glass tube 1 cm diam. and 6-7 cm
963.21 Arsenic in Food
Kjeldahl Flask Digestion
First Action 1963
Final Action 1965
A. Reagents
(a) Bromine water. — Half satd. Dil. 75 mL satd Br-H 2
with equal vol. H 2 0.
(b) Sodium hypobromite soln. — Place 50 mL 0.57V NaOH
in 200 mL vol. flask, and dil. to vol. with half-satd Br-H^O,
(a).
(c) Ammonium molybd ate -sulfuric acid soln. — Dissolve
5.000 g (NH 4 ) 6 Mo 7 2 4.4H 2 in H 2 and slowly add 42.8 mL
H 2 S0 4 . Dil. to 100 mL with H 2 0.
(d) Arsenious oxide std solns. — (J) Stock soln. — 1 mg/mL.
Dissolve 1.000 g As 2 3 (Caution: See safety notes on arsenic
FIG. 963.21 —Arsenic apparatus
244
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
Jong, with addnl constricted end to facilitate connection. Place
small wad of glass wool in constricted bottom end of tube and
add 3.5-4 g sand, taking care to have same amt in each tube.
Moisten sand with 10% Pb(OAc) 2 soln and remove excess by
light suction. Clean sand when necessary by treatment (do not
remove sand from tube) with HN0 3 followed by H 2 rinse
and suction. Treat with Pb(OAc) 2 soln. if sand has dried thru
disuse, clean and remoisten it as directed. Connect tube by
means of rubber stopper, glass tube, and rubber sleeve to bent
capillary tubing (7 mm od, 2 mm id) tapered at end to slide
easily into connecting tube and later into neck of 25 mL vol.
flask. Other end of capillary is sealed to Pyrex 1" 19/38 female
joint. To transfer contents of trap, attach bulb aspirator to male
¥ 19/38 joint and place in top of trap.
Clean traps between detns without removing beads by flush-
ing with H 2 0, followed by HN0 3 , soaking for 30 min or until
HN0 3 becomes colorless. Remove all traces of acid with H 2 0,
rinse with acetone, and dry with air current applied by suction
to tip of trap.
C. Preparation of Sample
(Caution: See safety notes on wet oxidation, nitric acid, and
sulfuric acid.)
(For details of convenient churn-type washer that will remove
arsenical spray residues from firm fruits or vegetables with an
aq. NH4NO3-HNO3 soln, see J AOAC 26, 150(1943). Digest
aliquot of "strip" soln and proceed as in (a).
All digestions can be greatly facilitated by following optional
method, JAOAC 47, 629(1964): Proceed as in (a) until mixt.
no longer turns brown or darkens. Cool, add 0.5 mL 70% HC10 4
(Caution: See safety notes on perchloric acid.) and heat until
fuming occurs and digest is clear. Cool, and add 2 addnl 0.5
mL portions HC10 4? heating each time as above. Finish diges-
tion with H 2 and satd NH 4 oxalate as in (a).
Conduct > I blank detn with samples. Blanks should not show
>1 fxg As.)
(a) For fresh fruits (apples, pears, or similar products). —
Weigh and peel representative sample (1-5 lb; 0.5-2 kg). At
blossom and stem ends cut out all flesh thought to be contam-
inated with As compds and include with peelings, if desired.
Place peelings in 1 or more 800 mL Kjeldahl flasks. (As-free
Pyrex glassware and "wet ashing" app. of Duriron are avail-
able.) Add 25-50 mL HN0 3 ; then cautiously add 40 mL H 2 S0 4
(20 mL if Gutzeit method is used). Place each flask on as-
bestos mat with 5 cm hole. Warm slightly and discontinue
heating if foaming becomes excessive.
When reaction has quieted, heat flask cautiously and rotate
occasionally to prevent caking of sample upon glass exposed
to flame. Maintain oxidizing conditions in flask at all times
during digestion by cautiously adding small amts of HN0 3
whenever mixt. turns brown or darkens. Continue digestion
until org. matter is destroyed and SO3 fumes are copiously
evolved. (Final soln should be colorless, or at most light straw
color.) Cool slightly, and add 75 mL H 2 and 25 mL satd
NH 4 oxalate soln to assist in expelling oxides of N from soln.
Evap. again to point where fumes of S0 3 appear in neck of
flask. Cool, and dil. with H 2 to 500 or 1000 mL in vol. flask.
(b) For dried fruit products. — Prep, sample by alternately
grinding and mixing 4-5 times in food chopper. Place 35-70
g portions in 800 mL Kjeldahl flasks, and add 10-25 mL H 2 0,
25-50 mL HNO3, and 20 mL H 2 S0 4 . Continue digestion as
in (a). Dil. digested soln to 250 mL.
(c) For small fruits, vegetables, etc. — Use 70-140 g sam-
ple and digest as in (a) or (b).
(d) For materials other than (a), (b) ? or (c). — Digest 5-
50 g, according to moisture content and amt of As expected,
as in (a) or (b). Dil. to definite vol. detd by As concn ex-
pected.
(e) For products containing stable organic As compounds,
products liable to yield incompletely oxidized organic deriv-
atives that inhibit arsine evolution, or products that are dif-
ficult to digest. — Shrimp, tobacco, oils, and some other prod-
ucts require special treatment to complete oxidn of org. As to
inorg. As 2 5 , or to destroy org. interferences previous to As
detn. For details consult: Ind. Eng. Chem., Anal. Ed. 5,
58(1933); 6, 280, 327 (1934); JAOAC 20, 171(1937); 47,
629(1964).
Dil. As solns obtained by these special methods of prepn to
definite vol.
(f ) For ultra-micro quantities of As, very labile forms of As,
and vacuum-accelerated Gutzeit reduction system for mercuric
bromide spot filtration.— Consult Ind. Eng. Chem., Anal. Ed.
16, 400(1944).
D. Isolation of Arsenic
Before making detns, isolate As, when interfering sub-
stances are present in digests (e.g., pyridine from tobacco), or
when samples contain excessive amts of salts, or H 2 S0 4 from
digestions. Consult first ref. of 963.21C(e) for method of iso-
lating As after digestion, or isolate As by AsCl 3 distn (JAOAC
16, 75, 325(1933); 17, 202(1934). Gelatin may be hydrolyzcd
with HC1 and As isolated as in first ref. of 963.21C(e).
Ref.: JAOAC 46, 245(1963).
CAS-7440-38-2 (arsenic)
91 2.01 *
Arsenic in Food
Gutzeit Method
Final Action
Surplus 1970
See 25.006-25.009, 11th ed.
942.17
Arsenic in Food
Molybdenum Blue Method
Final Action
A. Determination
Transfer 20 mL aliquots of sample and blank digest solns
to generator bottles. Add, swirling after each addn, 10 mL
H 2 0, 5 mL dil. HC1, (h), 5 mL KI soln, (f ), and 4 drops SnCl 2
soln, (g), 963.21A. Let stand >15 min.
Place 4 g sea sand over small glass wool wad in sulfide
absorption tube and cap with glass wool. Place 3 mm diam.
solid glass beads in trap over small glass wool pad until \/ 4
full and add 3.0 mL NaOBr soln, (b). Assemble app. except
for generator bottle. Add 4 g Zn, (j), to generator bottle, at-
tach immediately, and let react 30 min.
Disconnect trap and transfer contents to 25 mL vol. flask
with aspirator assembly. Rinse trap with six 2 mL portions
H 2 and aspirate into flask. Add, with swirling, 0.5 mL NH 4
molybdate-H 2 S0 4 soln, (c), and 1.0 mL N 2 H 4 .H 2 S0 4 soln, (e).
Dil. to vol., mix, and let stand 75 min. Mix, and read in spec-
trophtr or colorimeter at 845 nm against blank prepd similarly.
Alternatively, heat vol. flask and contents 10 min at 50° and
cool in tap H 2 to room temp, before reading. Det. As 2 3 (or
As) in aliquot from std curve.
AOAC Official Methods of Analysis (1990)
Arsenic 245
B. Preparation of Standard Curve
Place 0.0, 1.0, 2.0, 3.0, 5.0, 6.0 mL std soln contg 10 jig
As 2 3 /mL in 25 mL vol. flasks. Add 3.0 mL NaOBr soln,
(b), and H 2 to 15 mL. Add, with swirling, 0.5 mL NH 4 mo-
lybdate~H 2 S0 4 soln, (c), and 1.0 mL N 2 H 4 .H 2 S0 4 soln, (e).
Dil. to vol., mix, and let stand 75 min or heat 10 min at 50°
as for samples. Mix, and read at 845 nm. Plot A against )xg
As 2 3 (or As).
Refs.: Ind. Eng. Chem. Anal. Ed. 14, 442(1942). JAOAC
46, 245(1963).
CAS-7440-38-2 (arsenic)
952.13 Arsenic in Food
Silver Diethyldithiocarbamate Method
Final Action
A. Determination
Transfer aliquot of sample digest, 963.21 C (usually 2-5 mL),
and same vol. blank to generator bottles. Add H 2 to 35 mL;
then add, with swirling, 5 mL HO, 2 mL Kf soln, (f), and 8
drops SnCL soln, (g), and let stand >15 min. Evolve AsH 3
as in 942. 17 A, except add 4.0 mL Ag diethyldithiocarbamate
soln, (m), to trap.
Disconnect trap and mix trapping soln by gently drawing
back and forth 5 times with aspirator assembly. Transfer soln
directly to spectrophtr cell (g-s preferred) and read at 522 nm.
Det As 2 3 (or As) in aliquot from std curve.
B. Preparation of Standard Curve
Place 0.0, 1.0, 3.0, 6.0, 10.0, and 15.0 mL std soln contg
1.00 jxg As 2 3 /mL in generator bottles. Add H 2 to 35 mL
and proceed as in 952. 13A. Read at 522 nm and plot A against
jxg As 2 3 (or As).
Chem. 31,
Refs.
Chem. Listy 46, 341(1952). Anal.
1589(1959). JAOAC 46, 245(1963).
CAS-7440-38-2 (arsenic)
973.33 Arsenic in Meat and Poultry
Molybdenum Blue Method
First Action 1973
Final Action 1975
A. Principie
Sample is ashed in presence of Mg(N0 3 ) 2 at 600°. Ash is
dissolved in dil. HO; Zn is added to generate AsH 3 , which is
trapped with I soln in cell. Heteropoly blue compd is devel-
oped and read at 840 nm in same cell. Chief source of error
is often contamination. Always perform reagent blank and, when
possible, std sample.
B. Reagents
(Glassware should not be subjected to routine washing with
soap or detergents, which are often source of As contamina-
tion. When soap or detergent is used, clean with aqua regia
before use. Rinse delivery tubes by holding in slanted position
with crook up and squirting jet of H 2 up and over inside
crook until tube is filled; then rinse outside while tube drains.
Repeat rinsing 3 times. Rinse funnels in each direction alter-
nately by filling end that is up and placing funnel on 1-hole
rubber stopper in mouth of vac. flask to pull H 2 thru frit by
vac . )
(a) Tissue solvent. — CHC1 3 (or benzene) -acetone- absolute
alcohol (1 + 1 + 2).
(b) Dilute hydrochloric acid. — Mix 175 mL HCI and 280
mL H 2 0.
(c) Potassium iodide soln. — \5%. See 963.21A(f).
(d) Stannous chloride soln. — 40% in dil. HCI, (b). Store
in contact with metallic Sn.
(e) Zinc. — Shot of uniform size and shape, ca 0.5 g each.
(f) Lead acetate soln. — Prep, satd aq. Pb(OAc) 2 .3H 2 soln
in dropping bottle. Prep, fresh weekly or when soln becomes
cloudy.
(g) Iodine solas.— (1) 0.02N. —Dissolve 8 g KI and 2.54
g I in small amt H 2 and dil. to 1 L with H 2 0. Store in dark
bottle. (2) 0.001N.— Dil. 5 mL 0.02N I to 100 mL with H 2 0.
Prep, fresh daily.
(h) Ammonium, molybdate soln. — Dissolve 7.0 g
(NH 4 ) 6 Mo 7 24 .4H 2 in warm mixt. of 70 mL H 2 S0 4 and 300
mL H 2 0, cool, and dil. to 500 mL with H 2 0.
(i) Hydrazine sulfate soln. — Dissolve 0.3 g N 2 H 4 .H 2 S04 in
H 2 and dil. to 200 mL.
(j) Arsenious oxide std solas. — (/) Stock soln. — 1 mg As/
mL. Dissolve 0.1320 g As 2 3 in 50 mL H 2 contg 0.7 mL
50% NaOH. Neutze with 50% H 2 S0 4 and dil. to 100 mL. (2)
Working solns. — Dil. sep. 1.0 mL portions stock soln to 100,
200, and 500 mL with H 2 (10, 5, and 2 |xg As/mL, resp.).
(k) Arsanilic acid std solns. — (J) Stock soln. — 1 mg As/
mL. Dissolve 0.2897 g arsanilic acid (based on label assay)
in H 2 and dil. to 100 mL with H 2 0. (2) Working solns. —
Prep, in same concns as in (j).
C. Apparatus
(a) Cell rack. — Metal rack capable of holding eight 19 x
105 mm cells in 600 mL beaker.
(b) Distilling apparatus. — Kings ley-Schaffert As distg app.
(Corning Glass Works, No. 33680), consisting of 125 mL flask,
funnel trap, and bent dispersion tube.
(c) Absorbent cotton.— See 945.58B(i).
D. Preparation of Sample
Assure absence of interferences arising from laboratory and
reagent contamination. Recoveries thru method of added compds
should be ^90%. Conduct one or more reagent blanks and std
samples along with samples.
To obtain representative aliquot of large sample (^100 g),
grind entire sample ^2 times, using fine plate (grind liver sample
only once or blend). Mix thoroly and weigh calcd amt into 50
mL Vycor crucible. Add 4 g Mg(NO 3 ) 2 .6H 2 O/10 g sample
and mix, using stainless steel spatula or glass rod, until all
Mg(N0 3 ) 2 is dissolved. Spread mixt. in even layer around sides
of crucible.
For smaller samples (<100 g), weigh known amt or entire
sample into homogenizer or blender, add 4 g Mg(N0 3 ) 2 .6H 2 0/
10 g sample and enough tissue solv. to aid blending, weigh,
and blend 1 min. {Caution: Use explosion-proof blender when
benzene-acetone-alcohol is used as tissue solv.) Weigh aliquot
equiv. to desired amt sample into 50 mL Vycor crucible and
carefully evap. excess solv. and H 2 from tissue on steam bath
or in 95° oven.
E. Determination
Place crucible in cool furnace, gradually increase temp, to
600°, and ash sample at 600° until most visible C is burned.
Cool crucible and cool furnace. Dampen ash with H 2 and
add 3 mL HN0 3 (1 + 4). Place in cooled furnace (100°) and
heat gradually to 600°. Hold at 600° ca 1 hr until all HN0 3
fumes are evolved. Repeat dil. HN0 3 treatment if necessary
to obtain white ash.
Remove crucibles and let cool. Dampen ash with H 2 and
246
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
dissolve in 10 mL dil. HO, (b), delivered from all-glass hy-
podermic syringe without needle. Quant, transfer to 125 mL
flask, 973.33C(b), with aid of two 10 mL portions dil. HC1
and wash sides of flask with fourth 10 mL portion. (Use same
vol. liq. for each sample because vol. of air space above liq.
affects efficiency of distn of H and AsH 3 .) Cool to room temp.
Add 2.0 mL 15% KI soln and mix thoroly by swirling. Add
1.0 mL 40% SnCl 2 soln and mix thoroly by swirling. Let stand
>15 min (but <30 min). Place 7.0 mL 0.0017V I soln in cell.
Place small ball absorbent cotton in top of funnel and dampen
with satd Pb(OAc) 2 soln. Lubricate ground glass joint with H 2
and join delivery tube to funnel firmly. This union must be
firm enough to hold together under wt of funnel, flask, and
contents. Union must not be so firm as to prevent disassembly.
Do not dry parts by heating before use. Teflon sleeves are not
satisfactory.
Fill 600 mL beaker with finely crushed ice layered between
levels of cell holder. Add ice-H 2 to ca 2 / 3 ht of beaker and
place cell in holder. (It may be necessary to make path thru
ice for cell.) Lubricate lower ground glass joint of funnel with
H 2 0, add ca 12.5 g Zn to flask, join flask and funnel firmly,
and place delivery tube in cell as quickly as possible. Let distn
continue 1 hr (without added heat).
Carefully and slowly remove delivery tube from cell, letting
liq. drain as tube is removed. Add 0.5 mL NH 4 molybdate
soln and mix thoroly. Add 0.3 mL N 2 H 4 .H 2 S0 4 soln and mix
thoroly. Place cell (in cell holder) in moderately boiling H 2
bath or on medium (not vigorous) steam bath 10 min. Remove
from bath, wipe cell dry with soft lintless material, and place
in cool, dark place ca 1 hr (to ensure that samples reach same
temp, and full color development). Read in precalibrated spec-
trophtr or colorimeter at 840 nm against C0 2 -free H 2 0. Cor-
rect for blanks.
F. Preparation of Standard Curve
Prepare stds of 10 g As -free liver, 4 g Mg(N0 3 ) 2 , and suit-
able amts arsanilic acid working solns in definite progression,
such as 2, 4, 6, 8, and 10 u,g As. Repeat analysis of stds >3
times. Det. mean for each level and prep, curve; or fit line by
method of least sqs, in Definitions of Terms and Explanatory
Notes, if desired.
Ref.: JAOAC 56, 1144(1973).
CAS-7440-38-2 (arsenic)
945.58
Cadmium in Food
Dithizone Method
Final Action 1976
A, Principle
Sample is digested with H 2 S0 4 and HN0 3 . All reactive met-
als are extd from soln (after adjustment to pH ca 9) with dithi-
zone-CHCl 3 . Cu, Hg, and most of any Ni or Co present are
removed by stripping CHC1 3 soln with dil. HC1. Aq. layer,
adjusted to 5% NaOH, is extd with dithizone-CCl 4 . At this
alky, Zn, Pb, and Bi do not ext, whereas Cd dithizonate is
relatively stable. Stripping with dil. HO and development of
Cd dithizonate in 5% NaOH are repeated. Cd is finally estd
photometrically as dithizonate. Zn constitutes chief interfer-
ence.
B. Reagents
(a) Citrate. — Diammonium salt or citric acid.
(b) Chloroform. — Distil from hot H 2 bath, collecting dis-
tillate in absolute alcohol in proportion of 10 mL alcohol to 1
L distillate. Intermittently shake receiver during distn.
(c) Diphenylthiocarbazone {dithizone), twice purified.—
Purify as in 934.07 A(e), but make only 3 dil. NH 4 OH extns
of CHC1 3 soln. Carry thru, including H 2 0- washing steps, and
then repeat purification with 3 NH 4 OH extns, pptn with dil.
acid, etc. Instead of heating ext to dryness, evap. sponta-
neously, and complete drying under vac. in bell jar overnight.
(d) Carbon tetrachloride. — {Caution: See safety notes on
distillation, and carbon tetrachloride.) Reflux vigorously on
steam bath 1 hr with V20 vol. 20% KOH in MeOH. Cool, add
H 2 0, drain off CC1 4 layer, and wash >3 times with copious
vols of H 2 until alkali-free. Dry over CaCl 2 , filter, and distil
on hot H 2 bath. (Unless reagent is so purified, erratic Cd
results may be obtained with some lots of CC1 4 .)
(e) Dithizone in carbon tetrachloride. — 20 mg/L CC1 4 , (d).
Prep, daily, as dil. solns of dithizone are unstable. (When many
detns are to be made, dithizone reagent may be prepd by diln
from 300 mg/L soln. Store coned reagent under 0.1M S0 2
soln in refrigerator.)
(f) Dithizone in chloroform. — 1000 mg/L CHC1 3 , (b), prepd
as needed.
(g) Sodium hydroxide soln. — 28%. Dissolve 28 g NaOH
pellets in H 2 and dil. to 100 mL.
(h) Thymol blue indicator. — Triturate 0. 1 g indicator in ag-
ate mortar with 4.3 mL 0.05 N NaOH. Dil. to 200 mL in g-s
flask with H 2 0.
(i) Absorbent cotton. —Metal-free. If traces of metal are
present, remove by digesting cotton several hr with 0.2 N HO,
filtering on buchner, and finally washing with copious vols of
redistd H 2 until acid-free.
(j) Cadmium std solns. — (7) Stock soln. — 1 mg/mL. Dis-
solve 1 g Cd (Fisher Chemical Co., certified 99.9% pure, C-
565, or equiv.) in 20-25 mL HNO3 (1 + 9), evap. to dryness,
add 5 mL HO (1 + 1), evap. to dryness, and then add several
mL H 2 and again evap. to dryness. Dil. to 1 L. (2) Inter-
mediate soln. — 100 (xg/mL. Dil. 10 mL stock soln to 100
mL. (J) Working soln. — 2 jxg/mL. Transfer 20 mL inter-
mediate soln to 1 L vol. flask, add 15 mL HC1, and dil. to
vol. to give final acidity of ca 0.2 N.
C. Preparation of Standard Curve
Prep, in duplicate 6 stds contg 0, 5, 10, 15, 20, and 25 u>g
Cd as follows: Add appropriate vols std soln to Squibb- type
separators (125 mL size is convenient), adjust to 40 mL with
0.27V HC1, add 10 mL NaOH soln (soln is then 5% with re-
spect to NaOH) and 25 mL dithizone soln, (e), shake vigor-
ously exactly 1 min, let stand exactly 3 min, and filter org.
layer thru pledget of absorbent cotton, discarding first 5 mL.
Fill absorption cell (1 cm length is convenient) and det. A at
510 nm. Plot std curve or calc. ref. equation by method of
least squares, Definitions of Terms and Explanatory Notes , Item
(24).
D. Preparation of Sample
Use sample equiv. to 5-10 g of product, calcd to dry basis.
(Sample size is of concern only when comparatively large amts
of Mg and P are present.) Digest with 10 mL H 2 S0 4 (1 + 1)
and HNO3 as needed. If sample tends to char rather than to
oxidize evenly, add 5 or 10 mL addnl H 2 S0 4 . Continue diges-
tion, adding HN0 3 as required, until digestion is complete and
S0 3 is evolved. Cool, add 15 mL satd NH 4 oxalate soln, and
again heat to fumes.
Fat in biological materials, such as liver and kidney, may
cause bumping and frothing during digestion. If comparatively
large samples of such materials are available, make partial
digestion with warm HN0 3 until only fat remains undissolved.
Cool, filter free of solid fat, wash residue with H 2 0, make
combined filtrate to suitable vol., and digest appropriate ali-
quots as above.
AOAC Official Methods of Analysis (1990)
Cadmium
247
E. Determination
Oil. digest, 945.58D, with 25 mL H 2 0, filter free from ex-
cessive insoL matter (sulfates or silica) if present, and transfer
to separator marked at 125 mL, using addnl 10 mL portions
H 2 for rinsing and completing transfer. Add 1-2 g citrate
reagent, (a), and 1 mL thymol blue indicator, (h), and adjust
to ca pH 8.8 by adding NH 4 OH slowly, while cooling inter-
mittently, until soln changes from yellowish green to greenish
blue. Oil . to 125 mL mark with H 2 0. Ext vigorously with 5
mL portions dithizone soln, (f ), until CHC1 3 layer remains green.
Then ext with 3 mL CHC1 3 .
Transfer all CHC1 3 exts to second separator previously wet-
ted with 2-3 mL CHC1 3 . Add 40 mL 0.2N HC1 to combined
dithizone exts, shake vigorously ^1 min, and after layers sep.,
carefully drain CHC1 3 phase contg any Cu, Ni, Co, or Hg that
may be present, and discard. Remove remaining droplets of
dithizone by extg with 1-2 mL CC1 4 , (d), carefully conduct-
ing draining operation so that no acid enters bore or stem of
separator, as its presence there would in part decompose Cd
dithizonate subsequently formed and extd in next step.
Adjust aq. phase to 5% alky by adding 10 mL NaOH soln,
(g). Ext Cd with 25 mL dithizone soln, (e), shaking vigorously
>1 min, and transfer to third separator previously wetted with
2-3 mL same dithizone soln. Repeat extn with addnl 10 mL
portions dithizone soln until CC1 4 layer becomes colorless. Amts
of Cd usually found in foods or biological materials (ca 100
(xg) are completely removed by third extn.
To verify assumption that pale pink persisting after third extn
is due to Zn, transfer questionable ext to fourth separator contg
5% NaOH soln, add several mL dithizone soln, (e), and shake
vigorously. If CC1 4 layer becomes colorless, original pink was
due to Zn and no further extns are necessary. If, however, pink
persists, indicating presence of Cd, add ext to contents of third
separator, and continue extn.
Convert Cd and Zn dithizonates in third separator to chlo-
rides by adding 40 mL 0.2N HO and shaking vigorously >1
min. Carefully drain CC1 4 layer, which may contain traces of
Co and Ni not removed in second step, and discard. Remove
droplets of dithizone from aq. phase by rinsing with 1-2 mL
CC1 4 and drain off as completely as possible, but do not permit
any acid to pass bore of separator. Again adjust alky to 5%
by adding 10 mL NaOH soln, (g). Wipe separator stems dry
with cotton, (i). Det. Cd present by adding exactly 25 mL
dithizone soln, (e), shaking vigorously exactly I min, per-
mitting layers to sep. exactly 3 min, and continuing as in
945. 58C, beginning "... filter org. layer ..." Calc. Cd in
jmg by substituting A in linear equation or from std curve.
Note: If photometric measurement indicates >25 |mg Cd, make
first approximation by dilg dithizonate soln with CC1 4 and
evaluating A. For best results repeat analysis with wts or ali-
quots of samples contg <25 (xg Cd; 30 |ag is upper limit of
solubility of Cd dithizonate in 25 mL CC1 4 . Therefore amts
>30 |xg are incompletely extd.
Refs.: JAOAC 28, 257(1945); 32, 349(1949). Anal. Chem.
21, 300(1949).
CAS-7440-43-9 (cadmium)
973.34 Cadmium in Food
Atomic Absorption Spectrophotometric Method
First Action 1973
Final Action 1974
(Caution: See safety notes on AAS, wet oxidation, nitric acid,
sulfuric acid, and peroxides.)
A. Principle
Sample is digested with HN0 3 , H 2 S0 4 , and H 2 2 . All re-
active metals are extd from soln, after adjustment to ca pH 9,
with dithizone-CHCl 3 . Cd is removed by stripping CHCl 3 soln
with dil. HC1 and detd by AA spectrophotometry at 228.8 nm.
B. Reagents and Apparatus
(Thoroly wash all new glassware and glassware which has
contained high Cd concn with 8 N HN0 3 , and rinse with H 2 0.
Cover beakers with watch glasses during all operations.)
(a) Nitric acid. — Low in Pb and Cd (G. Frederick Smith
Chemical Co., No. 63).
(b) Hydrogen peroxide. — 50% (Fisher Scientific Co., No.
H-341).
(c) Citric acid. — Monohydrate, fine crystal.
(d) Thymol blue indicator. — See 945.58B(h).
(e) Dithizone solns. — (J) Concentrated soln. — 1 mg/mL.
Prep. 200 mL in CHC1 3 . (2) Dilute soln. — 0.2 mg/mL. Dil.
coned soln 1+4 with CHC1 3 . Prep, fresh daily.
(f) Cadmium std solns. — (J) Stock soln. — 1.0 mg/mL.
Dissolve 1.000 g Cd, 945.58B(j), in 165 mL HC1 in 1. L vol.
flask. Dil. to vol. with H 2 0. (2) Intermediate soln. — 10 |xg/
mL. Dil. 10 mL stock soln with 2N HC1 to 1 L. Prep, just
before use. (J) Working solns. — Dil. 0, 1, 5, 10, and 20 mL
intermediate soln to 100 mL with 2N HC1 (0, 0.1, 0.5, 1.0,
and 2.0 |xg Cd/mL, resp.).
(g) Atomic absorption spectrophotometer . — With hollow-
cathode Cd lamp and 10 cm burner head for air-C 2 H 2 flame;
wavelength 228.8 nm, range 0-2.0 u.g/mL.
C. Digestion
Weigh 50.0 g sample into 1 .5 L beaker. Add several boiling
chips or beads, and cover. Carefully add 25 mL HN0 3 , cover,
and warm gently with flame to initiate reaction. (Meker-type
burners are preferred thruout for their versatility and speed.)
When reaction subsides, add 25 mL HN0 3 , warm again, and
continue until 100 mL HN0 3 has been added. (Alternatively,
add 100 mL HN0 3 all at once, with caution, and let stand at
room temp, overnight.) Heat until most NO fumes have evolved;
control excessive frothing by cooling or quenching with H 2
from wash bottle. Only some cellulose and fatty materials, if
any, remain undissolved.
To remove any fat visible in hot soln, proceed as follows:
Cool beaker in ice, and decant clear, aq. soln from coagulated
oils and solids thru glass wool pad into 1 L beaker. Add 100
mL H 2 to 1.5 L beaker with fat, heat, swirl vigorously to
rinse fat, chill, and filter as before. Wash funnel and glass
wool pad with ca 20 mL H 2 0.
Add 20 mL H 2 S0 4 to sample, dil. to ca 300 mL with H 2 0,
and evap. over flame until charring begins. When charring be-
comes extensive, cautiously add 50% H 2 2 , 1 mL at time. Let
reaction subside before adding next portion of oxidant, and
never add >1 mL at a time. Continue addns of H 2 2 until soln
is colorless. Heat vigorously to S0 3 fumes, adding more H 2 2
as required to remove char. Heat vigorously to expel excess
H 2 2 . Cool colorless digest to room temp.
Prep, reagent blank of 100 mL HN0 3 , 20 mL H 2 S0 4 , and
same amts of H 2 as added to sample. Cautiously add same
amts 50% H 2 2 , as above, and remove all HN0 3 from blank.
Carry blank thru same operations as sample.
D. Extraction
Add 2 g citric acid to cooled digest and cautiously dil. to
ca 25 mL with H 2 0. Add 1 mL thymol blue indicator and
adjust to ca pH 8.8 by slowly adding NH 4 OH while cooling
in ice bath, until soln changes from yellowish green to green-
248
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
ish blue. Transfer quant, to 250 mL separator, using H 2 0, and
diL to ca 150 mL.
Cool soln, and ext with two 5 mL portions coned dithizone
soln, shaking 1-2 min each time. Continue extn with 5 mL
portions dil. dithizone soln until last 5 mL portion dithizone
ext shows no change in color. Combine dithizone exts in 125
mL separator; wash with 50 mL H 2 0, and transfer sol v. to
another 125 mL separator. Ext H 2 wash with 5 mL CHC1 3
and add this to dithizone exts. Add 50 mL 0.27V HC1 to com-
bined dithizone exts, shake vigorously 1 min, and let layers
sep.; discard dithizone layer. Wash aq. soln with 5 mL CHC1 3
and discard CHC] 3 . Quant, transfer aq. soln to 400 mL beaker,
add boiling chips, and evap. carefully to dryness. Carefully
rinse down sides of beaker with 10-20 mL H 2 and again
evap. to dryness.
£ Determination
Set instrument to previously established optimum condi-
tions, using air-C 2 H 2 oxidizing flame and 228.8 nm resonant
wavelength. Dissolve dry residue in 5.0 mL 2N HC1 and det.
A of sample and std solns against 2N HC1 as blank. Flush
burner with H 2 between readings. Use scale expansion con-
trols to obtain 4-10x expansion, as convenient. Det. Cd from
curve of A against jxg Cd/mL:
ppm Cd = (jxg Cd/mL) X (mL 2N HCl/g sample)
For concn >2.0 \xg Cd/mL, dil. soln with 2N HC1.
Ref.: JAOAC 56, 876(1973).
CAS-7440-43-9 (cadmium)
960.40 Copper in Food
Colorimetric Method
First Action 1960
Final Action 1965
International Union of Pure and Applied Chemistry-AOAC
Method
A. Principle
Sample is digested with HN0 3 and H 2 S0 4 . Cu is isolated
and detd colorimetrically at pH 8.5 as diethyldithiocarbamate
in presence of chelating agent, EDTA. Bi and Te also give
colored carbamates at pH 8.5 but are decomposed to colorless
compds with 17V NaOH. Cu complex is stable. Range of color
development is 0-50 u,g. Blank is ca 1 |xg Cu.
B. Precautions
Clean glassware with hot HN0 3 . Use white petrolatum to
lubricate stopcocks of separators, and do not use brass chains.
Purify H 2 and HN0 3 by distn in Pyrex.
C. Reagents
(a) Sodium diethyldithiocarbamate (carbamate soln). —
Dissolve 1 g of the salt in H 2 0, dil. to 100 mL, and filter.
Store in refrigerator and prep, weekly.
(b) Citrate-EDTA soln. — Dissolve 20 g dibasic NH 4 citrate
and 5 g Na 2 EDTA (Eastman Kodak Co.) in H 2 and dil. to
100 mL. Remove traces of Cu by adding 0.1 mL carbamate
soln and extg with 10 mL CC1 4 . Repeat extn until CC1 4 ext is
colorless.
(c) Copper std solns. — (7) Stock soln. — 1 mg/mL. Place
0.2000 g Cu wire or foil into 125 mL erlenmeyer. Add 15 mL
HN0 3 (1 + 4), cover flask with watch glass, and let Cu dis-
solve, warming to complete soln. Boil to expel fumes, cool,
and dil. to 200 mL. (2) Intermediate soln. — 100 fxg/mL. Dil.
20 mL stock soln to 200 mL. (3) Working soln. — 2 juig/mL.
Prep, daily by dilg 5 mL intermediate std soln to 250 mL with
2.0N H 2 S0 4 .
(d) Ammonium hydroxide. — 6N. Purify as in (b).
(e) Thymol blue indicator. — 0.3%. Dissolve 0.1 g thymol
blue in H 2 0, add enough 0.1/V NaOH to change color to blue,
and dil. to 100 mL.
D. Preparation of Sample
(Caution: See safety notes on nitric acid and sulfuric acid.)
Weigh sample contg <20 g solids, depending upon expected
Cu content. If sample contains <75% H 2 0, add H 2 to obtain
this diln. Add initial vol. HNO3 to equal ca 2 times dry sample
wt and 5 mL H 2 S0 4 , or as many mL H 2 S0 4 as g dry sample,
but >5 mL. Digest as in 963.21C.
When sample contains large amt of fat, make partial diges-
tion with HNO3 until only fat is undissolved. Cool, filter free
of solid fat, wash residue with H 2 0, add H 2 S0 4 to filtrate, and
complete digestion as above. After digestion, cool, add 25 mL
H 2 0, and remove nitrosyl sulfuric acid by heating to fumes.
Repeat addn of 25 mL H 2 and fuming. If after cooling and
dilg, insol. matter is present, filter thru acid- washed paper,
rinse paper with H 2 0, and dil. to 100 mL.
Prep, reagent blank similarly.
E. Isolation and Determination of Copper
Pipet 25 mL sample soln into 100 or 250 mL short-stem
separator and add 10 mL citrate-EDTA reagent. Add 2 drops
thymol blue indicator, (e), and 67V NH 4 OH dropwise until soln
turns green or blue-green. Cool, and add 1 mL carbamate soln
and 15 mL CC1 4 . Shake vigorously 2 min. Let layers sep. and
drain CC1 4 through cotton pledget into g-s tube or flask. Det
A or T in suitable instrument at ca 400 nm.
If >50 (Jig Cu is present in 25 mL aliquot, use smaller ali-
quot and dil. to 25 mL with 2.0 TV H 2 S0 4 . Highest accuracy
is obtained at ca 25 fxg Cu level (A ca 0.3 in 1 cm cell).
To test for Bi and Te, return CC1 4 soln to separator, add 10
mL 5% KCN soln, and shake 1 min. If CC1 4 layer becomes
colorless, Bi and Te are absent.
If test is pos., develop color in another 25 mL aliquot as
above (without KCN). Drain CC1 4 layer into second separator,
add 10 mL IN NaOH, and shake 1 min. Let layers sep. and
drain CC1 4 into third separator. Again wash CCJ 4 ext with 10
mL IN NaOH. Det. A or T of CC1 4 layer and convert to u,g
Cu.
F. Preparation of Standards and Calibration Curves
Transfer 0, 1, 2.5, 5, 10, 15, 20, and 25 mL of Cu std soln
(2 (xg/mL) to separators and add 2.07V H 2 S0 4 to make total
vol. of 25 mL.
Add 10 mL citrate-EDTA reagent and proceed as in 960. 40E,
beginning "Add 2 drops thymol blue indicator, ..."
Plot A against juug Cu on ordinary graph paper. If readings
are in % T, use semilog paper, and plot T on log scale. Since
there is usually some deviation from linearity, read sample val-
ues from smoothed curve.
Ref.: JAOAC 43, 695(1960).
CAS-7440-50-8 (copper)
944.07 Fluorine on Apples and Pears
Colorimetric Method
Final Action
A. Principle
Add filtrate from strip soln of apples and pears prepd with
HC1 rinse and acidification, 935. 51 A, is used. Aliquot of fil-
AOAC Official Methods of Analysis (1990)
Fluorine 249
trate is oxidized colorless with KMn0 4 , soln is then reduced
with H 2 NOH, and subaliquot is backtitrd in Nessler tubes;
Zr(N0 3 ) 4 is used in titrn, with purpurin (1,2,4-trihydroxyan-
thraquinone) as indicator (Ind. Eng. Chem., Anal. Ed. 6,
118(1934)). Principle of back-titrn, as applied here, is similar
to that used in general method where Th(N0 3 ) 4 and alizarin
occupy similar roles. Provision is made for removal of inter-
fering anions, and high acidity used in titrn minimizes inter-
ference of metals that would otherwise lake with indicator.
S. Apparatus
Nessler tubes. -^50 mL g-s, tall -form, matched for ht and
color (see944.W7E).
C. Reagents
(a) Mixed nitrate soln. — Dissolve 3.0 g Ba(N0 3 ) 2 and 2.0
g Th(N0 3 ) 4 .4H 2 in H 2 0, and dil. to 100 mL.
(b) Potassium permanganate soln. — Satd; ca 6%.
(c) Hydroxylamine hydrochloride soln. — 5%.
(d) Ferrous chloride soln. — Dissolve ca 1.0 g Fe powder
or wire in 50 mL HC1 (1 + 1), dil., and filter into 500 mL
vol. flask. Add few mL 5% H 2 NOH.HCl soln and dil. to vol.
Dil. still further before use, if desired.
(e) Purpurin indicator. — 0.01% in alcohol. Dissolve 25 mg
pure 1,2,4-trihydroxyanthraquinone in alcohol, heating if nec-
essary, and dil. to 250 mL with same solv. Prep, fresh weekly.
(f) Zirconium nitrate soln. — Dissolve 1.50 g Zr(N0 3 ) 4 .5H 2
in H 2 0, acidify with 20 mL HC1, and dil. to 1 L. Filter if not
clear.
(g) Fluorine std soln. — 54.5 |xg F/mL. Dissolve 0.1464 g
NaF in H 2 and dil. to 1 L.
D. Determination
Place 20 mL well mixed acid strip filtrate, 935.51 A, in 50
mL vol. flask. Add 2.0 mL mixed nitrate soln, then 4.0 mL
KMn0 4 soln. Rinse down neck of flask with little H 2 and
place on active steam bath 5 min. Remove flask, and while
still hot, add 5% H 2 NOH.HCl soln from buret, slowly and
with swirling, until Mn0 2 is dissolved and soln is colorless;
then add ca 0.5 mL in excess. (Appreciable phosphate is re-
vealed as flocculent Th 3 (P0 4 ) 4 , and sulfate as ppt with Ba.
Sometimes KMn0 4 is occluded in sulfate and/or phosphate
ppt, and pink color tends to persist but does not interfere.)
Cool, dil. to vol., and filter. (Filtrate must be clear. If there
is perceptible turbidity, return filtrate thru filter several times
if necessary, until filtrate is brilliant.) Pipet 25 mL clear fil-
trate into Nessler tube and mark "S."
For blank or comparison tube use 25 mL "blank" soln, contg
reagents used in method, prepd as follows:
Dil. 50 mL 10% Na oleate soln, 934.07A(k), 50 mL 30 g/
100 mL NaOH soln, and 15 mL HCl to 1 L. Acidify portions
with Vio vol. HCl as if soln were an actual "strip," and filter,
refiltering until filtrate is perfectly clear. (Chilling soln and
shaking vigorously will "churn" pptd oleic acid and aid in ob-
taining clear filtrate.) Carry 20 mL portions of acidified filtrate
thru method exactly as above. (In order more closely to du-
plicate conditions of actual detn, use 50 mL vol. flasks and
20 mL aliquots in preference to using larger aliquots with cor-
respondingly larger amts of reagents. After being dild to vol.
and filtered, blank soln may be combined to form supply of
"blank"; 10 portions worked up as above yield ca 500 mL
"blank," or enough for ca 20 detns.)
Add 25 mL of this "blank" to second Nessler tube, "B,"
and to both tubes "S" and "B" add 15.0 mL HCl measured
as carefully as possible from graduate. (Always add acid to
soln instead of vice versa.) Mix, and match tubes for color.
"S" tube will usually be found to have slight greenish tint in
comparison with "B" tube, due presumably to traces of Fe.
Balance both tubes to same shade by adding FeCl 2 soln drop-
wise to appropriate tube and mixing. This operation must be
done carefully. When tints are indistinguishable, add exactly
1.00 mL purpurin indicator to each tube. Mix; then add 1.50
mL Zr soln to each tube from 10 mL buret, and mix. Do not
shake tubes violently when mixing in reagents; 4 or 5 gentle
inversions are enough. Observe color difference, if any, be-
tween tubes when looking down their length toward white re-
flecting surface. If there is no appreciable difference after 5
min, F content of sample is negligible. If color of tube "S" is
yellower, presence of F is indicated. In this case, add addnl
amts of Zr(N0 3 ) 4 soln to tube "S" until its color about matches
that of tube "B" (to nearest 0.5 mL Zr soln). Dil. "S" to mark
and mix.
Now add to "B" exactly same total vol. Zr soln as was added
to tube "S," mix, and let tube stand 2 min for lake to develop
fully. Back-titr. std F soln into "B" from 10 mL buret until
tubes match, mixing frequently, and dilg nearly to vol. as end
point approaches. Add NaF soln in increments of ca 0.1 mL
at this stage, and observe usual precautions of transposing and
letting bubbles subside when making comparisons. Dil. to mark
for final comparison. Check end point by adding 0.1-0.2 mL
std F soln in excess. Distinct overbleach should develop.
For sample wt of 1 kg and aliquots specified above, each
mL std F soln consumed in back-titrn is equiv. to F content
on fruit sample, removable by solv. treatment, of 3.0 ppm.
Correct result obtained in titrn by sample wt ratio. (Thus, titer
of 3.27 mL std F soln, with 1.40 kg sample (ca 10 fruit),
represents F content of 7.0 ppm. Vol. restrictions of 50 mL
Nessler tube will allow estn of spray residue content up to ca
11 ppm F.) If calibration mark is exceeded in back-titrn, use
10 mL aliquot of acid filtrate in tube "S," and dil. to 25 mL
with "blank" soln, correcting titer of std F soln by appropriate
factor.
E. Notes on Rapid Method
G-s Nessler tubes are almost essential with coned acid pre-
scribed in this detn. Analysts familiar with Th-alizarin back-
titrn method should have no difficulty with Zr-purpurin titrn.
With latter, however, color changes are not so apparent and
titrn is less sensitive. However, with careful work, results ac-
curate to >0.5 ppm may be expected.
Indicator color at prescribed acidity is yellow, and fully laked
indicator is orange-red. This contrasts with Th titrn where cor-
responding range is from yellowish green to reddish purple.
Hence, in rapid method, choice of end point involves discrim-
ination between varying shades of orange. Addn of 1 .50 mL
Zr soln to tube "B" at start is merely to provide intermediate
shade of orange to guide analyst in amt of Zr to be added to
tube "S. " Analysts may prefer to work with redder or yellower
end point shade. In any event, make number of titrns by add-
ing varying amts of std F soln as unknowns to Nessler tubes
and carrying thru back-titrn as above, for purpose of learning
color changes involved. Pure aq. solns instead of "blank" may
be used, with acidities of 20 mL HC1/50 mL.
Accuracy of results with rapid method presupposes complete
removal of spray residue F by solv. process and good accuracy
(not necessarily precision) in titrn. These conditions may not
always hold; unless carefully done, solv. method may not be
entirely effective, and result on strip solns contg known amts
of F have tended to be slightly low. Hence accuracy >95% is
not to be expected with this method.
Refs.: JAOAC 27, 90, 246(1944); 28, 277(1945); 33,
587(1950).
CAS-7782-41-4 (fluorine)
250
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
944.08 Fluorine in Food
Distillation Method
Final Action
A. Principle
Sample is ashed with Ca(OH) 2 as F fixative; F is isolated
by Wi Hard-Winter distn (Ind. Eng. Chem., Anal. Ed. 5,
7(1933)) from HC10 4 , and estd in distillate by Th(N0 3 ) 4 back-
titrn method (J AOAC 27, 246(1944)). Technic and reagent
concns are designed to handle <10.0 mg F conveniently.
Modifications applicable to specific products are described.
B. Precautions and Interferences
Control magnitude of detn blank by careful choice and pu-
rification of reagents (see 944. 08D). With care, blank will be
low (1-3 (xg F), but with low-F foods it may represent con-
siderable part of total F detd; hence, it must be stable. Large
part of it will be "distn blank" apparently resulting from F
leached from glassware of still during distn. This blank can be
minimized by preliminary treatment of stills, 944. 08F, and av.
distn blank detd if stills of same material and design are rou-
tinely used; otherwise, each still must bear its special blank.
New, unused stills will usually be found to exhibit high blank,
which will diminish to const low figure after several detns.
They should not be used until several consecutive blank detns
yield const, low amt of F.
Check ashing utensils by blank detns with fixative soln to
det. if they contribute appreciable F. Even Pt vessels may be-
come contaminated (owing presumably to slight Ca content)
if they have been used recently for HF volatilization of Si0 2 .
In addn, such blank detns are useful for testing reagents and
app. used in method and also evaporators, hoods, furnaces,
and laboratory atm. for presence of F fumes and dust. If HF
bottles are permitted in same laboratory, seal immediately af-
ter use; avoid contamination from roach powders.
Ordinary tap H 2 may be source of F contamination, since
1 mL H 2 6 contg 2 ppm F will contribute 2 jxg F if allowed
to remain or to dry in still. Therefore, routinely rinse all glass-
ware (stills, flasks, burets, etc.) with H 2 0, preferably redistd
from alk. KMn0 4 . Filter papers may contribute |xg amts of F,
and glass filters are preferred if filtration is required in micro
detns.
Interferences are gelatinous Si0 2 , Al, and B compds, which
repress evolution of F as H 2 SiF 6 in distn; materials such as
nitrates, nitrites, peroxides, CI, S0 2 , and H 2 S, which act upon
indicator in titrn or otherwise interfere; halides (CI), which
distil to give excessive acidity in distillate; and phosphates and
sulfates, which react with Th in titrn to give high results. Method
is so designed that most of these interferences are automati-
cally eliminated, but analyst should be on guard against their
possible occurrence under unusual circumstances.
C. Apparatus
(a) Fluorine still. — Claisen 100-125 mL distg flask is most
practical for general work. It must be of Pyrex glass with aux-
iliary neck sealed off immediately above side arm to prevent
pocketing and refluxing of distillate. Still should be as small
and simply designed as practicable; ordinary distg flasks can
be used for some work and they are slightly more efficient than
Claisen type, except that danger of spraying over of distg acid
is greater.
Equip still with dropping funnel and 0-150° thermometer,
latter extending to within 6 mm of bottom of flask, so that
bulb is immersed in boiling acid mixt. Acid-alkali washed beads,
preferably Pyrex, should be on hand. Clean rubber stoppers
by boiling in 10% NaOH soln. All-glass app. with J acces-
sories is convenient, especially in routine work, and eliminates
need for rubber stoppers.
While not entirely necessary for heating still, use of Wood
metal (50 Bi, 25 Pb, 12.5 Sn, 12.5 Cd) bath, adequately
shielded, will prevent undue decomposition of HC10 4 and aid
materially in securing low blank and low-acid distillate; hence,
its use is strongly urged. If metal bath is used, do not immerse
flask so deeply that bath level is above that of liq. in flask; if
bath is not used, transite or asbestos shielding boards are es-
sential, and flask should be heated thru small hole in such shield
by low "clean" flame. (Bath and shielding boards prevent over-
heating of upper still walls.)
At analyst's option, distg H 2 may be added as steam in-
stead of thru dropping funnel; elec. boiler, Fig. 938.09, is con-
venient steam generator. If steam is used, inlet tube should dip
below surface of liq. in still. One advantage in adding distg
H 2 thru funnel is that last portions of rinse H 2 used in trans-
ferring an ash can be used in distn. If funnel plug is thinly
notched with sharp file on either side of bore, dropping rate
can be more easily controlled, and end of funnel stem need
not extend into liq. in still. Still is used in conjunction with
clean straight-tube condenser no longer than necessary for ad-
equate cooling. (Vertical arrangement of condenser will con-
serve bench space.)
(b) Nessler tubes. — Tall-form, 100 and 50 mL, g-s type
preferred. Matched in sets of >6. (100 mL size is used most
frequently in general method.)
(c) Additional apparatus.— (See 944. 08B.) Carefully cleaned
and tested Pt, or well-glazed porcelain, dishes of >100 mL
size; 150 mL vol. flasks, or if these are not available, 200 mL
size; and 10 mL burets (conveniently automatic) to deliver var-
ious solns required in distn and titrn. Overhead radiant heater
will be found invaluable for drying and preliminary charring
of samples, especially those of high- sugar type.
D. Reagents
(Caution: See safety notes on distillation, hydrofluoric acid,
perchloric acid, and sulfuric acid.)
(a) Lime suspension. — Carefully slake ca 56 g (1 mole) low-
F CaO (ca 2 ppm F) with ca 250 mL H 2 0, and slowly add 250
mL 60% HC10 4 with stirring. Add few glass beads and boil
down to copious fumes of acid; cool, add 200 mL H 2 0, and
boil down again. Repeat diln and boiling down once more;
cool, dil. considerably, and filter thru fritted glass filter, if
pptd Si0 2 appears. Pour clear soln, with stirring, into 1 L NaOH
soln (100 g/L), let ppt settle, and siphon off supernate. Re-
move Na salts from ppt by washing 5 times in large centrf.
bottles, shaking mass thoroly each time. Finally, shake ppt
into suspension and dil. to 2 L. Store in paraffined bottles.
(100 mL of this suspension should give no appreciable F blank
when evapd, distd, and carried thru titrn, (1).) Always shake
suspension well before use.
(b) Perchloric acid soln. —60%. Dil. HC10 4 with 3-4 vols
H 2 and boil down to original vol. Do not fume strongly. Re-
peat, and store in Pyrex. (Prepd acid should be Cl-free by test.)
(c) Sulfuric acid soln. —Carefully mix equal vols H 2 S0 4 and
H 2 0, boil down to fumes, cool, dil. carefully, boil down once
more, and dil. to 1 + I vol.
(d) Silver per chlorate soln. — 50 g/100 mL.
(e) p-Nitrophenol indicator. — 0.5% ale. soln.
(f) Potassium hydroxide soln. — Exactly 0.05 N.
(g) Potassium chloride soln. — 0.05N. 3.728 g/L.
(h) Hydroxy [amine hydrochloride soln. — 1.0%,
(i) Hydrochloric acid soln. — Exactly 0.05 N.
(j) Alizarin indicator. — 0.01% aq. soln of sodium alizarin
sulfonate (Alizarin Red S).
(k) Potassium \ fluo silicate std solns. — (/) Stock soln. — 0.5
mg F/mL Dissolve and dil. 0.9661 g (corrected for purity as
AOAC Official Methods of Analysis (1 990)
Fluorine
251
indicated below) K 2 SiF 6 to 1 L (much more will not dissolve).
Soln keeps indefinitely in paraffined bottle. (2) Working soln. —
10 |xg F/m'L. Prep soln used in titrn, 944.08G, by dilg 20 mL
stock soln to 1 L. Soln is stable several weeks in ordinary
volumetric ware.
If pure K 2 SiF 6 is not obtainable, prep, as follows: Add, thru
dropping funnel, satd soln of NaF, or suspension of crude
K 2 SiF 6 , into 500 mL Claisen distg app. contg 60 mL H 2 S0 4
(1 + 1), some glass beads, and 10-20 g powd Si0 2 (or glass)
kept at boiling temp, of 120-125°. Distil into 25% soln of
KC1, held at simmering temp, on hot plate so that vol. of dis-
tillate does not become excessive. If necessary, add more H 2
to mixt. from dropping funnel in side-neck of still. Regulate
rate of addn of NaF to still and temp, of condensing H 2 so
that side arm and condenser do not become clogged with evolved
H 2 SiF 6 , which tends to lodge as gelatinous mass. K 2 SiF 6 is
formed in receiver, and altho entirely cryst. it assumes ap-
pearance of gelatinous mass.
When substantial amt collects, pour contents of receiver into
large centrf. bottle and wash repeatedly by centrfg (shaking up
ppt thoroly each time), until washings are Cl-free by test. Col-
lect on buchner and either air dry or bring to const wt in vacuo
at 50-70°.
Det. purity by Travers titrn, 921. 04B, at boiling temp, with
0.2 N NaOH (1 mL - 0.01 101 g K 2 SiF 6 ); also by conversion
to K 2 S0 4 by treating 0.3-0.4 g in deep Pt dish with little H 2 0,
then H 2 S0 4 plus little HF, fuming off excess acid carefully (if
overheated, mixt. has tendency to spatter), and heating to const
wt of K 2 S0 4 at 650°. With glass app., entirely pure product
is not usually obtained, as some contamination with Si0 2 re-
sults from leaching effect of vapors on condenser. Pure prod-
uct can be obtained by use of Pt still. Prep, stock soln, cor-
recting wt of 0.9662 by purity factor of the K 2 SiF 6 (figure for
purity obtained from av. of 2 above methods of assay).
(I) Thorium nitrate soln.— 0.25 g Th(N0 3 ) 4 .12H 2 or 0.20
g Th(N0 3 ) 4 .4H 2 0/L. Check titer against std (10 jig/mL) F
soln as follows: Measure 10, 20, 30, etc., up to 80 jxg F into
100 mL Nessler tubes, and add 4.00 mL 0.05AJ HC1 (2.00 mL
if 50 mL Nessler tubes are used, and limiting range to only
50 u.g F) (JAOAC 24, 350(1941)). Oil. mixt. to ca 80 (or 40)
mL mark and add 1.00 mL 1.0% NFLOH.HC1 soln. Mix; then
add exactly 2.00 mL alizarin indicator (or 1 .00 mL for smaller
tube) and add Th soln from buret, mixing frequently until,
when sighting down tube toward white reflecting surface, in-
cipient pink or salmon pink color is observed. Add little H 2
occasionally so that soln is nearly to mark as end point is ap-
proached. Finally, dil. exactly to mark and mix thoroly before
checking final end point. Do not shake tube vigorously (5-6
gentle inversions are enough).
Make effort to secure end point shade intermediate between
yellowish green of acid indicator and reddish purple of fully
developed Th lake. Complete series and plot mL Th soln against
mL std fluoride to obtain rough equivalence curve for 2 solns.
Depending upon amt of F known to be present, add Th soln
in 1-2 mL portions at first, with final addns of 0.25 mL.
E. Preparation of Sample
(Caution: See safety notes on distillation and perchloric
acid.)
Methods of sample prepn are designed to furnish represen-
tative sample in workable amt of material and to obtain sample
in condition for final distn. Mineralization by ashing is usually
involved. Some mineral food products can be dissolved in and
distd from HC10 4 , 944. 08F, provided no interferences appear
in final distillate.
In general, >20 g dry material, 50-100 mL liq. samples,
and 50-100 g undried food products or plant material can be
taken for analysis, depending upon expected F content and in-
terferences, such as excessive CI, which use of large samples
may introduce. For reasonable precision in analysis of low-F
foods, sample should be sufficient to yield titer of >0.5 mL
for aliquot taken in final titrn. However, it may not always be
possible to handle this amt of material. If adequate grinding
and mixing equipment is available, it is often feasible to prep,
large amts of material (vegetables, mixed foods) and to take
aliquot portions for analysis (Ind. Eng. Chem., Anal. Ed. 13,
93(1941)).
Dry plant materials, feeds, bone meal, etc., can be ground
to convenient size in Wiley mill and thoroly mixed before sam-
ple is taken. Following special methods for certain products
are indicated:
(a) Direct ashing. — Applicable to fibrous (not highly fatty)
foods, liq. samples and, in general, to all foods that can be
thoroly wet with aq. fixative soln. This method will apply to
majority of food products.
Weigh suitable portion of prepd sample into clean Pt dish
and add 25 mL Ca(OH) 2 suspension. (Porcelain casseroles or
dishes are second choice because they may contribute small
amts of F and Al 2 3 to sample.) Mix in Ca(OH) 2 suspension
with glass rod, adding addnl H 2 if necessary; rinse and re-
move rod. Dry thoroly on steam bath or in hot air oven; then
slowly char sample by heating over low flame or elec. plate
with thermostat. Overhead radiant heater is convenient for both
drying and charring sample. Control excessive swelling of high
sugar foods by playing small flame over surface of sample
from time to time, and char these products slowly so that ex-
cessive acidity is not generated. When sample is charred past
danger of catching fire, ash in furnace at 600°. (For very small
samples and min. blanks, it may be advisable to cover ashing
vessel with inverted Pyrex petri dish while ashing.)
For plants high in silica, fusion with NaOH may be nec-
essary (Anal. Chem. 25, 450, 1061(1953)).
When clean ash is obtained, cool dish and wet ash with ca
10 mL H 2 0. (Small amt of unburned C does not interfere but
if much is apparent, dry down and repeat ashing.) Cover dish
with watch glass and cautiously introduce under cover just
enough HC10 4 soln to dissolve ash. Rinse down cover with
little H 2 and transfer soln to freshly prepd F still, 944.08F,
thru long-stem funnel. Rinse dish with remainder of distg acid,
using ca 20 mL in all, and adding and transferring in several
small portions. Do not prolong transferring operation. Finally
rinse funnel and stirring rod into dish, assemble still, and com-
plete rinsing of dish with several small portions H 2 0, pouring
these into dropping funnel of still. If distg FLO is added as
steam, 944.08C(a), rinse dish with little addnl FLO and add
directly to acid mixt. in still, but avoid excessive initial vol.
Add ca 6 Pyrex beads and enough AgC10 4 soln, 944.08D(d),
to ppt all CI. (Reasonable excess of AgC10 4 does no harm;
enough solid Ag 2 S0 4 may also be used.) Proceed as in 944.08F.
(b) Preliminary distillation. — (Necessary with certain
products high in phosphate, such as Ca phosphate and bone
meal, in order to eliminate distd H 3 P0 4 that may be present
in appreciable amts in first distillates. Also advisable with cer-
tain excessively fatty materials that may not be thoroly wet
with Ca(OH) 2 fixative, thus causing F loss in direct ashing
method . )
(7) For inorganic phosphatic materials, such as Ca phos-
phate. — Weigh sample, usually 10 g, into still; add few glass
beads, enough AgC10 4 to ppt possible CI, and ca 20 mL HC10 4
soln, (If inorg. phosphatic material does not contain excessive
Ca (enough to cause heavy ppt of CaS0 4 in still), use similar
amt of 1 + 1 H 2 S0 4 .) Distil at 135-140°, collecting ca 200
mL distillate. (For this preliminary distn, extreme care in se-
252
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
curing low-acid distillate is not essential.) Evap. distillate to
dryness in Pt dish after addn of excess Ca(OH) 2 suspension,
assuring aik. conditions by testing with drop of phthln. (If H 2 S0 4
is used in this preliminary distn, add few drops F-free 30%
H 2 2 to distillate to oxidize possible sulfites.) Heat dried res-
idue at 600° few min to destroy indicator residues and possible
Cl-contg compds. Transfer contents of dish to freshly prepd
still, 944.08F, with 20 mL distg HC10 4 soln as in (a), and
proceed with final distn as in 944. 08F,
Take 20 mL samples of sirupy H 3 P0 4 and collect >300 mL
first distillate at 135°, letting H 3 P0 4 function as its own distg
acid. (More distillate is necessary because H 3 P0 4 is less ef-
fective as F distg acid.) Neutze with Ca(OH) 2 suspension, evap.
to dryness, transfer to prepd still as above, and proceed as in
944.08F
(2) For organic phosphatic materials, such as bone meal,
feed supplements, etc. — As preliminary ashing treatment to
destroy most org. matter, moisten sample with enough Ca(OH) 2
suspension, dry, char, and heat 2-3 hr at 600°. Transfer ashed
material to still, which contains several beads and enough
AgC10 4 to ppt CI, with 20 mL distg acid (HC10 4 or H 2 S0 4 ,
depending on Ca content of sample) as in (a), and continue as
in (b)(7), beginning, "Distil at 135-140°, . . ."
Certain org. phosphatic materials (small samples of bone,
2-5 g, such as entire bones of small test animals) in which
amt of org. matter is not excessive, may be distd directly as
in (b)(7) without preliminary ashing. If sample contains ap-
preciable Ca (bone samples), use HC10 4 with reasonable pre-
caution; if org. phosphatic material does not contain excessive
Ca, use 1 + 1 H 2 S0 4 . In either case, add more Ca(OH) 2 to
first distillates and ash for longer periods to completely destroy
distd org. matter (fatty acids). Transfer contents of dish to freshly
prepd still, 944.08F, with 20 mL HC10 4 soln as in (a) and
proceed with final distn, 944. 08F.
Baking powders (Ca phosphate and combination types): Place
10 g sample in deep, covered Pt dish or casserole and slake
cautiously with ca 20 mL Ca(OH) 2 suspension. After action
subsides, rinse cover, dry contents of dish thoroly, and ash 2-
3 hr at 600°. Cool dish and, because of excess of carbonate
in ash, treat it with several small portions of warm H 2 0, break-
ing up with flat-end stirring rod, and transfer leachings to still.
Transfer remaining contents of dish with 20 mL HC10 4 soln,
avoiding excessive effervescence when acid is added to car-
bonate soln in still. Add several glass beads and enough AgC10 4
soln, and proceed as in (b)(7), beginning, "Distil at 135-140°,
..." With combination or Na Al sulfate baking powders, col-
lect >400 mL preliminary distillate, (b)(4).
Use of special still trap makes possible analysis of highly
phosphatic inorg. or thoroly ashed materials, and phosphoric
acids, with single distn. Special trap, or scrubber, consists of
12-15 g small, hollow glass beads supported in side-neck of
the 125 mL Claisen flask by several indentations punched in
side wall, and capped by glass disk or inverted bottom of 15
mm test tube. After construction of glass-bead scrubber, side-
neck is sealed off immediately above outlet tube. (Beads in
scrubber must be wet with little H 3 P0 4 (by tipping flask) be-
fore distn to furnish liq. acid phase.) Take 20 mL sirupy H 3 P0 4 ,
by itself, and 10 g samples Ca phosphate with 20 mL HC10 4
soln, for distn, and collect >400 mL distillate at 135°. With
single distn, observe precautions in 944.08C(a), and also in
944. 08F, regarding neutzn of final distillates. (Distillates should
show practically no acidity.) Presence of only traces of distd
H 3 P0 4 will vitiate titrn; as little as 20 |utg P 2 5 will definitely
interfere. Accordingly, if single distn procedure is to be ap-
plied with confidence, it is necessary to test distillates obtained
from phosphatic materials, by means of the special still, for
presence of this interference.
For convenient test utilizing Schricker reagent (J AOAC 22,
167(1939)), add 5 mL of 1 + 9 diln of this reagent to 45 mL
distillate in 50 mL graduate or Nessler tube, mix, and immerse
in steam bath 5-10 min. Compare against blank by sighting
down tube. Blue or blue-green color indicates phosphate, and
as little as 5 |xg (as P 2 5 ) is readily detected. If distillate shows
traces, make sure that such amts are below interference level
of 15 |xg in titrn aliquot before titrg addnl portions of distillate.
(Test with Schricker reagent is also useful in usual double distn
where phosphate interference is possible. Use of special trap
will save time where highly phosphatic materials are handled
routinely, but it is not justified in ordinary work because of
poor efficiency owing to excessive refluxing in distn.)
(3) For excessively fatty and oily food materials (oil-packed
foods, certain meats, etc.; also entire undried and un ground
organs of test animals). — If there is danger of F loss thru in-
complete wetting with Ca(OH) 2 fixation soln, handle as fol-
lows: Weigh appropriate amt of sample, usually 10-25 g, into
still, and add Ag (preferably 0.1-0.2 g solid Ag 2 S0 4 ), several
glass beads, and 20-25 mL H 2 S0 4 (1 + 1). Distil at 130-
1 35° and collect 200-250 mL distillate in beaker or open ves-
sel. If foaming is excessive, increase vol. of distg acid, and
where necessary, use larger (250-300 mL) still. If larger still
or more acid is used, collect proportionately more of first dis-
tillate. (Oil or fat of many of these products will tend to pre-
vent foaming, and, in some instances, use of ca pea-size piece
of pure paraffin is addnl aid.)
Oxidize distillate in cold by cautious addn of 2-3 mL F-
free 30% H 2 2 to remove sulfites, let stand few min, and evap.
portionwise in Pt dish contg excess (10—15 mL) Ca(OH) 2 sus-
pension. Ash residue at 600° until clean. Proceed as in (b)(7),
beginning "Transfer contents of dish to freshly prepd still, ..."
Handle pure oils by similar procedure: Use 10 g sample with
25 mL H 2 S0 4 (1 + 1) and carry temp, at first to ca 170° to
saponify; then carefully bring temp, down to 140° with distg
H 2 and collect >250 mL distillate. (It will probably be nec-
essary to use higher reading thermometer for this procedure.)
Oxidize distillate with 30% H 2 2 and evap. to dryness after
adding excess Ca(OH) 2 suspension. Ash at 600° and after brief
preliminary ash period remove dish, add little H 2 plus addnl
1-2 mL of the H 2 2 to remove sulfides, dry, and complete
ashing. Proceed as in (b)(7), beginning "Transfer contents of
dish to freshly prepd still, ..."
(4) For aluminum and boron compounds . — Al and B re-
press evolution of F. Isolate F by preliminary distn at elevated
temp. For this purpose, weigh sample, usually 5-10 g, into
still, add 25 mL H 2 S0 4 (1 + 1), and conduct first distn at 160-
165° (special thermometer), collecting 300 mL distillate. Ox-
idize distillate with 30% H 2 2 as above, evap. in Pt dish with
excess Ca(OH) 2 suspension, ash briefly at 600°, and proceed
as in (b)(7), beginning, "Transfer contents of dish to freshly
prepd still, ..."
F. Final Distillation
(Caution: See safety notes on distillation and perchloric
acid.)
Always make final distn from HC10 4 , and take precautions
to secure low-acid distillate, 944.08C(a). Since interferences,
such as org. matter, phosphate, sulfate, etc., must be absent
from distillate, make distn with careful temp, control in pres-
ence of enough Ag salt to repress HC1 evolution (944. 08B).
It is well to check distillates for presence of possible phosphate
as in 944.08E(b)(2), and where advisable, as in (b)(4), to test
for sulfate with little dil. BaCl 2 soln. HC10 4 used in final distn
is usually used in transferring ash to still, 944.08E(a). Few
AOAC Official Methods of Analysis (1990)
Lead
253
acid-alkali washed beads are used to control bumping. (Use of
powd Si0 2 does not appear necessary for microdetn.)
To promote better recoveries, and to minimize and render
const distn blank discussed in 944.08B and 944. 08G, prep.
still by special cleaning process before this transfer by treating
it with hot 10% NaOH soln after each detn, flushing out with
tap H 2 0, and then rinsing with distd H 2 0. Occasionally (at
least once daily, and especially after it has stood idle for any
length of time), give still addnl treatment by boiling down 15-
20 ml H 2 S0 4 (1 + 1) until still is filled with fumes. Cool,
pour off acid, treat with the 10% NaOH soln, and thoroly rinse
out. (Cleaning should be especially meticulous after high-F or
high-Si0 2 samples have been distd, and in such cases con-
denser should also be cleaned.)
At this stage, prepd sample has been transferred to specially
treated still, as directed above, for final isolation of F. Begin
distn, and when temp, reaches 137°, keep at this point (±2°)
by adding H 2 from dropping funnel, 944.08C(a). Heat still
at such rate that all distns require ca same time. (Time pro-
motes uniformity in blank correction.) Collect distillate in 150
or 200 mL vol. flask. After few mL distillate collects, add 1-
2 drops /7-nitrophenol indicator, (e), and keep distillate alk. to
this indicator (faintest perceptible yellow) by occasionally add-
ing 1-2 drops 0.05 N KOH from 10 mL buret during distn
while swirling receiver. So regulate this addn of alkali that
distillate is neutzd (within 1 drop of alkali) as it approaches
mark. Carefully note vol. alkali used. Dil. distillate to vol. and
mix thoroly. Do not let F distillate stand more than few min
before neutzg.
If sample contains such large amts of CI that bumping in
still cannot be controlled, dissolve ash of another sample, and
acidify slightly with H00 4 . Dil. considerably and ppt CI in
dish with AgC10 4 soln, avoiding large excess. Filter thru glass
filter, wash ppt thoroly with hot H 2 0, and evap. filtrate and
washings to dryness after adding excess (to alky) of Ca(OH) 2
suspension. Transfer residue to still with H00 4 soln and re-
peat distn as above.
G. Titration
Place aliquot of final distillate in Nessler tube and mark "S"
(sample). (Optimum F content for titrn is 60-70 jxg for 100
mL Nessler tubes and 30-40 fxg for 50 mL size, and it is well
to make exploratory titrn on small aliquot to check approx. F
content of distillate. Larger tubes are necessary for precise re-
sults on low-F foods.)
Add 0.05 N HC1, 4.00 mL for 100 mL tubes and 2.00 mL
for 50 mL size, and 1.00 mL H 2 NOH.HCl soln. (For routine
work with 100 mL tubes, dissolve 1.0 g H 2 NOH.HCl in 500
mL 0.04 W HC1 and dil. to 500 mL. Then proper amt of both
reagents can be added in single operation with 5 mL pipet.)
Dil. to ca 90 (or 40) mL, mix well, then add alizarin indicator
(2.00 or 1.00 mL), and mix again. Always add and mix in
H 2 NOH.HCl before adding indicator.
Prep, blank tube "B" by adding proper amt HCt and
H 2 NOH.HCl, and amt 0.05 N KC1 soln representing same pro-
portion of total vol. of 0.05 N KOH used to neutze distillate
as aliquot vol. taken for sample tube represents of total dis-
tillate vol. (Thus, if 1.50 mL 0.05 N KOH was used to neutze
distillate of 150 mL and aliquot taken for tube "S" was 75
mL, add 0.75 mL 0.05 TV KCl to tube "B.") Dil. and mix,
allowing slightly more headspace than in sample tube. Then
add proper vol. alizarin indicator and mix.
Measure Th soln into tube "S," mixing between addns, until
end point of about proper shade is reached. Dil. to mark, mix,
and check this end point shade. Note from curve, 944.08D(1),
approx. vol. std F soln corresponding to this vol. Th soln, and
add ca 0.5 mL less than this amt of std F soln to "B." Mix;
then add exactly same vol. Th soln as was added to "S," du-
plicating approx. increments in which it was added and num-
ber of mixings. Dil. nearly to mark and compare colors of "S"
and "B." (If vol. std F soln added to "B" was properly chosen,
this tube should be only slightly pinker in shade than sample
tube.)
Bleach "B" tube to exact match with tube "S" by adding
more std F soln to "B" in increments of 1-2 drops, mixing
gently between addns, Dil. to mark for final comparison and
observe usual precautions of letting bubbles subside and of
transposing tubes when final comparisons are made. (At match-
point, F content of tube "S" equals amt added to tube "B.")
Check this end point by adding 1-2 drops excess std F soln
to tube "B." Distinct overbleach should develop.
Repeat titrn on aliquots of different size to obtain total amt
of F distd. If time is available, repeat entire detn with different
wt sample.
For precise work, evaluation of reagent and of distn blank
is necessary, 944. 08B. Det. distn blank by making several distns
with prescribed amts HC10 4 and AgC10 4 solns from freshly
cleaned still, titrg distillate as above with as large aliquot as
practicable. Av. of values found should be <3 jxg F. If amts
found by individual blank detns are too small to be detd ac-
curately, make >5 sep. distns and evap. distillates, 150 mL
each time, successively in same Pt dish for final distn and av-
erage blank figure. Distn and total detn blanks can usually be
combined by carrying run (with same amts of reagents and
similar evapn and ashing treatment) thru entire detn. Reagents
and manipulations should increase distn blank but little.
Calc. total amt F distd from amt found in aliquot titrd, sub-
tract proper blank, and refer net figure to wt sample taken. If
double distn procedure was used, make appropriate blank cor-
rection.
Refs.: JAOAC 27, 90, 246(1944); 28, 277(1945); 33, 587
(1950).
CAS-7782-41-4 (fluorine)
935.50 Lead
Suitability of Methods and Precautions
A. Principle
Instrumental methods, polarography and atomic absorption
(AA) spectrophotometry, are generally more reliable than col-
orimetric method at lower concns. Method 972.25 is partic-
ularly applicable to samples contg high Ca concn. Special in-
strumental methods optimized for evapd milk and fish are given
in 973.35, 974.13, 979.17, 972.23, and 972.24.
General colorimetric method calls for ashing, 934. 07B, sepn
of Pb, either as dithizone complex, 934. 07D, or as sulfide,
934.07E, followed by colorimetric dithizone detn, 934.07F,
in comparator tubes, or with spectrophtr. Interference is treated
sep., 934.07G-I, and analyst should become familiar with de-
tails of these sections before applying method. Special meth-
ods of sample prepn are given in 934.07 J and K.
B. Precautions
Analyst should decide whether nature of detn requires un-
usual care in purification of reagents, or whether blank detn
will suffice. Smaller the amt of Pb to be detd, greater the care
required in reduction of blank (see also 934. 07F).
To test suitability of reagents, place 10-15 g solid reagents
dissolved in redistd H 2 or 15-20 mL coned acids previously
neutzd with redistd NH 4 OH in separator and add enough Pb-
free citric acid to prevent pptn by NH 4 OH of Fe, Al, alk. earth
phosphates, or other substances. Make soln ammoniacal and
254
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
add 2-3 mL 10% KCN soln. Shake soln with ca 5 ml dithi-
zone soln, 934.07A(e) (5-10 mg/L). If lower layer is green,
transfer it to another separator and ext excess dithizone with
NH 4 OH (1 + 99) to which has been added drop of KCN soln.
If C.H.CI3 layer is colorless, consider test neg. for use with
dithizone methods.
When special purification becomes necessary, redistil H 2
(distd H 2 stored in Sn-lined tanks usually contains Pb and
Sn), HNO3, HC1, HBr, Br, and CHCI3 in all-glass (pyrex) or
quartz stills (preferably quartz). Prep. NH 4 OH by distg ordi-
nary reagent into ice-cold redistd H 2 0. If stills are new, steam
them out with hot HC1 or HN0 3 vapors to remove "surface"
Pb. (Subsequent distillates may not be totally Pb-free.)
Pb(N0 3 ) 2 may be purified as follows: Dissolve 20-50 g in
min. of hot H 2 and cool with stirring. Filter crystals with
suction on small buchner, redissolve, and recrystallize. Dry
crystals at 100-1 10° to const wt. Cool in desiccator and store
in tightly stoppered bottle. (Product has no H 2 of crystn and
is not appreciably hygroscopic.)
Purify citric acid, NaOAc or NH 4 OAc, A1(N0 3 ) 3 , Ca(N0 3 ) 2 ,
and Na 2 S0 4 by pptg Pb from their aq. solns with H 2 S (Cau-
tion: See safety notes on hydrogen sulfide.), using 5-10 mg
CuS0 4 as coprecipitant (citric acid and A1(N0 3 ) 3 solns require
adjustment with NH 4 OH to pH 3.0-3.5, bromophenol blue in-
dicator). Filter (fritted glass filter is most convenient), boil fil-
trates 20 min to expel excess H 2 S, and re filter if necessary to
obtain brilliantly clear solns. Purify other reagents by recrystn.
Store redistd acids or purified solns of reagents in Teflon or
conventional polyethylene containers carefully cleaned of sur-
face Pb with hot HNO3. Paraffin-lined bottles may be used for
alk. reagents.
Carefully clean new glass, plastic, and chemical ware with
hot 10% NaOH soln followed by hot HNO3, and use only for
Pb detns.
In prepn of samples for analysis, avoid Pb contamination.
If mixing or grinding is necessary, use porcelain mortar if pos-
sible. Avoid use of metal food grinders unless previous ex-
periment has shown that no contamination of sample with Pb
or Sn results. If product to be analyzed cannot be thoroly mixed
in its own container, or if composite sample of number of con-
tainers is desired, empty into large glass jar or porcelain dish
and mix thoroly with wooden spoon or porcelain spatula. If
liq. portion of sample cannot be incorporated into ground solid
material to obtain homogeneous mixt., analyze sep. If food is
packed in tins having soldered seams (sardines and meats),
open tins from bottom to avoid contaminating sample with bits
of solder. Avoid sifting in prepn of samples to prevent metallic
contamination or segregation of Pb.
CAS-7439-92-1 (lead)
935.51 Lead on Apples and Pears
Colorimetric Method
Final Action
(Efficiency of 95% expected)
(For rapid detn of Pb spray residue on apples and pears; ppm
x 0.007 = grains/lb; (grains/lb) x 143 - ppm)
A. Preparation of Sample
Weigh >10 units and pull or cut out stems with narrow-
blade knife, cutting no more of flesh than necessary. Trim off
sepals (dried residue of blossom) and discard sepals and stems.
To 25 mL 30% NaOH soln in 600 mL beaker, add 175 mL
H 2 and 25 mL Na oleate soln, (k), and bring to gentle boil.
Have ready in wash bottle 250 mL hot HN0 3 (2 + 98) or hot
HC1 (3 + 97). (Reasonably accurate figure for As 2 3 can be
obtained by using the HO rinse and applying Gutzeit As detn,
912.01*, to portion of filtrate, after acidifying part of the 500
mL alk. strip soln with V10 vol. HC1 instead of HN0 3 (see
later in this section). Rapid method for F, 944. 07D, likewise
specifies HC1 rinse and acidification.)
Impale each fruit in turn upon pointed glass rod; immerse
in the alk. soln, with occasional rotation, until skin begins to
check; then remove to large funnel inserted in 500 mL vol.
flask and rinse with stream of the hot acid, being careful to
flush out stem and calyx ends thoroly. When all fruit has been
so treated, cool alk. soln and add it thru funnel to acid soln
in flask. Rinse beaker and funnel with any remaining acid and
with H 2 0, using entire 250 mL rinse acid. Cool, and dil. to
vol.
In dry 200 mL erlenmeyer place exactly 10 mL HN0 3 (10
mL HO for As or F). Thoroly mix contents of vol. flask and
immediately add 100 mL to acid in erlenmeyer while swirling
vigorously. Filter on rapid paper. If first portion of filtrate is
cloudy, refilter until clear. Det. Pb as in 935. 51B or C.
(See JAOAC 26, 150(1943)) for details of churn-type washer
for removing Pb spray residues from apples and pears.)
B. Determination with Messier Tubes
(At least 15 tall-form tubes matched for uniformity in color
and diam., are necessary. Caution: See safety notes on cya-
nides.)
(a) Stds. — To each of two 1 L vol. flasks add 47.5 mL 30%
NaOH soln. If HNO3 was used in rinsing and acidification,
935.51A, add 100 mL HNO3 to each flask. If HC1 (3 + 97)
was used in rinsing, add 91 mL HNO3 and 13.6 mL HO to
each flask. Do not mix in the acids unless solns are cold and
dil. To one flask add stock reagent, (a), equiv. to 25.45 mg
Pb. Mark this flask "std" and other "blank." Dil. both solns
to vol. at room temp, and mix. These 2 soJns contain reagents
as they occur in acidified and filtered sample soln. The "std"
is equiv. in Pb content to acidified soln from sample of 1400
g carrying Pb load (removable by "stripping" operation) of 10
ppm. By combination of the 2 solns in suitable proportions,
equiv. of any Pb load from to 10 ppm may be obtained.
Std tubes made up in intervals corresponding to 1.0 ppm
may be interpolated to 0.5 ppm. Following table gives vols of
"std" and "blank" to be added to Nessler tubes for each in-
terval; measure into tube by burets:
Pb,
"Standard,"
"Blank,"
ppm
mL
mL
0.0
0.0
10.0
1.0
1.0
9.0
2.0
2.0
8.0
3.0
3.0
7.0
4.0
4.0
6.0
5.0
5.0
5.0
6.0
6.0
4.0
7.0
7.0
3.0
8.0
8.0
2.0
9.0
9.0
1.0
10.0
10.0
0.0
Working with 1 tube at time, add 10 mL NH 3 -cyani de-ci-
trate soln, (1), to each tube followed by 30 mL std dithizone
soln, 30 mg/L, 934.07A(e). Shake vigorously 1 min and let
sep. The pH of aq. phase should be c a 9.4 regardless of whether
HC1 or HNO3 is used in rinsing. Stopper each std tube securely
with new cork stopper. It is unnecessary to make up entire
series of stds if only portion of range, e.g. 5.0—10.0 ppm, is
of quant, interest.
AOAC Official Methods of Analysis (1990)
(b) Comparison. -^Transfer 10 mL portions of clear filtrate
from 935. 51 A to each of 3 Nessler tubes. First add 10 mL
NH 3 -cyanide-citrate soln, (1), to each tube; to one tube add 30
mL std dithizone soln, 30 mg/L, 934.07 A(e), and to other 2
tubes 30 mL clear CHC1 3 . Shake vigorously 1 min and let sep.
With tube of clear CHC1 3 backing sample tube (contg the dithi-
zone) and 1 sample tube contg CHC1 3 backing each of 2 std
tubes, compare color in lower layer of sample with that of
stds, looking thru tubes at right angles to their lengths toward
strong diffused light. (Comparator box similar to boxes used
in col ori metric pH measurements but of larger size is conve-
nient. When working with apple strip solns, slight turbidity is
produced in sample tube, which slightly changes color ob-
served. To compensate for this effect, same turbidity is intro-
duced in field of view of std tubes made up exactly as sample,
except that CHC1 3 is substituted for the dithizone soln.)
If color produced by sample is redder than 10 ppm std, re-
peat with smaller aliquot of filtrate, dilg to 10 mL with "blank"
soln. If, for example, 5 mL aliquot is taken, indicated reading
must be doubled. After match is obtained, calc. result to basis
of 10 mL aliquot and 1400 g sample.
C. Determination with Photometer
(This method is suitable for photometric measurement of "mixed
color," 934.07F(b). Changes in 935. 51B are introduced here
to prevent formation of colors too dense for measurements.
Use 5 mL instead of 10 mL aliquots of acidified wash soln,
935.51A.)
Lead
255
(a) Stds. — Measure following proportions of "std"
* blank" solns, 935.51B(a), into separators:
and
Pb,
"Standard,"
"Blank,"
ppm
mL
mL
0.0
0.0
10.0
2.0
1.0
9.0
4.0
2.0
8.0
6.0
3.0
7.0
8.0
4.0
6.0
10.0
5.0
5.0
Add 10 mL NH 3 -cyanide-citrate soln, (I), and working with
1 separator at time, immediately develop color by shaking 1
min with 50 mL pure dithizone soln of 10 mg/L strength. Let
stand few min to cool, filter CHC1 3 layers thru specially washed
papers, (m), and fill cell of appropriate length (1 cm is con-
venient). Det. A and plot against ppm Pb to obtain std curve.
(b) Comparison. — Place appropriate size aliquot of acidi-
fied strip soln in separator and dil. to 10 mL with "blank"
soln. Add 10 mL NH 3 -cyanide-citrate soln, (I), and ext with
50 mL 10 mg/L std dithizone soln. Let stand few min to cool,
filter, and read as above. Det. amt of Pb from std curve prepd
as in (a) and calc. to basis of 5 mL aliquot and 1400 g sample.
CAS-7439-92-1 (lead)
973.35 Lead in Evaporated Milk
Atomic Absorption Spectrophotometry Method
First Action 1973
Final Action 1974
(Caution: See safety notes on AAS.)
A. Principle
Sample is dry-ashed; Pb is extd as the 1-pyrrolidinecarbo-
dithioate into BuOAc, and detd by A A spectrophotometry at
283.3 nm.
B. Apparatus
(a) Atomic absorption spectrophotometer . — Equipped with
4" single slot burner head.
(b) Ashing vessels. — Approx. 100 mL, flat-bottom Pt cru-
cible or dish, Vycor or quartz tall-form beaker, or evapg dish
(Corning Glass Works, No. 13180, or equiv.). Discard Vycor
vessels when inner surfaces become etched.
(c) Centrifuge. — Capable of holding 15 mL conical tubes
and centrfg at 2000 rpm.
(d) Furnace. — With pyrometer to control range of 250-600°
with variation <10°.
C. Reagents
(a) Nitric acid. — IN. See 972.25C(c).
(b) Butyl acetate. — Spectral grade, H 2 0-satd.
(c) Ammonium 1 -pyrrolidinecarbodithioate (APDC) .—2%.
Dissolve 2.00 g APDC in 100 mL distd or deionized H 2 0.
Remove insol. free acid and other impurities normally present
by 2-3 extns with 10 mL portions BuOAc.
(d) Lead std solns. — (1) Stock soln. — 1 mg Pb/mL IN
HN0 3 . See 972.25C(d)(7). (2) Intermediate soln.— 5.0 |xg
Pb/mL. Pipet 5 mL stock soln into I L vol. flask, add 1 mL
HNO3, and dil. to vol. with H 2 0. (Soln is stable several months
if stored in polyethylene bottle.) (3) Working solns. — Pipet
20, 10, 5, and 2 mL intermediate soln into sep. 100 mL vol.
flasks, and dil. to vol. with \N HN0 3 (1.0, 0.50, 0.25, and
0.10 u,g Pb/ mL, resp.). Pipet 10 and 5 mL soln contg 0.50
|xg Pb/mL into sep. 100 mL vol. flasks, and dil. to vol. with
IN HNO3 (0.05 and 0.025 \xg Pb/mL, resp.).
(e) Citric acid soln. — 10%. Weigh 10.0 g Pb-free citric acid
into 100 mL vol. flask, dissolve in H 2 0, and dil. to vol. Stop-
per flask and shake thoroly. If necessary, remove Pb impurity
as in 972.24D.
(f) Bromocresol green. — 0.1%. pH range, 3.8 (yellow) to
5.4 (blue). Transfer 0.100 g bromocresol green, Na salt, to
100 mL vol. flask, and dil. to vol. with H 2 0. Use 1 drop/10
mL anal. soln.
D. Ashing
(Clean all glassware thoroly in HNO3 (1 4- 1).)
Weigh ca 25 g (to nearest 0.1 g) sample into ashing vessel.
Dry samples overnight in 120° forced-draft oven. (Sample must
be absolutely dry to prevent flowing or spattering in furnace.)
Place sample in furnace set at 250°. Slowly (50° increments)
raise temp, to 350° and hold at this temp, until smoking ceases.
Increase temp, to 500° in ca 75° increments (sample must not
ignite). Ash 16 hr (overnight) at 500°. Remove from furnace
and let cool. Ash should be white and essentially C-free. If
ash still contains excess C particles (i.e., ash is gray rather
than white), proceed as follows: Wet with min. amt H 2 fol-
lowed by dropwise addn of HN0 3 (0.5-3 mL). Dry on hot
plate. Transfer to furnace at 250°, slowly increase temp, to
500°, and continue heating 1-2 hr. Repeat HN0 3 treatment
and ashing if necessary to obtain C-free residue. (Note: Local
overheating or deflagration may result if sample still contains
much intermingled C and especially if much K is present in
ash (see 934.07B).)
Dissolve residue in 5 mL 17V HNO3, warming on steam bath
or hot plate 2-3 min to aid soln. Filter, if necessary, by de-
cantation through S&S 589 black paper into 50 mL vol. flask.
Repeat with two 5 mL portions \N HNO3, filter, and add
washings to original filtrate. Dil. to vol. with IN HN0 3 .
Prep, duplicate reagent blanks for stds and samples, includ-
ing any addnl H 2 and HN0 3 , if used for sample ashing. Note:
Do not "ash" HNO3 in furnace, since Pb contaminant will be
lost. Dry HN0 3 in ashing vessel on steam bath or hot plate,
and then proceed as above.
256
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
E. Extraction
(Complete analysis on same day.)
Pipet 20 mL each working soln, reagent blank for stds (if
different from that used for samples), sample soln, and ap-
propriate reagent blank(s) for samples into sep. 60 mL sepa-
rators. Treat each soln as follows: Add 4 mL citric acid soln,
(e), and 2-3 drops bromocresol green indicator, (f). (Color of
soln should be yellow.) Adjust pH to ca 5.4, using NH 4 OH
initially and then NH 4 OH-H 2 (1 + 4) in vicinity of color
change (first permanent appearance of light blue) . Add 4 mL
APDC soln, (c), stopper, and shake 30-60 sec. Pipet in 5 mL
BuOAc, (b). Stopper separator and shake vigorously ca 30-
60 sec. Let stand until layers sep. cleanly; drain and discard
lower aq. phase. If emulsion forms or solv. layer is cloudy,
drain solv. layer into 15 mL centrf. tube, cover with Al foil
or Parafilm, and centrf. ca 1 min at 2000 rpm.
F. Determination
Set instrument to previously detd optimum conditions for
org. solv. aspiration (3-5 mL/min), using 283.3 nm Pb line
and air-C 2 H 2 flame adjusted for max. Pb absorption. Flame
will be somewhat fuel-lean. Optimum position in flame for
max. absorption should be just above burner top. If using re-
corder, DCR, etc., adjust to manufacturer's specifications.
Depending upon signal-to-noise ratio, scale expansion up to
10x may be used. Check point while aspirating H 2 0-satd
BuOAc. Aspirate sample and std solns, flushing with H 2 and
then BuOAc between measurements. Record A of each soln.
Prep, std curve by plotting A of each std corrected for blank
against concn of that std in |mg Pb/mL BuOAc. Concn of std
in BuOAc is 4 times that in aq. std. Det. Pb concn from std
curve, using A corrected for sample reagent blank, if used.
ppm Pb — [(jxg Pb/mL from curve)
x 5 (mL BuOAc)] /(g sample x 20/50)
Ref.: JAOAC56, 1246(1973).
CAS-7439-92-1 (lead)
974.13 Lead in Evaporated Milk
Anodic Stripping Voltammetric Method
First Action 1974
Final Action 1976
A. Principie
Evapd milk is dry-ashed and residue is dissolved in dil. HN0 3 .
Pb is electroanal. coned on hanging Hg drop or Hg film elec-
trode, and detd by reversing potential sweep and measuring
anodic current peak.
B. Apparatus
See 982.23B(a), (b), (c), and (d).
C. Ashing
Proceed as in 973.35D.
D. Preparation of Standard Curve
Prep, stds as in 973.35C(d) and 17V HN0 3 reagent blank.
Transfer 10-20 mL std soln to cell, depending on cell capac-
ity. Adjust to 25 ± 1°, and bubble N thru soln 5 min. Adjust
gas inlet tube to let N flow gently above and across soln sur-
face. If hanging drop Hg electrode is used, add fresh drop Hg
to capillary tip with micrometer (Hg drop must be reproducible
for each measurement), turn on stirrer motor, and electrolyze
soln 1-10 min at -0.6 v against satd calomel electrode (time
depends on manufacturer's instructions). Stop stirring and let
soln stand 30 sec. Linearly increase applied voltage (pos. volt-
age scan). Use manufacturer's instructions for rate of scan,
e.g., 2-6 mv/sec. Measure wave ht at half-wave potential
(-0.45 ± 0.05 v against satd calomel electrode). Plot fxg Pb/
mL std soln against wave ht x sensitivity factor (SF). Repeat
for each std soln and reagent blank soln. Prep, new curve with
each batch of samples.
E. Determination
Transfer to cell same vol. sample soln as used in stdzn. Bub-
ble N, add Hg drop, and apply voltage as in stdzn. Measure
wave ht at appropriate potential and det. Pb concn by com-
paring wave ht of sample soln with that of std curve or analyze
std soln immediately before or after sample soln (preferable
when instrument scale factor must be changed).
Prep, reagent blank soln as in 973. 35D, last par., and ana-
lyze as above.
F. Calculations
Calc. u,g Pb/mL as follows:
C = C'{\{WH X SF) - (WH B X SF B )]/
l(WH' x SF') - (WH' B x SF' B )]}
where C and C — (xg Pb/mL sample and std, resp.; SF, SF' ,
SF B , and SF' B = sensitivity factor settings of sample, std, and
reagent blank (may be different for sample (SF B ) and std (SF' B ))
solns, resp.; and WH, WH 1 ' B , WH B , and WH' = wave hts.
ppm Pb = (C x 50) /g sample
Ref.: J AOAC 56, 1246(1973).
CAS-7439-92-1 (lead)
979.17 Lead in Evaporated Milk
and Fruit Juice
Anodic Stripping Voltammetric Method
First Action 1979
Final Action 1984
A. Apparatus
(a) Voltammetric analyzer. — With staircase anodic strip-
ping ramp and graphite electrode coated with thin film of Hg.
Capable of measuring 5 ng Pb in presence of dissolved O.
(Solns cannot be deaerated). Peak area integration desirable.
ESA Model 3010A Trace Metals Analyzer (ESA, Inc., 45
Wiggins Ave, Bedford MA 01730), or equiv.
(b) Micropipets. — 50, 100, 200, and 300 jxL, pos. dis-
placement type. (SMI or Drummond, available from supply
houses, or equiv.).
B. Reagents
(Use deionized H 2 to prep, std solns. Prep, and store solns
in same Pyrex vol. flasks. Do not wash flasks with strong acids
between use; just rinse 3 times with deionized H 2 0. Always
prep, same soln in same flask.)
(a) Lead releasing reagent. — Contg <1 ppb Pb. Acid soln
of cation able to displace Pb from sample. Metexchange Re-
agent (ESA, Inc.), or equiv.
(b) Lead std solns. — (1) Stock soln. — 1 mg/mL. Prep, as
in 972.25C(d)(7). (2) Intermediate soln. — 10 jmg/mL. Pipet
1 mL stock soln into 100 mL vol. flask contg 1.0 mL HN0 3
and ca 50 mL H 2 0. Mix, and dil. to vol. Prep, each week.
After soln is prepd 6 times in same flask, it is stable 1 month.
(3) Working soln for fruit juice detn. — 3 u^g/mL. Pipet 30 mL
soln (2) into 100 mL vol. flask contg 0.7 mL HN0 3 and ca
50 mL H 2 0. Mix, and dil. to vol. (4) Working soln for evap-
AOAC Official Methods of Analysis (1990)
Lead
257
orated milk detn. — 1 |xg/mL. Prep, as in (3), using 10 mL
soln (2) and 1.0 mL HN0 3 . Working solns are stable 3 days.
After being prepd 5 times in same flask, they are stable 2 weeks.
(5) Calibration solns. — Evapd milk or fruit juice of type being
detd, and contg > 0.5 ppm added Pb.
C. Determination
Calibrate instrument according to manufacturer's directions.
Mix aliquot of sample with releasing reagent, (a), and perform
detn according to manufacturer's instructions. Data for ESA
analyzer are as follows:
Calibration
Juice
Milk
Initial potential, v
Final potential, v
Sweep rate, mv/step
Integration set points,
Sample size, |xl_
-1.025 ± 0.005
-0.100 ± 0.005
14.0 ± 0.05
-0.490 ± 0.005
300
-1.090 ± 0.005
-0.100 ± 0.005
10.50 ± 0.05
-0.490 ± 0.005
200
Run control or spiked sample with each 15-20 analyses in
a series.
Refs.: JAOAC 66, 1409, 1414(1983).
CAS-7439-92-1 (lead)
972.23 Lead in Fish
Atomic Absorption Spectrophotometric Method
First Action 1972
Final Action 1976
(Caution: See safety notes on AAS.)
A. Apparatus
(a) Atomic absorption spectrophotometer. — See 973.35B(a);
range 0-10 u,g/mL.
(b) Lead lamp. — Hollow cathode Pb lamp.
(c) Crucible. — Porcelain, ca 50 mL capacity and 5 cm deep;
or tall-form Vycor or quartz beaker, 100 mL.
B. Reagents
(a) Hydrochloric acid. — IN. Oil. 82 mL HC1 to 1 L with
H 2 0.
(b) Lead std solns. — (I) Stock soln. — 1 mg Pb/mL 17V
HN0 3 . See 972.25C(d). (2) Working soln. — 10 jxg Pb/mL.
Pipet 10 mL stock soln into 1 L vol. flask, add 82 mL HC1,
and dil. to vol. with H 2 0.
(c) Buffer soln.— Disperse 163 g EDTA in 200 mL H 2 in
2 L vol. flask and add enough NH 4 OH to dissolve. Dil. 60
mL 70.5% HCIO4 (Caution: See safety notes on perchloric
acid) by pouring carefully into ca 500 mL H 2 and cool. Dis-
solve 50 g La 2 3 in HCIO4 soln. Add 8 drops Me orange in-
dicator to ammoniacal EDTA soln and add La 2 3 soln to EDTA
soln while stirring vigorously, if necessary, add NH 4 OH to
maintain alky of above soln to Me orange. Dil. to 2 L.
C. Reagent Blank
(Caution: See safety notes on nitric acid.)
Before proceeding with analysis, test purity of reagents as
follows: Evap. 4 mL HNO3 in crucible to dryness on hot plate
or steam bath, dissolve residue in 17V HC1, and transfer to 25
mL vol. flask. Heat residue again successively with two 5 mL
portions 17V HC1 and add to flask. Cool, dil. to vol. with 17V
HO, and mix. Proceed with detn. Total reagent blank should
be <10 fxg Pb (equiv. to 0.4 ppm in sample) for detns at levels
>1 ppm. For detns at <1 ppm, purify reagents as in 973. 50B
to attain blank <50% of limiting level of concern.
D. Preparation of Sample
Weigh ca 25 g (to nearest 0.1 g) sample into crucible, (c),
and dry 2 hr at 135-150°. Transfer to cold, temp. -controlled
furnace and slowly raise temp, to 500°. Set control and check
for maintenance of 500°. (Temp, as low as 550° may cause
loss of Pb.) Ash overnight (16 hr). Remove sample, let cool
to room temp., cautiously add 2 mL HNO3, and swirl. Evap.
carefully just to dryness on warm hot plate or steam bath.
Transfer to cooled furnace, slowly raise temp, to 500°, and
hold at this temp. 1 hr. Remove dish and cool. Repeat HN0 3
ashing, if necessary, to obtain clean, practically C-free ash.
Add 10 mL 17V HO and dissolve ash by heating cautiously on
hot plate. Transfer to 25 mL vol. flask. Heat ash residue again
successively with two 5 mL portions 17V HO and add to flask.
Cool, dil. to vol. with 17V HO, and mix.
E. Preparation of Standard Curve
Transfer 0, 1, 3, 5, 15, 25, and 50 mL Pb working soln,
972.23B(b)(2), to sep. 50 mL vol. flasks and dil. to vol. with
17V HO (0, 0.2, 0.6, 1.0, 3.0, 5.0, and 10.0 \xg Pb/mL, resp.).
Set spectrophtr to previously established optimum conditions
for max. signal at 283.3 nm. Use air-C 2 H 2 flow rates rec-
ommended by manufacturer for std conditions for Pb. For dig-
ital concn readout, calibrate in concn mode with solns contg
0.2 and 10.0 jxg Pb/mL. Record concn directly after calibra-
tion of instrument. For strip chart readout, set amplification to
give >1% absorption reading for 0.2 |mg/ mL working soln
and prep, std curve of A against jxg Pb/mL.
F. Determination
Use aliquot of sample soln, 972. 23D, and proceed as in (a)
or (b). Treat reagent blank, 972. 23C, as sample and subtract
reading from A of samples.
(a) Clear solns. — Det. A of sample and std solns as in
972. 23E, using following sequence 3 times: Read std soln first,
then sample soln, alternating until all sample and std solns
have been read. When many samples are to be analyzed, std
solns may be read after series of 3 samples instead of after
each.
ppm Pb - [(|mg Pb/mL sample soln) x 25] /g sample
(b) Cloudy solns. — Proceed as in (a), but add 1 mL buffer
soln, (c), to aliquots of sample and std solns before reading.
If addnl dilns are necessary or if buffer is added:
ppm Pb = (|xg Pb/mL dild sample)
x (mL dild sample/mL original aliquot) x (25 /g sample)
Ref.: JAOAC 55, 727, 733(1972); 56, 406(1973).
CAS-7439-92-1 (lead)
972.24
Lead in Fish
PoBarographic Method
Final Action 1974
A. Apparatus
Polarograph. — Any voltammetric or polarographic instru-
ment with necessary accessories (cells, electrodes, Hg, cap-
illaries, etc.) capable of effectively scanning up to 3.0 volts
in either pos. or neg. direction, starting at selected initial po-
tential, and of measuring >1.0 ppm Pb.
B. Preparation of Standard Curve
Transfer 0, 1 , 3, 5, 15, 25, and 50 mL Pb std working soln,
972.23B(b)(2), to sep. 50 mL vol. flasks and dil. to vol. with
17V HC1 (0, 0.2, 0.6, 1.0, 3.0, 5.0, and 10.0 u,g Pb/mL, resp.).
Transfer 5 mL soln to polarographic cell, adjust to 25 ± 1°,
258
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
and bubble N thru soln 5 min. Polarograph between —0.2 and
-0.7 v against Hg pool ref. electrode.
Peak potential for Pb at 25° is -0.45 ± 0.05 v. Plot u,g Pb/
mL cell soln against wave ht X sensitivity factor.
C. Determination
Transfer 5 mL sample soln to polarographic cell, adjust to
25 ± 1°, bubble N thru soln 5 min, and polarograph as in
972. 24B. Measure ht of wave whose potential corresponds to
that of Pb and det. concn from newly prepd std curve or, pref-
erably, by comparing wave ht of sample soln with that of std
soln polarographed immediately before or after sample. Use
latter method for greater accuracy, particularly when it is nec-
essary to change instrument scale factor.
C u (u.g Pb/mL) = (C s x WH U x SF U )/(WH S x SF S )
where subscripts s and u refer to std and sample, resp.; C =
jxg Pb/mL cell soln; WH = wave ht; and SF = sensitivity
factor setting.
ppm Pb = (C u x 25)/g sample
D. Interference from Tin
Sn polarograph s at same peak potential as Pb. If presence
of Sn is suspected, add 1 mL NH 4 OH and 0.4 g tartaric acid
to cell soln, bubble N thru soln, and polarograph as in 972. 24B.
Treat std in same manner. Peak potential for Pb is 0.54 v. Sn
does not polarograph at this peak potential.
Ref.: JAOAC 55, 727, 733(1972).
CAS-7439-92-1 (lead)
972.25 Lead in Food
Atomic Absorption Spectrophotometric Method
First Action 1972
Final Action 1976
A. Principle
(Caution: See safety notes on AAS, wet oxidation, nitric acid,
perchloric acid, and sulfuric acid.)
Org. matter is digested and Pb released co-ppts with SrS0 4 .
Sol. sulfate salts are decanted, and ppt is converted to car-
bonate salt, dissolved in acid, and detd by AAS at 217.0 or
283.3 nm.
B. Apparatus
(a) Atomic absorption spectrophotometer. — Operated at 217
or 283.3 nm.)
(b) Stirring motor. — With eccentric coupling for stirring
centrf. tubes (Sargent-Welch Scientific Co. Model S-76509-
21 C (Vortex, Jr.), or equiv.).
C. Reagents
(Age all new glassware and all glassware which has contained
high Pb concn in boiling HN0 3 before washing. Never let used
glassware dry before washing, and always include final HN0 3
rinse followed by deionized H 2 rinse.)
(a) Strontium soln.— 2%. Dissolve 6 g SrCl 2 .6H 2 in 100
mL H 2 0.
(b) Ternary acid mixture. — Add 20 mL H 2 S0 4 to 100 mL
H 2 0, mix, add 100 mL HN0 3 and 40 mL 70% HC10 4 , and
mix.
(c) Nitric acid. — Add 128 mL redistd HN0 3 to 500-800
mL distd or deionized H 2 and dil. to 2 L. Redistd HN0 3 (G.
Frederick Smith Chemical Co., No. 63) may be dild and used
without redistn.
(d) Lead std so Ins. — (J) Stock soln. — 1000 juig/mL. Dis-
solve 1.5985 g Pb(N0 3 ) 2 , recrystd as in 935.50B, in ca 500
mL IN HN0 3 in 1 L vol. flask and dil. to vol. with 17V HN0 3 .
(2) Working solns. — Prep. 100 jxg Pb/mL by dilg 10 mL stock
soln to 100 mL with IN HN0 3 . Dil. 1, 3, 5, 10, 15, and 25
mL aliquots of this soln to 100 mL with IN HN0 3 (1, 3, 5,
10, 15, and 25 |xg Pb/mL, resp.).
D. Separation of Lead
Accurately weigh sample contg <10 g dry matter and >3
[jig Pb. Place in 500 mL boiling or Kjeldahl flask and add 1
mL 2% Sr soln, (a), and several glass beads. Prep, reagent
blank and carry thru same operations as sample. Add 15 mL
ternary acid mixt., (b), for each g dry matter and let stand >:2
hr. Heat under hood or H 2 vac. manifold system until flask
contains only H 2 S0 4 and inorg. salts. (Note: Take care to avoid
sample loss from foaming when heat is first applied, and when
foaming occurs soon after sample chars. Remove heat and swirl •
flask before continuing digestion. Add HN0 3 , if necessary.)
Cool digest few min. (Digest should be cool enough to add
ca 15 mL H 2 safely, but hot enough to boil when H 2 is
added.) Wash while still hot into 40-50 mL tapered-bottom
centrf. tube and swirl. Let cool, centrf. 10 min at 350 x g 7
and decant liq. into waste beaker. (Film- like ppt on surface
may be discarded.) Dislodge ppt by vigorously stirring with
eccentric-coupled stirring motor. To complete transfer, add 20
mL H 2 and 1 mL IN H 2 S0 4 to original flask and heat. Do
not omit this step even though it appears transfer was complete
in first wash. Wash hot contents of original digestion flask into
centrf. tube contg ppt. Swirl to mix, cool, centrf., and decant
liq. into waste beaker.
Dislodge ppt by stirring vigorously, add 25 mL satd
(NH 4 ) 2 C0 3 soln (ca 20%), and stir until all ppt is dispersed.
Let stand 1 hr, centrf., and decant liq. into waste beaker. Re-
peat (NH 4 ) 2 C0 3 treatment.
After decanting, invert centrf. tube on paper towel and drain
all liq. Add 5 mL \N HN0 3 (use larger vol. 17V HN0 3 in both
sample and blank if >25 |xg Pb is expected), stir vigorously
to expel C0 2 or use ultrasonic bath 2-3 min, let stand 30 min,
and centrf. if ppt remains. (Use same technic for all samples.)
E. Determination
Set instrument to previously established optimum condi-
tions, using air-C 2 H 2 oxidizing flame and 217 or 283.3 nm
resonant wavelength. Det. A of sample and blank solns and
>5 stds within optimum working range (10-80% T) before
and after sample readings. Flush burner with IN HN0 3 and
check point between readings. Det. Pb from std curve of A
against |xg Pb/mL:
ppm Pb - [(|xg Pb/mL) x (mL 1W HN0 3 )]/g sample
Ref.: JAOAC 55, 737(1972).
CAS-7439-92-1 (lead)
934.07 Lead in Food
General Dithizone Method
Final Action
(Sn and Bi Absent)
(Applicable to such materials as carbohydrates, cereals and cereal
products, cacao and dairy products, feeds, meats, fish, plant
material, fruit and fruit products, fresh vegetables, etc., and
in general to all org. materials (except fats) in which no Sn
AOAC Official Methods of Analysis (1990)
Lead
259
and Bi are encountered. For products contg Sn (canned foods)
or Bi, proceed as in 934.07G-I.)
A. Reagents
(Caution: See safety notes on bromine.)
(a) Lead std solns. — (/) Stock soln. — 2 mg Pb (3.197 mg
Pb(N0 3 ) 2 )/mL in 1% HN0 3 . Prep, from Pb(N0 3 ) 2 purified as
in 935. SOB. (2) Working solns. — Prep, as needed by dilg stock
soln with 1% HN0 3 .
(b) Nitric acid.—\%. Dil. 10 mL fresh, colorless HN0 3
(sp gr 1.40) to 1 L with redistd H 2 0. If acid has been redistd,
boil off nitrous fumes before dilg.
(c) "Ash-aid" jo/n.— Dissolve 40 g A1(N0 3 ) 3 .9H 2 and 20
g Ca(N0 3 ) 2 .4H 2 in 100 mL H 2 0.
(d) Citric acid soln. — Coned Pb-free soln. 1 mL - 0.5 g
citric acid (reagent partially neutzd with NH 4 OH during pu-
rification, 935.50B, fifth par.).
(e) Diphenylthiocarbazone (dithizone). — Dissolve ca 1 g
com. reagent in 50-75 mL CHC1 3 and filter if in sol. material
remains. Ext in separator with four 100 mL portions metal-
free (redistd) NH4OH (1 + 99). (Dithizone passes into aq. phase
to give orange soln.) Filter aq. exts into large separator thru
cotton pledget inserted in stem of funnel. Acidify slightly with
dil. HO and ext pptd dithizone with two or three 20 mL por-
tions CHCI3. Combine exts in separator and wash 2 or 3 times
with H 2 0. Drain CHC1 3 into beaker and evap. with gentle heat
on steam bath, avoiding spattering as soln goes to dryness.
Remove last traces of moisture by heating 1 hr at <50° in
vacuo. Store dry reagent in dark in tightly stoppered bottle.
Prep, reagent solns for extn to contain 100, 50, and 10 mg/
L in freshly redistd CHC1 3 (JAOAC 21, 695(1938); 26,
26(1943)) and store in dark at 5-10°. (Stock soln of dithizone
in CHCI3 contg 1 mg/mL will keep long time and is conve-
nient for use in making dilns.) Soln of 30 mg/L CHC1 3 stored
in dispensing app. is required for use in rapid method, 935.51B.
(f) Ammonia-cyanide mixture. — To 100 mL 10% recrystd,
P0 4 -free KCN (JAOAC 20, 191(1937)) in 500 mL vol. flask,
add enough redistd NH 4 OH to introduce 19.1 g NH 3 , and dil.
to vol. with redistd H 2 0. (Concn of redistd NH 4 OH can be
detd by sp gr or titrn.)
(g) Pure metallic tin. — Purest obtainable, such as NIST
Sample No. 42. Granulate Sn as finely as possible by melting
and pouring very slowly into H 2 0. Det. Pb content as follows:
Dissolve 1-2 g sample in HBr or HC1 and volatilize Sn by
evapg soln to dryness and treating with several 5 mL portions
of the HBr-Br mixt., (h), evapg to dryness on steam bath after
each treatment. Take up with 2-3 mL HN0 3 , evap. to dryness
to expel Br, and take up with ca 50 mL hot H 2 0. Filter, and
proceed as in 934. 07D and F.
(h) Hydrobromic acid-bromine mixture. — To 250 mL 40%
redistd HBr add 35 mL redistd liq. Br.
(i) Sodium poly sulfide soln. — Dissolve 480 g Na 2 S.9H 2
and 40 g NaOH in H 2 0, add 16 g powd S, shake until S dis-
solves, filter, and dil. to 1 L.
(j) Hydrochloric-citric acid soln. — Add vol. reagent (d)
equiv. to 50 g citric acid to 50 mL HC1 and dil. to 250 mL.
(k) Sodium oleate soln,— 10%. To 45 mL 30% NaOH soln
and 400 mL H 2 in 1.5 L beaker, add slowly, while heating
and stirring, 90 g (by difference from separator) oleic acid.
Heat mixt. on steam bath until soap is entirely dissolved. (Small
flocculent ppt of impurities may remain.) Cool, dil. to 1 L,
mix, and filter.
(I) Ammonia-cyanide-citrate soln. — Dissolve 10 g phos-
phate-free KCN and 10 g citric acid in 250 mL NH 4 OH (sp
gr 0.90) and dil. to 1 L. Reagent is conveniently preserved in
dispensing app. that causes min. volatilization of NH 3 .
(m) Washed filter paper. — Soak 9 cm quant, papers over-
night in 1% HNO3. Wash with large vols H z O on buchner to
remove acid and any traces of Pb.
B. Preparation of Sample (Ashing)
(Caution: See safety notes on hydrofluoric acid and perchloric
acid.)
Accurately weigh representative sample of 5-200 g, de-
pending upon amt sample available and expected Pb content,
into suitable porcelain dish or casserole. Dry wet samples on
steam bath or in oven. Add 2-5 mL "ash-aid" soln, (c), to
products difficult to ash (meats), or to furnish ash bulk to low-
ash products (candies, and jellies low in fruit content); mix
well, and dry.
Char gelatin, carbohydrate foods such as jam, and other
products that tend to swell excessively by carefully heating
over burner. (Swelling can be controlled by playing small flame
from glass jet over surface of material in dish, but metallic
burner must not be used because of possible metallic contam-
ination.) Do not let material ignite. Milk, candies, etc., may
be charred without ignition by adding sample little at time to
casserole heated over burner or hot plate. (Overhead radiant
heater is often very convenient.) When samples are dry or
charred, place in temp. -controlled furnace and raise temp, slowly
to 500° without ignition.
If sample contains fat, "smoke" it away by heating long
enough at ca 350°. Cover floor of furnace with piece of as-
bestos board or Si0 2 plate so that sample receives most of its
heat by radiation from sides and roof and not by conduction
from hotter floor of furnace.
If furnace has automatic control, ash overnight at <500°. If
sample is not completely ashed next morning or if day-time
ashings at 500° are not proceeding satisfactorily, remove cas-
serole, cool, and moisten char with 2-5 mL ash-aid. Dry con-
tents of casserole past danger of spattering (no free liq.) and
replace in furnace. If ashing is not complete or proceeding
rapidly after 30 min, remove casserole, cool, and cautiously
add 2-3 mL HNO3. Dry, place in furnace, and continue ashing
until practically C-free. Avoid excessive use of ash-aid, and
particularly HNO3, if sample still contains much intermixed
C, because local overheating or deflagration may result, es-
pecially if much K is present in ash.
When clean ash is obtained, cool, cover casserole with watch
glass, and cautiously add 15-20 mL HO. Rinse down watch
glass with H 2 and heat on steam bath. If clear soln is not
obtained, evap. again to dryness and repeat addn of HC1. If
insol. matter persists, evap. HC1 and dehydrate Si0 2 by heat-
ing to fumes with 5-10 mL 60% HC10 4 (double distd pre-
ferred). If HC10 4 is used, considerable H 2 (200 mL) may be
necessary to completely dissolve KC10 4 later, as when KCN
is used in dithizone extn of Pb, 934. 07D.
Dil. with H.O and filter soln when necessary with suction
thru fine fritted glass filter. Catch filtrate in 500 mL g-s er-
lenmeyer under bell jar. Leach insol. material on filter suc-
cessively with few mL hot HO, hot HCl-citric acid soln, and
hot 40% NH 4 OAc soln.
In certain instances take following special precautions:
(/) If amt of insol. material (Si0 2 ) remaining on filter is
abnormal, flush it into Pt dish with H 2 0, evap., and treat res-
idue with one or two 5 mL portions HF. Evap. to dryness,
take up residue with H 2 and few drops of HO or H00 4 ,
and add to bulk of ash filtrate.
(2) When ashing is of long duration, no ash-aid has been
used, or natural ash is low with little ash bulk, Pb may be
baked on dish. To remove this Pb, add few pellets (2-3 g) of
NaOH and dissolve in few mL hot H 2 0. Tilt dish so that sirupy
260
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
soln completely wets that portion of interior originally occu-
pied by sample; then heat short time on steam bath, but do not
bring to dryness. (Overheating with coned NaOH may result
in extg few |xg Pb from casserole. Porcelain retains Pb to less
extent than does Si0 2 but may contain very small amts of Pb.)
Take up residue with H 2 and add directly to filtrate. Finally
rinse dish with few mL hot HCl followed by hot H 2 0.
C. Isolation of Lead: Principle
Method 934. 07D, while rapid and convenient, is limited to
those materials that, with aid of citric acid, yield clear am-
nion iacal soln required for quant, extn of Pb with dithizone.
Pb is readily occluded by many alk. ppts (Mg and Ca phos-
phates, Al and Fe hydroxides and silicates). Many food ma-
terials may be handled in this way because the naturally oc-
curring amts of these substances are not excessive. However,
some materials contain more of these substances than can be
kept in soln under alk. conditions with any reasonable amt of
citric acid (J AOAC 26, 26(1943)). In these cases proceed as
in 934. 07C. Difficulty of ammoniacal pptn may sometimes be
overcome by limiting sample size in cases where sampling is
no problem.
D. Dithizone Extraction
(Applicable to most carbohydrates and cereal foods, fruit and
fruit products, milk, fresh vegetables, plant materials, etc.)
Transfer ash soln to 300 mL short- stem separator and add
citric acid reagent, (d), equiv. to 10 g citric acid. Make slightly
alk. to litmus with NH 4 OH, keeping soln cool, and let stand
1-2 min. If ppt forms, redissolve with HCl and isolate Pb as
in 934.07C. If no ppt forms, add 5 mL 10% KCN soln (more
may be necessary if large amts of Zn, Cu, Cd, etc., are pres-
ent), and check pH of soln by adding drop of thymol blue soln
and observing color of drop (pH should be >8.5, blue-green
to blue with thymol blue).
If ash was highly colored with Fe, keep pH of soln com-
paratively low, because pH of >10 in presence of Fe may
cause oxidn of dithizone. Immediately ext with 20 mL portions
dithizone reagent, using more dil. solns unless exceptionally
large amts of Pb are present. Shake 20-30 sec, let layers sep.,
and note color of CHC1 3 phase. (Pb dithizone complex is red,
but color may be masked by excess green dithizone, giving
intermediate hues of purple and crimson. Color of CHC1 3 ext
gives first indication of amt of Pb present, and progress of extn
can be followed by noting color of successive exts.)
Drain exts directly into small separator contg 25 mL 1%
HN0 3 , (b). When extn is complete, shake combined exts in
smaller separator and drain green dithizone layer into another
separator contg addnl 25 mL portion 1% HN0 3 . Shake, let
layers sep., and discard CHC1 3 fraction. Filter acid exts contg
Pb in succession thru small pledget of wet cotton inserted in
stem of small funnel, into 50 mL flask or g-s graduate, using
second acid ext to rinse separator in which first acid extn was
made. (This procedure removes CHCJ 3 globules.) Make up any
slight deficiency in vol. with 1% HN0 3 and mix. Proceed as
in 934.07F.
E. Sulfide Separation
(Applicable to all products and usually necessary in case
of cacao products, tea, sardines, and all food products contg
high proportion of alk. earth phosphates, especially those of
Mg, which promote formation of ppts in ammoniacal citrate
solns.)
Cool acid soln of ash, add citric acid soln, (d), equiv. to
10 g citric acid, and adjust to pH 3.0-3.4 (bromophenol blue)
with NH4OH. If enough Fe is present to color soln strongly,
make final adjustment with help of spot plate. (Phosphates pptd
by local action of NH 4 OH may usually be redissolved by shak-
ing and cooling.) If amt of Pb is small, add 5-10 mg pure
CuS0 4 .5H 2 to soln to act as coprecipitant. Ppt sulfides by
passing in H 2 S until soln is satd (3-5 min). (Caution: See safety
notes on hydrogen sulfide.) Immediately filter with suction into
flask in bell jar (fine fritted glass filter is preferred).
Dissolve sulfides, without previous washing, with 5 mL hot
HNO3, drawing soln thru into original flask; wash with hot
H 2 0, stopper, shake, and boil to remove H 2 S. Transfer to 200
mL separator, add citric acid soln equiv. to 5 g citric acid,
make ammoniacal, ext, and det. Pb as in 934.070 and F(a)
or (b).
F. Colorimetric Dithizone Determination
(Pb 0.001-0.200 mg)
Limiting factor in detn of minute amts of Pb by colorimetric
dithizone method is size of reagent blank, particularly when
amts of Pb of order of 1-5 u.g are being detd. With special
care in purification of reagents and by use of carefully cleaned
Pyrex ware, including separators, it is possible to reduce re-
agent blank to ^1 fig. Owing to Pb-bearing dust, vapors, etc. ,
it is necessary to expose blank detn in furnace or on steam
bath for same length of time as sample is exposed, and to use
exactly same amts of reagents (even H 2 0) for blank and actual
detns.
Pb is extd from aq. soln, under std conditions of vol. and
pH, with definite vol. of CHC1 3 soln of dithizone of std concn.
Optimum pH of operation is 9,5-10.0. Dithizone strengths are
so chosen that excess dithizone is always present in reaction
mixt. Pb is brought into CHCI 3 phase in form of red complex,
and uncombined green dithizone partitions between aq. and
CHC1 3 phases and modifies color of ext according to relative
amts of Pb and dithizone. Thus, series of colors from red to
green may be arranged with intermediate crimsons, purples,
and blues. Vols and strengths of CHC1 3 solns depend upon Pb
range it is desired to cover and are so chosen as to give same
general color progression from red to green for each range.
Limiting range increases accuracy at expense of flexibility.
Colors produced with std amts of Pb furnish basis for quant,
estn by comparison. Vols and conens of std dithizone for var-
ious ranges are as follows when 1 cm cell is used:
Pb Ranges, \xg
Concn, mg/L
Volume, mL
1-10
0-50
0-200
8
10
20
5
25
40
See Anal. Chem. 19, 684(1947), for modification operated
atpH 11.5.
(a) Simple color matching. — Prep. 10 stds covering in equal
steps the desired concn range, as follows: Use std Pb soln,
934.07A(a), in 1% HN0 3 , 1 mL of which equals some simple
fraction or multiple of 1 jxg Pb. Measure vols representing
various steps of range into series of separators and add 1%
HNO3 so that total vol. is always 50 mL. (Add acid first so
that Pb soln is not lost around stopcock of separator.) Add 10
mL NH 3 -cyanide mixt., (f), and mix. Resultant pH will be ca
9.7. Immediately add appropriate vol. std dithizone soln, which
depends on range to be covered (see table), and shake 1 min.
Drain lower layers into series of tubes or vials and arrange in
order. For lower ranges, i.e., <20 (xg Pb, matching is best
done by viewing longitudinally in small flat-bottom vials ca
75 mm long. For higher ranges, 20—50 |xg and above, depth
of column must be reduced, and matching is conveniently done
AOAC Official Methods of Analysis (1990)
Lead
261
by viewing transversely in Nessler tubes of matched diam.,
because even pure dithizone solns appear red by transmitted
light if concn or depth of column is increased beyond certain
point. If stds are kept covered when not in use, they should
last >1 day.
For detn, place aliquot part, or entire amt, of the 50 mL 1%
HN0 3 in which Pb has been isolated, 934.07D or E, in sep-
arator, and if aliquot is taken, dil. to 50 mL with 1% HN0 3 .
Add 10 mL NH^-cyanide mixt., (f), and mix. Immediately
develop color by shaking I min with proper amt std dithizone
soln. Drain lower layer into tube or vial similar to those used
with stds and compare. If range is exceeded, repeat with smaller
aliquot or re-ext with excess dithizone before draining from
separator, isolate once more in 50 mL 1% HN0 3 reagent, and
compare with stds covering higher range. Interpolation be-
tween steps of various ranges should be easily made.
If aliquot of the 50 mL 1% HNO3 in which Pb has been
isolated is taken, subtract only corresponding amt of total re-
agent blank from amt of Pb found.
(b) Photometric methods. — Absorption spectra of the 2
components in dithizone ext (Pb dithizone complex and free
dithizone) show marked difference in ability to absorb 510 nm
light, red Pb complex absorbing stongly and green dithizone
transmitting freely. Thus, when absorption of light of this
wavelength is detd photometrically, linear relationship is ob-
served between amt of Pb and A. In making measurements,
spectrophtr set at this wavelength or photometer equipped with
blue-green filter centered at about this point can be used.
Stdze dithizone solns as follows: Using appropriate vols and
concns of solns specified for various ranges (see above) in sep-
arators, prep, std colors as in visual color-matching procedure,
satg std Pb and 1 % HN0 3 solns with clear CHC1 3 before use,
and thereby eliminating differences in vol. of ext between stds
and unknowns. (It is unnecessary to prep, full 10 steps of the
range, and number of stds may be limited to 5 or 6.) Develop
colors by shaking separators 1 min, let stand few min, and
filter exts thru specially prepd papers, (m). (Fitting 9 cm paper
directly into mouth of 50 mL Pyrex beaker eliminates need of
funnel in filtering operation.) Fill cell with filtered exts and
det. A for various steps of range.
Plot against amt of Pb to obtain std curve for particular lot
of dithizone. Preferably calc. slope of line connecting std points
and intercept of line on A axis, making calcn by least squares
method as in Definitions of Terms and Explanatory Notes.
Det. Pb content of unknown falling within the range of detg
A, using std dithizone and same cell with which std readings
were made, and calc. Pb from equation X - (Y/h) — (a/b),
using values of a and b detd previously. If protected from evapn
and direct sunlight, std factors of dithizone solns should not
change appreciably for > 1 month (JAOAC 21, 695(1938); 26,
26(1943)).
For actual detn proceed as in (a), except to filter ext thru
prepd papers before photometric measurement. Det. A, using
stdzd dithizone with same cell used in making std curve, and
read amt of Pb from this std curve or calc. from factor of
dithizone soln. If range is exceeded, repeat with smaller ali-
quot, or re-ext and repeat with dithizone stdzd to cover higher
range. If aliquot of the 50 mL 1% HNO^ in which Pb has been
isolated is taken, subtract only corresponding amt of total re-
agent blank from amt of Pb found.
Refs.: JAOAC 19, 130(1936). Ind. Eng. Chem. Anal. Ed. 11,
400(1939).
G. Interferences
Interferences in col ori metric dithizone method are limited by
use of KCN to Sn +2 , Bi, and Tl. Rarity of Tl makes its in-
terference unlikely in ordinary work, and no method of re-
moval is given (JAOAC 26, 26(1943)). Dithizone itself is de-
stroyed by strong oxidizing agents, such as free halogens and
large amts of ferric Fe, under conditions of dithizone extn of
Pb.
H. Removal of Tin
(Caution: See safety notes on perchloric acid, bromine, and
hydrogen sulfide.)
Sn becomes problem in analysis of canned foods; in amts
>150 ppm it will usually appear in ash soln as milky suspen-
sion of Sn0 2 . It must be dissolved to facilitate filtration and
to release occluded Pb. Quantities of Sn of this order may cause
trouble by pptg under conditions of dithizone extn of Pb,
934.07D.
Two methods for elimination of larger amts of Sn are given:
(a) Volatilization as SnBr 4 from acid soln of ash, and (b)
leaching mixed sulfides with warm Na poly sulfide soln, when
sulfide method of isolation, 934. 07E, has been applied. These
methods may not eliminate Sn completely. Stannic Sn is not
extd with dithizone, and as small amts of residual Sn will be
in Sn +4 form after application of either (a) or (b), final iso-
lation of Pb by dithizone extn will eliminate Sn completely.
In general, amts <100 mg should not interfere in colori-
metric dithizone methods of Pb detn provided Sn is in Sn H " 4
form and preliminary isolation with dithizone is made; hence,
this method of isolation should be applied wherever possible.
(a) Volatilization as SnBr 4 from acid soln of ash. — After
almost C-free ash is obtained, 934.07B, add 15-20 mL 40%
redistd HBr. If nitrates were used as ash aids, cover casserole
with watch glass and heat on steam bath until Br evolution
diminishes; then rinse watch glass with H 2 and bring to boil
to complete expulsion of Br. (This process destroys undecom-
posed nitrates.) Add more HBr if necessary to dissolve ash,
and examine solns for clearness. If there is insol. residue of
Sn0 2 , add 50-100 mg pure Sn, (g), to simmering HBr soln
of ash and let it dissolve. (Metallic Sn is best agent to bring
ignited Sn0 2 into soln. To be effective, ash soln must be in
reduced state. Fe 2 3 sometimes becomes "noble" during ash-
ing and dissolves with difficulty, but treatment with metallic
Sn also brings it into soln. Treatment with Sn is necessary only
with contents of badly corroded cans.)
When soln of ash is free from milkiness due to Sn0 2 , add
20 mL 60% HCJO4 (double distd preferred), oxidize mixt. with
few mL HBr-Br mixt., (h), and then add addnl 15 mL of the
reagent portionwise, while soln is evapd to incipient fumes of
HCIO4 (ca 150°) on hot plate. Repeat with addnl 10 mL por-
tion HBr-Br mixt. if >100 mg Sn was used to dissolve ash.
(Hot HCIO4 helps keep ash salts in soln and with Br holds Sn
as volatile SnBr 4 .) When HBr and Br are completely volatil-
ized, cool, and take up with hot H 2 (200 mL may be nec-
essary if much KCIO4 is present). Filter off any small amts of
dehydrated Si0 2 , ext residue twice with 5 mL hot HC I -citric
acid reagent, (j), and hot H 2 0, treat dish if necessary with
NaOH as in 934.07B(2), and isolate Pb by dithizone extn as
in 934.07D, or by sulfide sepn, 934.07E, finally detg Pb as
in 934.07F(a) or (b).
(b) With sodium poly sulfide. — (Recommended for routine
work on canned foods.)
Isolate Pb by sulfide pptn, 934. 07E, filter, and wash flask
and filter with 3-6 portions of ca 5 mL each of warm Na
polysulfide soln, (i). (Sn, As, and Sb sulfides are dissolved;
CuS may be partially dissolved and repptd in filtrate.) Wash
flask and residual sulfides several times with 3% Na 2 S0 4 soln
adjusted to pH 3.0-3.4 and satd with H 2 S, and proceed as in
934. 07E, beginning "Dissolve sulfides, without previous
washing, . . .** When ash contains much Sn, as when metallic
262
Metals and Other Elements
AOAC Official Methods of Analysis (1 990)
Sn has been added to dissolve insol. metallic oxides, sulfide
ppt will be so bulky as to be difficult to handle, and it will be
necessary to use volatilization method (a) before sulfiding. For
colorimetric dithizone detn of Pb, ext HND 3 soln of dissolved
sulfides and proceed as in 934. 07E and F(a) or (b).
/. Detection and Removal of Bismuth
(Caution: See safety notes on nitric acid, bromine, cyanides,
and arsenic trioxide.)
(a) By dithizone at pH 2.0 after preliminary dithizone ex-
traction at pH 8-11 . — (This method completely removes small
amts of Bi.) (Ind. Eng. Chem. Anal. Ed. 7, 285(1935)).
Ext metals from CHC1 3 dithizone ext with 50 raL 1% HN0 3
as in 934. 07D. Adjust acid ext to pH 2.0 (metacresol purple
indicator) with 5% NH 4 OH soln and shake vigorously ca 1 min
with 10 mL CHC1 3 soln of dithizone (200-250 mg/L). Let
layers sep., and if CHC1 3 ext is orange red to red (Bi), drain
off and ext with addnl 10 mL dithizone soln. If shades of green
or purple are visible, indicating excess dithizone, drain CHC1 3
ext and ext aq. phase once more with 5 mL dithizone soln
(shaking should be prolonged (3-5 min) to ensure complete
extn of Bi). Continue extns until dithizone ext remains pure
green. Adjust aq. soln to pH .8.5 with NH 4 OH, add KCN, and
ext with dithizone as in 934. 07D. Det. Pb colori metrically as
in 934.07F(a) or (b).
(Method of Bambach and Burkey (Ind. Eng. Chem., Anal.
Ed. 14, 904(1942)) seps small amts of Bi from Pb by shaking
out CHC1 3 soln of their mixed dithizonates with aq. soln buff-
ered at pH 3.4; Bi remains as dithizonate in CHC1 3 phase,
while Pb enters aq. phase and can be sepd Bi-free. Only slight
excess of free dithizone should be present in CHC1 3 mixt. of
dithizonates, otherwise Pb does not strip out completely. Sys-
tem of photometric detection and evaluation of Bi interference
has also been outlined (J AOAC 26, 26(1943)).
(b) From acid soln of sulfides.— (Intended for small amts
of Bi, particularly when sulfide sepns may be necessary.) Dis-
solve mixed sulfides, 934. 07E, with hot HN0 3 and sep. Bi
and Pb as in (a).
Special conditions. — (Intended for products contg large amts
of Bi.) Dissolve inorg. Bi compds directly in HBr-Br, (h).
Prep. org. Bi compds or Bi prepns mixed with org. matter
contg little ash, as in 934. 07B, and dissolve residue in HBr-
Br. If sample contains org. matter with appreciable ash ma-
terial other than Bi compds, proceed as in 934. 07B or K, ap-
ply sulfide sepn, 934.07E, and dissolve mixed sulfides in HN0 3 .
Evap. HNO3 soln of sulfides to dryness in porcelain dish and
treat with small portions HBr-Br mixt. Evap. contents of dish
contg Bi dissolved in HBr-Br, after any of above methods of
prepn, on steam bath to volatilize Sn and to convert other met-
als to bromides. Evap. to dryness, place in furnace with temp,
control, and raise temp, gradually to 300°. (AsBr 3 and SbBr 3
volatilize first at 100° or above; BiBr 3 volatilizes as dense or-
ange fumes at 300°.) After 5 min, or when fumes cease, re-
move dish, cool and treat again with small portions HBr-Br.
Again evap. to dryness and heat addnl 5 min at 300-325° (PbBr 2
does not volatilize appreciably at <350°). Remove dish, cool,
and dissolve residue in hot HN0 3 . Proceed with removal of
last traces of Bi at pH 2.0 and det. Pb as in (a).
Special Methods of Sample Preparation
J. Solution in Acids
(Applicable to chemicals sol. in H 2 or acid, e.g., phosphates,
sulfates, etc., and org. products of type of tartrates and ci-
trates . )
Dissolve 5—100 g sample, according to its nature and amt
of Pb expected, in HC1 in 400 mL beaker. With Ca phos-
phates, use 10-50 g. Dissolve in smallest practicable vol. of
soln by warming and adding alternately small amts of hot H 2
and HC1. Filter soln with suction (fritted glass preferred) into
beaker or flask under bell jar and leach any residue with 10-
25 mL hot HCl-citric acid, (j), followed by 10-25 mL hot
40% NH 4 OAc soln. Rinse beaker and filter with hot H 2 and
cool soln.
Proceed as in 934.070. If interfering ppt forms, again acidi-
fy and isolate Pb by sulfide pptn, 934.07E. If it is difficult to
obtain clear soln with Ca phosphates at pH 3.0-3.4 (sulfide
ppt may be contaminated with excessive phosphates), redis-
solve ppt, add more citric acid soln, (d), readjust pH, and reppt
sulfides; or make one sulfide pptn, dissolve sulfides in hot
HNO3, boil off H 2 S, and ext Pb with dithizone, 934.07D.
Sometimes difficulty due to ppt formation in 934. 07D can be
avoided by using smaller sample for extn and colorimetric detn.
If Sn or Bi is suspected, remove by methods described in
934. 07H and I. Finally det. isolated Pb colorimetrically,
934.07F.
K. Complete Digestion
(Applicable to most food or biological products; with difficulty
to fats and oils, oily products, etc. Caution: See safety notes
on distillation.)
Digest representative sample in Kjeldahl flask as in 963. 21C.
Distil As, if desired, as AsCI 3 , 963. 21D. If As is not to be
distd, add 100 mL H 2 and enough HC1 to flask to dissolve
any CaS0 4 in residue. Filter on fritted glass filter, pulverizing
any insol. residue (anhyd. Si0 2 or BaS0 4 ) with flat-end stir-
ring rod. Dissolve any PbS0 4 in flask and leach residue on
filter with 10-20 mL hot HCl-citric acid soln, (j), followed
by 10-20 mL hot 40% NH 4 OAc. Finally rinse both flask and
filter with hot H 2 0. Isolate Pb by dithizone, 934. 07D, or sul-
fide pptn, 934. 07E, methods. (In general, sulfide method is
preferable, especially when BaS0 4 or excessive CaS0 4 is pres-
ent, as insol. sulfates readily occlude Pb.) If Bi and Sn are
present, remove them as in 934. 07H or I. After isolation, det.
Pb by colorimetric method, 934.07F.
Refs.: JAOAC 17, 108(1934); 18, 315(1935); 19, 130(1936).
CAS-7439-92-1 (lead)
930.34
Manganese in Food
Final Action
See 921.02 or 931.09*.
971.21 Mercury in Food
Flameless Atomic Absorption Spectrophotometric Method
First Action 1971
Final Action 1976
(Rinse all glassware before use with HNO3 (1 + 9). Caution:
See safety notes on wet oxidation, nitric acid, perchloric acid,
sulfuric acid, and mercury salts.)
A. Apparatus
(a) Atomic absorption spectrophotometer. — Instrumenta-
tion Laboratory, Inc., 1 13 Hartwell Ave, Lexington, MA 02173,
Model 153 (or successors), or equiv. Equipped with Hg hol-
low cathode lamp and gas flow-thru cell (Fig. 971.21), 25 (id)
AOAC Official Methods of Analysis (1 990)
Mercury 263
x 1 15 mm with quartz windows cemented in place. Operating
conditions: Wavelength 253.7 nm, slit width 160 |mm, lamp
current 3 ma, and sensitivity scale 2.5.
(b) Diaphragm pump. — Neptune Dyna-Pump, orequiv. Coat
diaphragm and internal parts of pump with acrylic-type plastic
spray. Use 16 gage Teflon tubing for all connections.
(c) Water condenser. — 12—18 (id) x 400 mm borosilicate,
24/40 1 joint, modified to hold 6 mm Raschig rings. Fill con-
denser with Raschig rings to ht of 100 mm; then place 20 mm
layer of 4 mm diam. glass beads on top of rings.
(d ) Gas inlet adapter.— 24/40 ¥ (Kontes Glass Co. No. K-
181000).
(e) Digestion flask. — 250 m'L flat-bottom boiling flask with
24/40 I joint.
S. Reagents
(a) Reducing soln. — Mix 50 mL H 2 S0 4 with ca 300 mL
H 2 0. Cool to room temp, and dissolve 15 g NaCl, 15 g hy-
droxylamine sulfate, and 25 g SnCI 2 in soln. Oil . to 500 mL.
(b) Diluting soln. — To 1 L vol. flask contg 300-500 mL
H 2 0, add 58 mL HN0 3 and 67 mL H 2 S0 4 . Dil. to vol. with
H 2 0.
(c) Magnesium perchlorate . — Drying agent placed in filter
flask (Fig. 971.21). Replace as needed. {Caution: Mg(C10 4 ) 2
is explosive when in contact with org. substances.)
(d) Mercury std solns. — (I) Stock soln. — 1000 p,g/mL.
Dissolve 0.1354 g HgCl 2 in 100.0 mL H 2 0. (2) Working soln.—
1 |xg/mL. Dil. 1 mL stock soln to 1 L with IN H 2 S0 4 . Prep,
fresh daily.
C. Determination
Weigh 5.0 g sample into digestion flask; add 25 mL 187V
H 2 S0 4 , 20 mL IN HN0 3 , 1 mL 2% Na molybdate soln, and
5-6 boiling chips. Connect condenser (with H 2 circulating
thru it) and apply gentle heat ca 1 hr. Remove heat and let
stand 15 min. Add 20 mL HN0 3 -HC10 4 (1 + 1) thru con-
denser. Turn off H 2 circulating thru condenser and boil vig-
orously until white fumes appear in flask. Continue heating 10
min.
Cool. Cautiously add 10 mL H 2 thru condenser while
swirling liq. in flask. Again boil soln 10 min. Remove heat
and wash condenser with three 15 mL portions H 2 0.
Cool soln to room temp. Completely transfer digested sam-
ple with H 2 to 100 mL vol. flask and dil. to vol. with H 2 0.
Transfer 25.0 mL aliquot from each sample to another diges-
tion flask. Adjust vol. to ca 100 mL with dilg soln, (b).
Adjust output of pump to ca 2 L air/min by regulating speed
of pump with variable transformer. Connect app. as in Fig.
971.21, except for gas inlet adapter. With pump working and
spectrophtr zeroed, add 20 mL reducing soln to dild aliquot.
Immediately connect gas inlet adapter and aerate ca 3 min.
(Adjust aeration time to obtain max. A.) Record A, disconnect
pressure on "out" side of pump, and open vent on filter flask
to flush system.
Prep, reagent blank and std curve by adding 0, 0.2, 0.4,
0.6, 0.8, and 1.0 fxg Hg to series of digestion flasks. To each
flask add 100 mL dilg soln. Finally, add reducing soln and
aerate stds as for sample.
Plot std curve from least squares linear regression of A against
|xg Hg. {See "Definitions of Terms and Explanatory Notes,"
or use calculator which performs linear regression.) Det. p,g
Hg in aliquot from curve. If jxg Hg detd falls outside range of
calibration, repeat detn with smaller aliquot of sample soln to
bring \xg Hg into region of std curve. From size of aliquot
used, det. total |mg Hg in original sample.
ppm Hg - p,g Hg/g sample
Ref.: JAOAC 54, 202(1971).
CAS-7439-97-6 (mercury)
977.15 Mercury in Fish
Alternative Flameless Atomic Absorption
Spectrophotometric Method
First Action 1977
Final Action 1978
(Rinse all glassware before use with HN0 3 (1 + 9). Caution:
See safety notes on wet oxidation, nitric acid, sulfuric acid,
and mercury salts.)
A. Apparatus
See 971.21A(a), (b), (e), and in addn:
(a) Boiling stones. — 6-8 mesh (Lurex Scientific, No. D-
7325).
(b) Gas inlet adapter. — 1" 24/40 (Kontes Glass Co., No.
K- 181000),. Cut offend of glass tube which extends downward
from adapter and affix gas dispersion tube with fritted cylinder
(Corning Glass Works, No. 39533, porosity 12C).
(c) Trap. — Construct from cut off bulb of 15 mL pi pet and
16 Gage Teflon Tubing
■10 g Mg(CI0 4 ) 2
FIG. 971.21 — Apparatus for flameless atomic absorption analysis
264
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
place between digestion flask and cell, replacing flask of Fig.
986. 15B, to trap overflow.
(d) Water condenser. — 12-18 (id) X 300 mm Liebig con-
denser with ¥ 24/40 joint. Modify by making indentations in
glass between lower std taper and H 2 jacket with pointed C
rod. Indent glass to hold 6 mm Raschig rings. Add 8-10 rings
to condenser and cover with Vs" (3.17 mm) id glass helices
(Lurex Scientific, No. 181-2012) to ht of 90 mm.
B. Determination
Weigh 5.0 g (wet wt) thoroly mixed fish sample into diges-
tion flask, 971.21A(e). Rinse neck of flask with <5 mL H 2 0,
if necessary. Add ca 20 boiling stones, (a), 10-20 mg V 2 5 ,
and 20 mL H 2 S0 4 -HN0 3 (1 + 1). Quickly connect flask to
condenser, (d), and swirl to mix. Circulate cold H 2 thru con-
denser during digestion. Apply sufficient heat (luminous flame
is suitable) to produce low initial boil (ca 6 min) and finish
digestion with strong boil (ca 10 min). Swirl flask intermit-
tently during digestion. No solid material should be apparent
except for globules of fat after ca 4 min.
Remove flask from heat and wash condenser with 15 mL
H 2 0. Add 2 drops 30% H 2 2 thru condenser and wash into
flask with 15 mL H 2 0. Cool digested fish soln to room temp.
by placing flask, still connected to condenser, in beaker of
H 2 0. Disconnect flask, rinse ground joint with H 2 0, and quant,
transfer digest to 100 mL vol. flask. Ignore solidified fat; it
does not interfere. Carefully rinse digestion flask with several
portions H 2 and dil. to vol. with rinse H 2 0.
Pipet 25 mL soln into original digestion flask and add ca 75
mL dilg soln, 971.21B(b). Proceed as in 971.21C, beginning
"Adjust output of pump . . .", except aerate ca 1 min. 1 juug
std should give A > 0.400.
Ref.: J AOAC 60, 833(1977).
CAS-7439-97-6 (mercury)
974.14 Mercury in Fish
Alternative Digestion Method
First Action 1974
Final Action 1976
{Caution: Do not change sample wt or acid vol. stated; oth-
erwise excessive pressure during heating may result in explo-
sion.)
A. Apparatus
Digestion vessel. — See Fig. 974.14. Stainless steel body
supporting Teflon crucible and screw-on cap with Teflon liner
to provide Teflon sealing surface. Teflon spout is snapped on
outside rim to permit quant, transfer of contents without con-
tact with metal parts. (Available from Uni-Seal Decomposition
Vessels, PO Box 9463, Haifa, Israel.)
B. Digestion
Accurately weigh 1 ± 0. 1 g sample (Caution: Do not use
>300 mg dry wt; for materials with high fat content do not
use >200 mg dry wt.) into digestion vessel, add 5.0 mL HN0 3 ,
and close vessel by tightening screw cap. Place vessel, without
tilting, into preheated 150° oven 30-60 min or until clear. Re-
move vesse] and let cool to room temp. Unscrew cap, snap
on spout, and transfer with aid of 95 mL dilg soln, 971.21B(b),
to 250 mL flask, 971.21A(e). Proceed as in 971.21C, begin-
ning "Adjust output of pump ..."
Refs.: JAOAC 55, 741(1972); 57, 568(1974). Anal. Chem.
40, 1682(1968).
CAS-7439-97-6 (mercury)
952.14 Mercury in Food
Colorimetric Dithizone Method
Final Action
A. Principle
Sample is digested with HN0 3 and H 2 S0 4 under reflux in
special app., Hg is isolated by dithizone extn, Cu is removed,
and Hg is estd by photometric measurement of Hg dithizonate.
B. Precautions
Critical step is digestion of sample, which must be almost
complete, otherwise residual org. matter may combine with
Hg and prevent or hinder extn with dithizone. Oxidizing ma-
terial in digest must also be destroyed or dithizone reagent is
decomposed and Hg is not quant, extd. Because of volatility
of Hg compds, careful heating of digest during sample prepn
is required. Acidity of final sample soln (after partial neutzn
with NH 4 OH) before extn should be ca IjV and not >\.2N.
Do not use silicone grease in stopcocks.
FIG. 974.14— Digestion vessel
AOAC Official Methods of Analysis (1990)
Mercury 265
C. Apparatus
(As Hg compds tend to adsorb on glassware, app. and partic-
ularly separators should be rinsed with dil. HN0 3 and then
with H 2 0.)
Special digestion apparatus. — See Fig. 952.14. App. is made
from Pyrex with 1" joints thruout. Unit A is modified Soxhlet
extractor, 5 cm od, 200 mL capacity to overflow, without in-
ner siphon tube but equipped with stopcock on tube leading to
digestion flask, D. With stopcock open, app. is in reflux po-
sition; when closed, unit serves as trap for condensed H 2 and
acids. Top of A is attached to Friedrichs condenser, 35 cm
long. Bottom of A is attached thru center neck of 2 neck ¥ 24/
40 r-b 500 mL flask, D. Necks are 3 cm apart to provide clear-
ance. Second neck is used for attaching 75 mL dropping fun-
nel, B.
D. Reagents
(a) Mercury std solns. — (7) Stock soln. — 1 mg/mL. Prep,
from dry, recrystd HgCl 2 (67.7 mg/50 mL). (2) Working soln. —
2 jjLg/mL is convenient. Prep, from stock soln and store in
Pyrex bottles. Add HC1 in proportion of 8 mL/L to all stds
before dilg to final vol.
(b) Chloroform.— See 945.58B(b).
viwt>vs
vv
24/40
FIG. 952.14— Special digestion apparatus for mercury residues
(c) Dithizone soln. — See 934.07A(e). Reagent as now dis-
tributed needs no purification for this method. Prep, stock soln
in redistd CHC1 3 (100 mg/L is convenient) and store in re-
frigerator. Prep, dilns as needed.
(d ) Sodium thiosulfate soln . — 1.5%. Prep . daily .
(e) Sodium hypochlorite soln, — Preferably 5% available CI
reagent. As distributed, reagent varies in available CI content.
Det. strength by 935. 07B. Store in refrigerator when not in
use and det. titer monthly. (Certain prepns of hypochlorite in-
tended for household use contain traces of Hg. If these prepns
are used, det. blank. Reagent with >0.1 |xg Hg/mL should
not be used.)
(f) Dilute acetic acid. — 30% by vol.
(g) Hydroxylamine hydrochloride soln. — 20% w/v. Ext with
dil. dithizone until CHC1 3 layer remains green, remove excess
dithizone with CHC1 3 , and filter.
E. Preparation of Sample
(Conduct acid digestion in hood.)
In all detns use wt sample equiv. to <10 g dry wt.
(a) Fresh fruits or vegetables and beverages. — Place weighed
sample in digestion flask with 6 glass beads, connect assem-
bly, and add, thru dropping funnel, 20 mL HN0 3 . Pass rapid
stream of H 2 thru condenser, adjust stopcock of Soxhlet unit
to reflux position, and apply small flame to flask. Use asbestos
board with 2-5 cm diam. hole between flask and flame. (Orig-
inal reaction must not proceed violently or evolved N0 2 will
carry vapors of digest mech. thru condenser and cause loss of
Hg.) After initial reaction is complete, apply heat so that digest
just refluxes. If mixt. darkens, add HN0 3 dropwise thru funnel
as needed. Continue refluxing 0.5 hr, or until digest does not
change consistency, and cool.
Slowly add 20 mL cold HN0 3 -H 2 S0 4 mixt. (1 + 1). (Use
10 mL acid mixt. for <5 g (dry wt) of sample.) Heat with
small flame, subsequently adding HN0 3 dropwise as needed
to dispel darkening of digest. Continue heating until fibrous
materia] (fruit skin, cellulose, etc.) is apparently digested. Turn
stopcock of Soxhlet unit to trap H 2 and acids, and continue
heating. Let digest become dark brown (not black) before add-
ing further increments of HN0 3 . (Fats and waxes cannot be
totally digested by the hot acids under reflux. Therefore no
attempt should be made to effect complete digestion in this
step.) When all except fat and wax is in soln, let digest cool,
and cautiously drain H 2 and acids into main digest. Cool,
and pour two 25 mL portions H 2 thru condenser and inter-
mediate unit. Remove reaction flask, chill under cold H 2 or
by surrounding with ice to solidify fats and waxes, and filter
off insol. matter on small pledget of glass wool. Rinse reaction
flask and filter pad successively with two 10 mL portions H 2 0.
Remove Soxhlet unit, and wash it and flask with hot H 2 to
remove insol. material. Pour hot H 2 thru condenser to re-
move volatile fats and oils. Discard all washings.
Connect flask contg filtered sample soln to assembled app.,
heat, and collect H 2 and acids in trap. Complete digestion,
using small addns of HN0 3 as needed. In final stage of diges-
tion, adjust flame until digest reaches incipient boiling (soln
simmers) and acid vapors do not rise beyond lower half of
condenser. Continue heating 15 min after last addn of HN0 3 .
Digest should now be colorless or pale yellow. Let digest cool,
drain trapped liqs carefully into reaction flask, and add two 50
mL portions H 2 thru condenser. Reflux soln until all N0 2 is
expelled from app. Add 5 mL 40% w/v urea soln and reflux
15 min. (Digest should be colorless or pale yellow.)
(b) Dried fruit, cereal, seeds, and grains. — Dil. sample with
50 mL H 2 before adding HN0 3 , and proceed with sample
prepn as in (a).
266
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
(c) Meats, fish, and biological, material. — Because of high
fat and protein content of these materials, conduct initial diges-
tion carefully to avoid foaming of digest into condenser. Add
20 mL HN0 3 to sample, swirl flask, and let stand 0.5 hr in
digestion assembly before heating. Add 25 mL H 2 and heat
cautiously with small rotating flame until initial vigorous re-
action is over and foaming ceases. Proceed as in (a).
F. Isolation of Mercury
Titr. 1 mL prepd sample soln, 952. 14E, with std alkali. Add
calcd amt of coned NH 4 OH to reduce acidity to 1.0 A/: swirl
flask during addn of the NH 4 OH to avoid local excess. (Soln
should never be ammoniacal to avoid formation of Hg com-
plexes.)
Transfer sample soln to 500 mL separator. Add 10 mL 4
mg/L dithizone and shake vigorously 1 min. (If characteristic
green of dithizone is visible in CHC1 3 layer, indicating excess
of dithizone, amt of Hg is within 0-5 |xg.) Let layers Sep.,
and drain CHC1 3 layer quickly to second separator contg 25
mL 0. \N HC1 and 5 mL H 2 NOH.HCl soln. (Small amt of ox-
idizing material may still be present. On long contact with
dithizone soln, oxidizing substances may destroy dithizone re-
agent and prevent extn of Hg.)
Repeat extn of sample soln with two 5 mL portions dithi-
zone soln, transferring CHC1 3 layer successively to second
separator. If first extn indicates >5 |xg Hg, add stronger conens
of dithizone, as indicated by table, 952. 14H, until, after 1 min
vigorous shaking, CHCI 3 layer contains dithizone in marked
excess. Drain CHC1 3 layer into second separator contg 0.17V
HC1 and again ext sample soln with two 10 mL portions 4 mg/
L dithizone soln, draining each successive ext into second sep-
arator.
Shake contents of second separator vigorously 1 min, and
drain CHC1 3 layer into third separator contg 50 mL 0. IN HC1.
(Shaking dithizone ext with dil. acid in second separator re-
moves entrained org. matter. With biological materials or those
of high protein content, aq. layer is usually light yellow be-
cause of nitrated org. compds. Small amts are carried into third
separator where they are destroyed by CI.) Ext soln in second
separator with 1-2 mL CHCI3 and transfer org. layer to third
separator.
To contents of third separator add 2 mL Na 2 S 2 3 soln, shake
vigorously 1 min, let layers sep., drain off CHC1 3 as com-
pletely as possible, and discard. (Cu if present is removed as
dithizonate.) Ext again with 1-2 mL CHC1 3 , drain carefully,
and discard. Add 3.5 mL NaOCl reagent (or enough soln of
different titer to furnish 175 mg available CI) to decompose
Hg thiosulfate complex and to oxidize excess thiosulfate, and
shake vigorously 1 min. Add 5 mL H 2 NOH.HCl reagent from
pipet, taking care to wet both stopper and neck of separator.
Shake vigorously 1 min. Hold mouth of separator in front of
air vent and blow out any remaining gaseous CI. Stopper sep-
arator and shake vigorously 1 min. (It is imperative that all
hypochlorite be reduced. Trace amts remaining would oxidize
dithizone, subsequently added, to yellow oxidized form which
would be measured in photometer as Hg.) Ext soln with 2-3
mL CHCI3, drain off org. layer carefully, and discard. Final
aq. soln should now be colorless.
G. Determination
To third separator add 3 mL 30% HO Ac and appropriate
vol. and concn of dithizone soln as indicated by table, 952.14H,
and proceed with colorimetric detn of Hg as in 952. 14H, con-
verting A, measured at 490 nm, to |mg Hg from working curve.
H. Preparation of Standard Curve
Following table is useful in prepg std curve and for estab-
lishing approx. Hg range in sample soln when 1 cm cells are
used:
Hg Range,
m-9
Dithizone
Concn, mg/L
Volume
Dithizone, mL
0-10
0-50
0-100
6
10
10
5
25
40
Prep, working curve of required range, starting with blank
and extending to final std of range, with 4 intermediate incre-
ments. Add appropriate amts of Hg to 50 mL 0.1 /V HC1 in
separator. Add 5 mL H 2 NOH.HCl reagent and 5 mL CHC1 3 ,
and shake vigorously 1 min. Let layers sep., drain off CHC1 3 ,
and discard, being careful to remove as completely as possible
all droplets of CHC1 3 . Add 3 mL 30% HOAc and appropriate
vol. dithizone soln, shake vigorously .1 min, and let layers sep.
(HOAc aids in stabilizing mercuric dithizonate.) Insert cotton
pledget into stem of separator and collect dithizone ext (dis-
carding first mL) in test tube for transfer to appropriate cell.
Make photometer readings at 490 nm. (Since both dil. dithi-
zone and mercuric dithizonate are somewhat unstable, read
immediately.) Plot A against |xg Hg.
Ref.: JAOAC 35, 537(1952).
CAS-7439-97-6 (mercury)
983.20 Mercury (Methyl) in Fish
and Shellfish
Gas Chromatographic Method
First Action 1983
Final Action 1988
(Caution: See safety notes on benzene and acetone.)
A. Principle
Org. interferences are removed from homogenized sample
by acetone wash followed by benzene wash. Protein-bound Me
Hg is released by addn of HCl and extd into benzene. Benzene
ext is coned and analyzed for CH 3 HgCl by GC.
B. Reagents
(a) Solvents. — Acetone, benzene, and isopropanol are all
distd in glass (Burdick & Jackson Laboratories, Inc.; EM Sci-
ence Omni Sol v reagents). Note: Benzene is a possible carcin-
ogen.
(b) Hydrochloric acid soln (1 + 7). — Add coned HCl to
equal vol. of distd or deionized H 2 and mix. Ext HCl soln
5 times with 1 / 4 its vol. of benzene by shaking vigorously 15
sec in separator. Discard benzene exts. Soln may be mixed in
advance but must be extd immediately before use.
(c) Carrier gas.— -GC quality Ar-CH 4 (95 + 5).
(d) Sodium sulfate. — Heat overnight in 600° furnace, cool,
and store in capped brown bottle. Line cap with Al foil to
prevent contamination from cap.
(e) Methyl mercuric chloride std solns. — Keep tightly stop-
pered. (J) Stock std soln.— 1000 \xg Hg/mL. Weigh 0.1252
g CH 3 HgCl into 100 mL vol. flask. Dil. to vol. with benzene.
(2) High intermediate std soln. — 40 |xg Hg/mL. Dil. 10.0 mL
stock soln to 250.0 mL with benzene. (J) Low intermediate
std soln. — 2.0 |xg Hg/mL. Dil. 10.0 mL high intermediate
std soln to 200.0 mL with benzene. (4) Working std solns. —
0.010-0.30 |xg Hg/mL. Prep, monthly by dilg with benzene
in vol. flasks as follows: Dil. 15 mL of 2.0 |xg Hg/mL std to
100.0 mL. 10. mL to 100.0 mL, and 10.0 mL to 200.0 mL
for 0.30, 0.20, and 0.10 ^g Hg/mL, resp. Dil. 20 mL of 0.10
|xg Hg/mL std to 25.0 mL, 10.0 mL to 25.0 mL, 10.0 mL
to 50.0 mL, and 10.0 mL to 100.0 mL for 0.080, 0.040, 0.020,
and 0.010 jxg Hg/mL, resp.
(f) Mercuric chloride column treatment soln. — 1000 ppm
HgCL. Dissolve 0.1 g HgCL in 100 mL benzene.
AOAC Official Methods of Analysis (1990)
Mercury 267
C. Apparatus
Wash all glassware with detergent (Micro Laboratory Cleaner,
International Products, PO Box 1 18, Trenton, NJ 08601-0118,
or equiv.) and rinse thoroly with hot tap H 2 followed by distd
or deionized H 2 0.
(a) Centrifuge.— Model JJV (replacement Model PR-7000)
(International Equipment Co.), or equiv.
(b) Centrifuge tubes. — 50 mL capacity with ground glass
or Teflon-lined stoppers.
(c) Kuderna-Danlsh (K-D) concentrators .—250 mL flask
(No. K5 70001, Kontes Glass Co.) and 10 mL graduated con-
centrator tube (No. K570050, size 1025, Kontes Glass Co.).
(d) Snyder distilling column. — Use No. K-503100, size 0003
(Kontes Glass Co.) as is or modify Kontes No. K503000, size
121, in either of 2 ways: (0 Shorten 3-section, 3-ball column
to 2-section, 2-ball column by cutting off top at uppermost
constriction, (a) Insulate 3-section, 3-ball column by wrapping
glass wool around top section and holding it in place with Al
foil. Glass wool and foil must surround only top section above
top ball.
(e) Carborundum boiling chips. — 20 mesh, HCl-washed.
(f) Graduated cylinders. — Class A, 25 mL capacity, with
ground-glass stopper (Kimble 20036, or equiv.).
(g) Transfer pipets. — Disposable glass, Pasteur-type 5 3 /4 in.
long (No. 13-678-6A, Fisher Scientific Co., or equiv.).
(h) Dropping pipets. — 5 mL capacity (No. 13-710B, Fisher
Scientific Co., or equiv.).
(i) Gas chromato graph. — Hewlett-Packard Model 5710A
or equiv., equipped with linear ft3 Ni electron capture detector
and 6 ft x 2 mm id silanized glass column packed with 5%
DEGS-PS on 100-120 mesh Supelcoport (Supelco, Inc., No.
1-1870). Pack column no closer than 2.0 cm from injection
and detector port nuts and hold packing in place with 2 cm
high quality, silanized glass wool at both ends. Install oxygen
scrubber and molecular sieve dryer (No. HGC-145, Analabs,
Inc., or equiv.) between carrier gas supply and column. Con-
dition column according to manufacturer's instructions as fol-
lows: Flush column 0.5 h with carrier gas flowing at 30 mL/
min at room temp. Then heat 1 h at 100°. Next, heat column
to 200° at programmed heating rate of 4°/min and hold at 200°
overnight. Do not connect column to detector during this con-
ditioning process. Maintain 30 mL/min carrier gas flow at all
times during conditioning, treatment, and use. Operating con-
ditions: column 155°; injector 200°; detector, 300°; carrier gas
flow 30 mL/min; and recorder chart speed 0.5-1 .0 cm/min.
Under these conditions and with HgCl 2 column treatment pro-
cedure described below, CH 3 HgCl peak will appear 2-3 min
after sample injection.
D. Mercuric Chloride Column Treatment
5% DEGS-PS conditioned according to manufacturer's in-
structions can be used to det. CH 3 HgCl only after treatment
by HgCl 2 soln, (f). Treat column any time column has been
heated to 200°. Because column performance degrades with
time, also treat column periodically during use. Perform ap-
propriate H.gCl 2 treatment procedures described below. Pro-
cedure (b) produces most stable baseline and is recommended
over procedure (c) for routine use.
(a) Following 200° column conditioning .—If column has
just been conditioned overnight at 200°, use this procedure.
Adjust column temp, to 160° and connect detector. When
baseline is steady, treat column by injecting 20 jjlL HgCl 2
treatment soln 5 times at 5-10 min intervals. (Change in col-
umn performance may be monitored by injecting 5 jjlL 0.010
(xg Hg/mL std soln before and between HgCl 2 treatment soln
injections.) During treatment procedure, large broad peaks will
elute. (CH 3 HgCl peak retention time will decrease and peak
ht will increase.) Approximately 1 V2— l 3 Ai h after last HgCl 2
treatment soln injection, a final large peak will elute. (CH 3 HgCl
peak ht and retention time will be stable.) This broad peak and
CH 3 HgCl peak ht stability signal completion of treatment pro-
cess. Adjust column temp, to 155° and wait for steady base-
line; then column is ready for use.
(b) On day preceding sample extract analysis. — If column
has been treated by procedure (a) or used at 155° to analyze
sample exts, column may be treated at end of working day for
next day's use as follows: Lower column temp, to 115° and
inject 20 |xL HgCL treatment soln one time. Broad peaks will
elute between 1 1 and 15 h after HgCl 2 injection. Next working
day, increase column temp, to operating temp. When baseline
is steady (ca 15-30 min), column is ready for use.
(c) During sample extract analysis at 155° . — If column has
been used at 155° for ext analysis and column performance
has degraded enough to require HgCl 2 treatment, increase col-
umn temp, to 160°, inject one 20 jxL aliquot of HgCl 2 treat-
ment soln, and monitor baseline. Large, broad peaks will elute
1-1 V2 h after HgCl 2 injection, signaling completion of treat-
ment process. Decrease column temp, to 155° and wait for
steady baseline; then column is ready for use.
E. Extraction of Methyl Mercury Chloride
Perform all operations, except weighing, in laboratory hood.
Accurately weigh 2 g homogenized sample into 50 mL centrf.
tube. Add 25 mL acetone, stopper, and shake vigorously 15
s. Remove stopper, cover with foil, and centrf. 2-5 min at
2000 rpm. Carefully decant and discard acetone. (Use drop-
ping pipet to remove acetone, if necessary.) Repeat 25 mL
acetone wash step twice more. Break up tissue with glass stir-
ring rod before shaking, if necessary. Add 20 mL benzene,
stopper, and shake vigorously 30 s. Remove stopper, cover
with foil, and centrf. 2-5 min at 2000 rpm. Carefully decant
(or draw off with dropping pipet) and discard benzene. Extra-
neous peaks in final GC anal, chromatograms indicate that more
vigorous shaking with acetone and benzene is required.
Add 10 mL HCI soln to centrf. tube contg acetone and ben-
zene-washed sample. Break up tissue with glass stirring rod,
and ext sample by adding 20 mL benzene and shaking gently
but thoroly 2 min. Remove stopper, cover with foil, and centrf.
5 min at 2000 rpm. If emulsion forms, add 2 mL isopropanol
and gently stir benzene layer to break emulsion, taking care
not to disturb aq. phase, and recentrf. Carefully transfer ben-
zene layer to K-D concentrator, using 5 mL dropping pipet.
Rinse centrf. tube walls with 3-4 mL benzene and transfer
rinse to K-D concentrator. Repeat extn step twice more, add-
ing 20 mL benzene and shaking 1 min each time. Combine
all 3 benzene exts in K-D concentrator.
Place 4-6 boiling chips in K-D concentrator, connect Sny-
der column, wet Snyder column bubble chambers with 3-4
drops of benzene, and immediately place tube in steam bath
or vigorously boiling H 2 bath. Evap. so that 8 mL remains
when cooled to room temp. Cool. Disconnect concentrator tube
and quant, transfer soln to 25 mL g-s graduate using Pasteur-
type transfer pipet. Dil. to 20.0 mL with benzene and mix.
Add 4 g Na 2 S0 4 and mix again. Na 2 S0 4 must be added to 20
mL coned sample ext within 10 h of first acetone wash. Tightly
stoppered exts may be held overnight at this point. Analyze
by GC.
F. Chromatography
Verify that system is operating properly by injecting 5 |ulL
vols of 0.01 [xg Hg/mL working std soln into chromatograph.
Difference between CH 3 HgCI. peak hts for 2 injections should
be <4%. Check detector linearity by chromatographing all 0.0 1 -
0.30 |xg Hg/mL working std solns.
Inject duplicate 5 p,L vols, (equiv. to 0.5 mg sample) of
ext. Difference between CH^HgCl peak hts for 2 injections
268
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
should be <4%. Next, inject duplicate 5 |ulL vols, of std soln
with CH 3 HgCl concn approx. equal to or slightly greater than
ext CH 3 HgCl concn. Because column performance and peak
ht slowly decrease with time, calc. each sample concn by com-
parison to std soln injected immediately after sample.
Calc. Me Hg content of homogenate in |xg Hg/g (ppm Hg)
by comparing av. CH 3 HgCl peak ht of duplicate sample in-
jections with av. CH 3 HgCl peak ht of duplicate std injections.
ppm Hg - (R/R') x (C'/C) X 20
where R = av. peak ht of duplicate sample injections; R' =
av. peak ht of duplicate std injections; C = g sample; C =
concn of Hg in CH 3 HgCl std soln (|xg Hg/mL).
Ref.: JAOAC 66, 1121(1983).
CAS-7439-97-6 (mercury)
988.11 Mercury (Methyl) in Fish
and Shellfish
Rapid Gas Chromatographic Method
First Action 1988
A. Principle
Org. interferences are removed from homogenized seafood
by acetone wash followed by toluene wash. Protein-bound Me
Hg is released by addn of HC1 and extd into toluene. Toluene
ext is analyzed for CH 2 HgCl by electron capture GC.
B. Reagents
Equiv. reagents may be used.
(a) Solvents. — Acetone, toluene, and isopropanoi, all distd
in glass (Burdick and Jackson Laboratories, Inc., or EM Sci-
ence OmniSolv® reagents). Caution: Toluene is harmful if in-
haled and is flammable; conduct all operations with toluene in
laboratory hood.
(b) Hydrochloric acid soln (7 + /). — Add coned HC1 to
equal vol. distd or deionized H 2 and mix. Use 2 vols toluene
to ext potential interferences from 1 vol. HC1 soln by vigor-
ously shaking mixt. 15 s in separator. Discard toluene ext.
Repeat extn step 4 times. Soln may be mixed in advance.
However, extn must be performed immediately before HC1 soln
is used to avoid formation of electron-capturing compds which
produce extraneous peaks in chromatograms.
Before beginning analysis, check quality of reagents by
chromatographing blank taken thru method. Do not use HC1
and solvs which produce extraneous peaks at retention time of
Me Hg.
(c) Carrier gas.— GC quality Ar-CH 4 (95 + 5).
(d) Sodium sulfate. — Anhyd. reagent grade. Heat overnight
in 600° furnace, let cool, and store in capped bottle. Line cap
with acetone- washed Al foil to prevent contamination from cap.
Peaks appearing at 14-15 min may be eliminated by refiring
Na 2 S0 4 (600° overnight).
(e) Methyl mercuric chloride std solns. — Keep tightly stop-
pered. Seal stopper with Teflon tape. (7) Stock std soln. —
1000 pg Hg/mL. Weigh 0.1252 g CH 3 HgCl (ICN-K&K Lab-
oratories, Inc., PO Box 28050, Cleveland, OH 44128-0250)
into 100 mL vol. flask. Dil to vol. with toluene. (2) High level
intermediate std soln. — 40 p.g Hg/mL. Dil. 10.0 mL stock
std soln to 250.0 mL with toluene. (3) Low level intermediate
std soln.— -2.0 fig Hg/mL. Dil. 10.0 mL high level inter-
mediate std soln to 200.0 mL with toluene. (4) Working std
solns. — 0.005-0.10 pg Hg/mL. Prep, monthly by dilg with
toluene in vol. flasks as follows: Dil. 10.0 mL of 2.0 pg Hg/
mL soln to 200.0 mL for 0.10 fig Hg/mL. Dil. 20.0 mL of
0.10 pg Hg/mL soln to 25.0 mL, 15.0 mL to 25.0 mL, 10.0
mL to 25.0 mL, 10.0 mL to 50.0 mL, 10.0 mL to 100.0 mL,
and 10.0 mL to 200.0 mL for 0.080, 0.060, 0.040, 0.020,
0.010, and 0.005 |xg Hg/mL, resp.
(f) Mercuric chloride column treatment soln. — 1000 ppm
HgCl 2 . Dissolve 0.1 g HgCL in 100 mL toluene.
(g) Fortification solns. — (7) Stock soln. — 1000 pg Hg/mL.
Weigh 0.1252 g CH 3 HgCl into 100 mL vol. flask. Dil to vol.
with H 2 0. (2) Working fortification soln. — 15 pg Hg/mL. Dil.
1500 pL stock fortification soln to 100.0 mL with H 2 0.
C. Apparatus
Wash all glassware with detergent (Micro Laboratory Cleaner,
International Products, PO Box 1 18, Trenton, NJ 08601-01 18)
and rinse thoroly with hot tap H 2 followed by distd or deion-
ized H 2 0. Then rinse 3 times with acetone and 3 times with
toluene. Dry in hood.
Equiv. app. may be used except use packed column speci-
fied.
(a) Centrifuge. —Model lEC CRU-5000 or CR6000 (Inter-
national Equipment Co.).
(b) Centrifuge tubes. — Glass, 50 mL capacity with Teflon-
lined screw caps (Cat. No. 9212-K78, Thomas Scientific).
(c) Graduated cylinders. — Glass, class A, 50 mL capacity,
with ground-glass stoppers (Kimble 20036).
(d) Transfer pipets. — Disposable glass, Pasteur-type.
(e) Dropping pipets . — Glass, 5 mL capacity (No. 13-71 0B,
Fisher Scientific Co.).
(f) Mechanical shaker. — Model S-500 shaker-in-the-round,
with timer (Glas-Col Apparatus Co., 71 1 Hulman St, PO Box
2128, Terre Haute, IN 47802.)
(g) Gas chromatograph. — Hewlett-Packard Model 5710A
equipped with linear 63 Ni electron capture detector, Model
71 31 A recorder, and 6 ft x 2 mm id silanized glass column
packed with 5% DEGS-PS on 100-120 mesh Supelcoport (Su-
pelco, Inc.). Pack column no closer than 2.0 cm from injection
and detector port nuts and hold packing in place with 2 cm
high quality, silanized glass wool at both ends. Install oxygen
scrubber and molecular sieve dryer (No. HGC-145, Analabs,
Inc.) between carrier gas supply and column. Condition col-
umn according to manufacturer's instructions as follows: Flush
column 0.5 h with carrier gas flowing at 30 mL/min at room
temp. Then heat 1 h at 50°. Next, heat column to 200° at 4°/
min and hold at 200° overnight. Do not connect column to
detector during this conditioning process. Maintain 30 mL/
min carrier gas flow at all times during conditioning, treat-
ment, and use. Operating conditions: column 155°, injector
200°, detector 300°; carrier gas flow 30 mL/min; recorder chart
speed 0.5-1.0 cm/min. Under these conditions and with HgCl 2
column treatment procedure described below, CH 3 HgCl peak
appears 2-3 min after injection of ext.
D. Mercuric Chloride Column Treatment
Column of 5% DEGS-PS, conditioned according to manu-
facturer's instructions, can be used to det. CH 3 HgCl only after
treatment by HgCL soln, (f). Because column performance
degrades with time, also treat column periodically during use.
Perform appropriate HgCl 2 treatment procedures described be-
low.
(a) Following 200° column conditioning and after every 2~
3 days of analyses. — If column has just been conditioned ac-
cording to manufacturer's instructions or has been used 2-3
days to analyze exts, proceed as follows: Adjust column temp,
to 200° and inject 20 pL HgCl 2 treatment soln 5 times at 5-
10 min intervals. Maintain 200° temp, overnight. Chromato-
gram will contain large, broad peaks. Adjust column temp, to
155° next morning and inject 20 pL HgCl 2 treatment soln 2
more times. Large, broad chromatgc peaks appearing at ca 1-
AOAC Official Methods of Analysis (1990)
Selenium
269
2 h signal completion of treatment process and that column is
ready for use.
(b) On day preceding analyses. — If column has been treated
by procedure (a) or used 1 day at 155° to analyze exts, column
may be treated at end of working day for next day's use as
follows: Lower column temp, to 115° and inject 20 |xL HgCl 2
treatment soln 1 time. After large, broad peaks appear in chro-
matogram (11-20 h), treatment process is complete. Next
working day, increase column temp, to 155° operating temp.
When baseline is steady, column is ready for use.
(c) During extract analyses at 155°. — If column has been
used at 155° to analyze exts or if column performance and peak
ht have degraded enough to require HgCl 2 treatment, inject
two 20 |xL aliquots of HgCl 2 treatment soln. Large, broad peaks
will appear in chromatogram 1-2 h after HgCl 2 injection, sig-
naling completion of treatment process. Wait for steady base-
line; then column is ready for use.
B. Extraction of Methyl Mercuric Chioride
Perform all operations except weighing in laboratory hood.
Take empty centrf. tube thru all steps for method blank detn.
Accurately weigh 1 g homogenized test sample into 50 mL
centrf. tube. Add 25 mL acetone; tightly cap and vigorously
shake tube by hand 15 s. Loosen cap and centrf. 5 min at 2000
rpm. Carefully decant and discard acetone. (Use dropping pi-
pet to remove acetone, if necessary.) Repeat 25 mL acetone
wash step 2 more times. Break up tissue with glass stirring
rod before shaking tube, if necessary. Add 20 mL toluene;
tightly cap and vigorously shake tube by hand 30 s. Loosen
cap and centrf. 5 min at 2000 rpm. Carefully decant (or draw
off with dropping pipet) and discard toluene. Extraneous peaks
in final GC chromatogram may indicate that more vigorous
shaking with acetone and toluene is required. In products for
which Me Hg recoveries are to be detd, fortify tissue at this
point by adding working fortification soln, (g), to centrf. tubes.
Add 2.5 mL HC1 soln, (b), to centrf. tube contg acetone-
and toluene-washed sample. Break up tissue with glass stirring
rod, if necessary. Ext CH 3 HgCl by adding 20 mL toluene and
shaking tube gently but thoroly 5 min on mech. shaker at set-
ting 5 (2 min by hand). Loosen cap and centrf. 5 min at 2000
rpm. If emulsion is present after centrifugation, add 1 mL iso-
propanol and gently stir into toluene layer with glass stirring
rod to reduce emulsion. Do not mix isopropanol with aqueous
phase. Add equal amts of isopropanol to blank and test solns.
If emulsion is not present, do not add isopropanol to blank or
test solns. Vigorous mixing of isopropanol with HC1 may pro-
duce interfering peaks in chromatograms. Recentrifuge. With
dropping pipet, carefully transfer toluene to graduated cylin-
der. Rinse walls of centrf. tube with 1 -2 mL toluene and transfer
rinse to graduated cylinder. Repeat extn step 1 more time.
Combine both exts in graduated cylinder, dil. to 50 mL with
toluene, stopper, and mix well. Add 10 g Na 2 S0 4 and mix
again. Tightly stoppered exts (sealed with Teflon tape) may
be refrigerated and held overnight at this point. Analyze by
GC.
F, Gas Chromatography
Verify that system is operating properly by injecting 5 |xL
std soln contg 0.005 |ULg Hg/mL into GC system. Diff. be-
tween CH 3 HgCl peak hts for 2 injections should be <4%. Check
detector linearity by chromatographing all working std solns.
Inject 5 |jlL std soln with concn approx. equal to or slightly
greater than concn of ext. Immediately after CH 3 HgCl peak
appears, inject another 5 jjiL ext. Immediately after CH 3 HgCl
and background peaks for ext appear, inject another 5 p,L ali-
quot of std soln. Because column performance and peak ht
slowly decrease with time, calc. Hg concn in each test sample
by comparing peak ht for each test ext to average peak ht for
std solns injected immediately after test ext.
Correct ht of CH 3 HgCl peak for test ext by subtracting ht
of peak for method blank obtained at same attenuation and
recorder sensitivity. Calc. Me-bound Hg content of test sample
expressed as p,g Hg/g (ppm Hg) by comparing ht of peak from
injection of test ext to av. ht of peak from dup. injections of
std soln as follows:
M£ Hg/g fish = (R/R') x (C'/C) x 50
where R = corrected ht of CH 3 HgCl peak from injection of
test ext, R f = av. ht of CH 3 HgCl peak from dup. injections
of std soln, C — wt (g) of test portion, C = concn (u,g/mL)
of Hg in std soln, and 50 = final vol. (mL).
Ref.: J AOAC 70, 24(1987).
CAS-7439-97-6 (mercury)
975.34 Nickel in Tea
Atomic Absorption Spectrophotometry Method
Final Action
See 971.20.
974.15 Seienium in Food
Fluorometric Method
First Action 1974
Final Action 1976
A. Apparatus
(a) Fluorometer. — Filter fluorometer or spectrophotofluo-
rometer capable of excitation at 366 nm and detection of flu-
orescence at 525 nm. (Caution: See safety notes on photo-
fluorometers.)
(b) Cuvets or tubes. — Pyrex culture tubes, 12 X 75 mm,
selected by matching, are suitable for fluorometer.
(c) Wrist-action shaker. — Model BB (Burrell Corp.), or
equiv., set at max. speed.
(d) Separators. — Glass, 250 and 125 mL, with Teflon
stopcocks.
&. Reagents
(Use anal, grade reagents and glass-distd H 2 thruout
except as noted.)
(a) Nitric acid. — Distil from glass, discarding first and fi-
nal 10%.
(b) Dilute sulfuric acid.—SN. Dil. 140 mL H 2 S0 4 to 1 L
with H 2 0.
(c) Ammonium hydroxide soln. — Approx. 6N. Dil. 400 mL
NH 4 OH to 1 L with H 2 (X
(d) Disodium EDTA soln. — 0.02 Af. Dissolve 7.445 g
Na 2 H 2 EDTA.2H 2 and dil. to 1 L with H 2 0.
(e) 2 ,3-Diatninonaphthalene (DAN) soln. — 1 mg/mL. Pul-
verize DAN (purest grade available; product from Aldrich
Chemical Co. has been found satisfactory) in clean mortar to
fine powder. Insert glass wool plug in stem of 250 mL sep-
arator and add 150 mL 57V H 2 S0 4 . Transfer 0.150 g DAN to
separator and place on shaker 15 min to dissolve. Add 50 mL
cyclohexane and shake 5 min. Let phases sep. 5 min, drain
lower phase into another separator, and discard cyclohexane
(upper) phase. Repeat cyclohexane extn twice more; after third
extn, drain lower phase into low-actinic g-s flask, add 1 cm
layer hexane, and store in cold. Soln is stable several weeks.
270
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
(f) Selenium std soln. — (./) Stock soln. — 100 |mg/mL. Dis-
solve 0.1000 g black Se (purity >99.9%) in ca 5 mL HN0 3 ,
(a), and warm to dissolve. Dil. with H 2 and 20 mL 5N H 2 S0 4
to 1 L. (2) Working soln. — Dil. stock soln with H 2 and 5N
H 2 S0 4 to give Se concns in 0.17V H 2 S0 4 appropriate for level
of Se expected in sample. Store all solns in all-glass con-
tainers. So Ins are stable indefinitely.
C. Preparation of Standard Curve and Fluorometric Blank
Conduct appropriate vols of Se std solns (<10 mL contg
<800 ng Se) and 10 mL H 2 each thru entire detn, including
digestion, along with samples. Zero fluorometer against blank
soln and read fluorescence at 525 nm or subtract blank fluo-
rescence from that of stds. Plot reading against ng Se/6 mL
cyclohexane soln. Prepare new std curve daily.
D. Determination
(To ensure adequate cleanliness for fluorometry, acid-wash all
glassware except cells. In particular, clean Kjeldahl flasks and
erlenmeyers, separators, centrf. tubes, and glass beads before
each detn. Rinse glassware with hot H 2 0, dry in oven, and
wash with hot HNO r H 2 S0 4 (1 + 1). Rinse with hot tap H 2
followed by distd H 2 and dry in oven or let air dry. Rinse
cells with alcohol followed by acetone. Do not use plastic ware
other than that mentioned. Caution: See safety notes on wet
oxidation; nitric acid, perchloric acid, sulfuric acid.)
Place accurately weighed sample contg <1.0 g dry matter
and <0.8 |xg Se with 3 glass beads into 100 mL Kjeldahl flask
contg 10 mL H 2 0, and swirl to wet sample. Add 10 mL HN0 3 ,
(a). (Alternatively, omit the 10 mL H 2 0, add 10 mL HN0 3 ,
or more if all HN0 3 is absorbed by sample, and let digest
overnight at room temp.) Heat cautiously to reduce vol. to ca
5 mL, taking care to prevent severe foaming or bumping, and
cool. Add 6.0 mL 70% HC10 4 and 5.0 mL H 2 S0 4 , return to
cool heater, and heat until soln first turns yellow and then be-
comes colorless. Avoid charring of sample during digestion
which may result in loss of Se. If charring occurs, repeat anal-
ysis with new sample, using higher HNCVHClC^/sample wt
ratio. If this fails, add small amts of HN0 3 at first signs of
darkening.
Remove flask from heat, swirl to wet entire bulb area and
lower neck of flask, replace flask on heater, and continue heat-
ing until soln becomes colorless and white fumes appear.
Remove flask from heat, swirl, add 1 .0 mL 30% H 2 2 , rins-
ing walls of flask, and swirl until fuming ceases. Resume heat-
ing until contents boil briskly and white fumes are again evolved.
Repeat addn of H 2 2 and heating twice more, and continue
final heating 5 min after appearance of white fumes. Let flask
cool, add 30 mL H 2 0, rinsing walls of flask, and mix thoroly.
Transfer quant, to 250 mL g-s erlenmeyer, using two 10 mL
and one 5 mL H 2 rinses. Add, successively with mixing,
10.0 mL EDTA soln, 25.0 mL 6N NH 4 OH, and 5.0 mL DAN
soln. Bring quickly to brisk boil and boil exactly 2 min.
Let reaction mixt. stand at room temp, for definite interval
between 1 and 2 hr. Use same interval for all samples, stds,
and blank in set. Accurately add 6.0 mL cyclohexane, stopper
flask, and place on shaker 5 min. Transfer to 125 mL sepa-
rator, and let phases sep, ca 5 min. Discard lower aq. phase
and drain cyclohexane soln into 15 mL centrf. tube. Centrf. 5
min to further sep. H 2 and transfer ca 5 mL to fluorometer
cell.
Zero fluorometer against reagent blank and read fluores-
cence of sample at 525 nm. Alternatively, subtract fluores-
cence of blank from that of sample. Det. Se content from std
curve. Altho fluorescence readings for both samples and blanks
increase with time, net readings (sample
stant with 1-2 hr complexing period.
Ref.: JAOAC 57, 368, 373(1974).
CAS-7782-49-2 (selenium)
blank) remain con-
939.09*
Selenium in Food
Titrimetric Method
Final Action
Surplus 1975
See 25.121-25.126, 12th ed.
915.02*
Tin in Food
Gravimetric Method
Final Action 1976
Surplus 1980
See 25.131-25.133, 13th ed.
912.02*
Tin in Food
Volumetric Method
Final Action
Surplus 1980
See 25.134-25.135, 13th ed.
980.19* Tin in Food
Atomic Absorption Spectrophotometric Method
First Action 1980
Surplus 1986
See 25.161-25.163, 14th ed.
985.16 Tin in Canned Foods
Atomic Absorption Spectrophotometric Method
First Action 1985
Final Action 1988
A. Principle
Samples are digested with HN0 3 and then HO and are dild.
Aq. KC1 is added to samples and stds to reduce pos. instru-
ment interference. Sn is detd by A AS at 235.5 nm with oxi-
dizing N 2 0-C 2 H 2 flame.
B. Reagents and Apparatus
(a) Atomic absorption spectrophotometer . — With simulta-
neous background correction and N 2 OC 2 H 2 burner.
(b) Tin std solns. — (/) Stock soln. — 1 mg Sn/mL. Dis-
solve 1.000 g Sn (reagent grade) in ca 200 mL coned HO,
add ca 200 mL H 2 0, cool to ambient temp., and dil. to 1 L
with H 2 C). (2) Working solns.— 0, 50, 100, 150, and 200 p,g
Sn/mL. Into each of five 100 mL vol. flasks, pipet 10 mL
coned HC1, 1.0 mL KC1 soln, (c), and 0, 5, 10, 15, or 20 mL
Sn stock soln. Dil. to vol. with H 2 0.
(c) Potassium chloride soln. — 10 mg K/mL. Dissolve 1.91
g KC1 and dil. to 100 mL with H 2 0.
(d) Nitric acid. — Coned. Test purity of lot by dilg portion
1:4 v/v with H 2 and aspirating into AA spectrophtr. Ab-
sence of Sn signal indicates suitability for analysis.
AOAC Official Methods of Analysis (1990)
Tin
271
C. Preparation of Sample
Accurately (±0.01 g) weigh sample into 250 mL erlen-
meyer: 30-40 g juices or drinks, 20 g foods contg 50-75%
H 2 0, and 5-10 g solids or semisolids. Limit fat or oil content
to 2-4 g and total organics to ca 5 g. Dry in oven at 120 D .
Do not add HN0 3 to samples unless there is time to com-
plete this stage of digestion in the same day. Add 30 mL coned
HN0 3 to flask and, within 15 min, heat gently in hood to ini-
tiate digestion, avoiding excessive frothing. Gently boil until
3-6 mL digest remains or until sample just begins to dry on
bottom. Do not let sample char. Remove flask from heat.
Without delay, continue as follows, including 2 empty flasks
for reagent blanks: Add 25 mL coned HC1, and heat gently ca
15 min until sample bumping from evolution of Cl 2 stops. In-
crease heat, and boil until 10-15 mL vol. remains, using sim-
ilar flask with 15 mL FLO to est. vol. Add ca 40 mL H 2 0,
swirl, and pour into 100 mL vol. flask, rinsing once with ca
10 mL H 2 0. When HCI is present in digest, samples may stand
overnight or longer.
Pipet 1 .0 mL KC1 soln into each vol. flask. Cool to ambient
temp, and dil. to vol. with H 2 0, adding addnl H 2 to approx.
compensate for vol. of fat in flask. Mix well and filter ca SO-
SO mL thru dry, medium porosity paper into dry, polypro-
pylene or polyethylene screw-cap bottle. Do not filter blanks.
Cap bottles until analysis. Solns are stable several months.
D. Determination
{Caution: Due to explosive nature of gases, take care when
igniting and using flame. Warming tape on N 2 regulator may
be needed to maintain steady gas flow.) Using 200 pig/mL std
and 235.5 nm Sn line, optimize spectrophtr, burner, and flame
according to manufacturer's instructions. Then increase N 2
flow or decrease C 2 H 2 flow to give oxidizing flame; red part
should be ca 4 mm above burner slot. This reduces sensitivity
but improves precision to ± 0.0004 A for blank and 0.201
± 0.001 A for 100 u,g/mL std. Periodically monitor sensitivity
of a std; if sensitivity decreases >20%, turn off flame and
carefully clean burner slot.
Zero spectrophtr while aspirating H 2 but do not adjust zero
until after detns; autozero reduces precision. Aspirate H 2 be-
fore and after each sample, std, and blank soln. Take three 5
s readings for each soln, average, and ref. all A measurements
to A of H 2 0.
Record A for stds, draw calibration curve, and visually check
for inaccurate stds. Two times blank-corrected A for 50 |xg/
mL std should not differ by more than 3% from blank-cor-
rected A for 100 |JLg/mL std.
Block std blank with 50 (xg/mL std, and using ratio of A 7
calc. concn of std blank:
Std blank (jmg/mL) = [Aj(A r - A )] X 50
where A Q and A ' refer to blank and mean of readings for 50
jxg/mL blocking std, resp.
Add std blank concn to nominal std concn s to obtain true
std conens.
Measure A of sample blanks as for std blank and calc:
Sample blank (|xg/mL)
= (A /A ! ) X true concn of 50 jxg/mL std
where A and A' refer to blank and 50 (xg/mL std, resp. Calc.
mean concn of sample blanks, B.
Det. sample soln conens by one of 2 ways: (7) Measure A
of sample solns (max. 3 samples) and 50 [xg/mL std (or 100
fxg/mL std, depending on sample concn level), blocking sam-
ples with stds. Calc. blank-corrected sample soln conens:
Sample concn (fxg/mL) = (A /A' x true std concn) — B
where A and A' refer to sample and std, resp.
When high accuracy is not required or when calibration cur-
vature is extensive, use procedure (2) after confirmation that
sensitivity changes and baseline drift are absent during ana-
lytical run. (2) Calibrate using blank and 50, 100, and 150
|xg/mL stds. Run sample blanks and samples, and calc. soln
conens using either instrument microprocessor or calibration
curve. Calc. mean of sample blank conens, B. Calc. blank-
corrected soln conens (|xg/mL) by subtracting B from soln
conens.
For both (J) and (2), calc. sample conens:
Sample (jxg/g)
= [blank-corrected soln concn/sample wt (g)] x 100
Ref.: JAOAC68, 209(1985).
CAS-7440-31-5 (tin)
973.36 Titanium in Cheese
Spectrophotometric Method
First Action 1973
Final Action 1976
(Caution: See safety notes on sulfuric acid.)
A. Standard Solution
Titanium dioxide std soln. — 0.1 mg/mL. Accurately weigh
50 mg Ti0 2 and transfer to 250 mL beaker; add 15 g anhyd.
Na 2 S0 4 and 50 mL H 2 S0 4 . Add boiling chips, cover with watch
glass, and heat to bp on hot plate to dissolve. Cool, and cau-
tiously add 100 mL H 2 with stirring. (Warm on steam bath
if soln becomes cloudy.) Cool, transfer soln to 500 mL vol.
flask contg 200 mL H 2 0, and dil. to vol. with H 2 0.
B. Preparation of Sample
Weigh, to nearest 0.1 g, 10 g prepd sample, 955.30, into
100 mL Pt dish and char under TR lamp. Place in cold furnace
and ignite at 850° to white ash.
Cool, add ca 1.5 g anhyd. Na 2 S0 4 and 10 mL H 2 S0 4 , cover
with watch glass, and bring to bp on hot plate to dissolve. Turn
heat off and let cool on hot plate. Cautiously rinse cover, care-
fully add ca 30 mL H 2 0, and mix with stirring rod to disperse
any insol. salts. Heat on steam bath if insol. material forms
cake on bottom of dish.
Transfer quant, to 100 mL vol. flask with aid of ca 40 mL
H 2 0. If soln is cloudy, heat on steam bath or in boiling H 2
bath to clarify. Cool, and dil. to vol. with H 2 0.
C. Preparation of Standard Curve
Transfer 0, 1,2, 3, 4, and 5 mL Ti0 2 std soln to sep. 5 mL
g-s graduates (or vol. flasks) and dil. to vol. with H 2 S0 4 (1
+ 9). Add 0.2 mL 30% H 2 2 , mix, and det. A on recording
spectrophtr in 1.0 cm cells from 650 to 325 nm against 0.2
mL 30% H 2 2 in 5.0 mL H 2 S0 4 (1 + 9). Det. A at max., ca
408 nm, and prep, std curve.
D. Determination
Transfer 3.0 mL sample soln to 5 mL g-s graduate (or vol.
flask), dil. to vol. with H 2 S0 4 (1 + 9), and continue as in
973.36C, beginning "Add 0.2 mL 30% H 2 2 , ..."
Det. mg Ti(>7 in sample from std curve, and calc. as %
Ti0 2 .
Ref.: JAOAC 56, 535(1973).
CAS-7440-32-6 (titanium)
272
Metals and Other Elements
AOAC Official Methods of Analysis (1990)
944.09
Zinc in Food
Colorimetric Method
Final Action 1976
A. Principle
Method involves wet oxidn of sample; elimination of Pb,
Cu, Cd, Bi, Sb, Sn, Hg, and Ag as sulfides with added Cu
as scavenger agent; simultaneous elimination of Co and Ni by
extg metal complexes of a-nitroso-p-naphthol and dimethyl-
glyoxime, resp., with CHC1 3 ; extn of Zn dithizonate with CC1 4 ;
transfer of Zn to dil. HCl; and final extn of Zn dithizonate for
color measurement.
B. Reagents
(All H 2 must be redistd from glass. Pyrex glassware should
be used exclusively and must be scrupulously cleaned with hot
HN0 3 . Purify HNO3 (usually unnecessary) and NH 4 OH by distn
in Pyrex if appreciably contaminated. Test H 2 S0 4 if Zn con-
tamination is suspected.)
(a) Copper sulfate soln. — 2 mg Cu/mL. Dissolve 8 g
CuS0 4 .5H 2 in H 2 and dil. to 1 L.
(b) Ammonium citrate soln. — Dissolve 225 g (NH 4 ) 2 HC 6 H 5 07
in H 2 0, make alk. to phenol red with NH 4 OH (pH 7.4, first
distinct color change), and add 75 mL in excess. Dil. to 2 L.
Ext this soln immediately before use as follows: Add slight
excess of dithizone and ext with CC1 4 until solv. layer is clear
bright green. Remove excess dithizone by repeated extn with
CHCI3, and finally ext once more with CC1 4 . (It is essential
that excess dithizone be entirely removed, otherwise Zn will
be lost during elimination of Co and Ni.)
(c) Dimethylglyoxime soln. — Dissolve 2 g reagent in 10 mL
NH4OH and 200-300 mL H 2 0, filter, and dil. to 1 L.
(d) Alpha-nitroso-beta-naphthol soln. — Dissolve 0.25 g in
CHCl 3 and dil. to 500 mL.
(e) Chloroform. — Redistd.
(f) Diphenylthiocarbazone (dithizone) soln. — Dissolve 0.05
g dithizone in 2 mL NH 4 OH and 100 mL H 2 0, and ext re-
peatedly with CO4 until solv. layer is clear bright green. Dis-
card solv. layer and filter aq. portion thru washed ashless pa-
per. (This soln is best prepd as needed, since it is only
moderately stable, even when kept in dark and under refrig-
eration.)
(g) Carbon tetrachloride . — Redistd.
(h) Dilute hydrochloric acid. — 0.047V. Dil. required amt of
HCl with H 2 (redistd acid may be used altho usually unnec-
essary).
(i) Zinc std solns. — (I) Stock soln. — 500 (xg/mL. Dissolve
0.500 g pure granulated Zn in slight excess of dil. HCl and
dil. to 1 L. (2) Working soln. — 5 jxg/mL. Dil. 10 mL stock
soln to 1 L with 0.04 tf HCl.
C. Preparation of Sample
(Caution: See safety notes on wet oxidation, nitric acid, per-
chloric acid, and sulfuric acid.)
Weigh, into suitable size erlenmeyer, representative sample
<25 g, estd to contain 25-100 |JLg Zn. If sample is liq., evap.
to small vol. Add HN0 3 and heat cautiously until first vig-
orous reaction subsides somewhat; then add 2-5 mL H 2 S0 4 .
Continue heating, adding more HN0 3 in small portions as
needed to prevent charring, until fumes of SO3 evolve and soln
remains clear and almost colorless. Add 0.5 mL HC10 4 and
continue heating until it is almost completely removed. Cool,
and dil. to ca 40 mL. (Wet digestion and subsequent sulfide
sepn may also be advantageously performed in small Kjeldahl
flask.)
D. Separation of Sulfide Group
(Caution: See safety notes on bromine and hydrogen sulfide.)
To H 2 S0 4 soln add 2 drops Me red and 1 mL CuS0 4 soln,
and neutze with NH 4 OH. Add enough HCl to make soln ca
0A5N with respect to this acid (ca 0.5 mL excess in 50 mL
soln is satisfactory); pH of soln as measured with glass elec-
trode is 1.9-2.1. Pass stream of H 2 S into soln until pptn is
complete. Filter thru fine paper (Whatman No. 42, or equiv.,
previously fitted to funnel and washed with HCl (1 +6), then
with redistd H 2 0). Receive filtrate in 250 mL beaker, and wash
flask and filter with 3 or 4 small portions FLO. Gently boil
filtrate until odor of H 2 S can no longer be detected; then add
5 mL satd Br-H 2 and continue boiling until Br- free. Cool,
neutze to phenol red with NH 4 OH, and make slightly acid with
HCl (excess of 0.2 mL 1 + 1 HCl). Dil. resultant soln to
definite vol. For optimum conditions of measurement, soln
should contain 0.2-1.0 p-g Zn/mL.
E. Elimination of Nickel and Cobalt
Transfer 20 mL aliquot of prepd soln to 125 mL separator;
add 5 mL NH 4 citrate soln, 2 mL dimethylglyoxime soln, and
10 mL a-nitroso-p-naphthol soln; and shake 2 min. Discard
solv. layer and ext with 10 mL CHC1 3 to remove residual a-
nitroso-p-naphthol. Discard solv. layer.
F. Isolation and Estimation of Zinc
To aq. phase following removal of Ni and Co, which at this
point has pH of 8.0-8.2, add 2.0 mL dithizone soln and 10
mL CC1 4 , and shake 2 min. Let phases sep. and remove aq.
layer as completely as possible, withdrawing liq. with pipet
attached to vac. line. Wash down sides of separator with ca
25 mL H 2 and without shaking again draw off aq. layer. Add
25 mL 0.04N HCl and shake 1 min to transfer Zn to acid-aq.
layer. Drain and discard solv., being careful to dislodge and
remove drop that usually floats on surface. To acid soln add
5.0 mL NH 4 citrate soln and 10.0 mL CCl 4 (pH of soln at this
point is 8.8-9.0).
Det. vol. dithizone to be added as follows: To separator contg
4.0 mL working Zn std (20 |xg), dild to 25 mL with 0.04N
HCl, 5.0 mL citrate buffer, and 10.0 mL CC1 4 , add dithizone
reagent in 0.1 mL increments, shaking briefly after each addn
until faint yellow in aq. phase indicates bare excess of reagent.
Multiply vol. dithizone soln required by 1.5 and add this vol.
(to nearest 0.05 mL) to all samples. Shake 2 min. Pipet exactly
5.0 mL solv. layer into clean, dry test tube, dil. with 10.0 mL
CC1 4 , mix, and det. T (or A) at 540 nm.
G. Preparation of Standard Curves
Prep, series of separators contg 0, 5, 10, 15, and 20 )jLg Zn
dild to 25 mL with 0.04 N HCl; add 5.0 mL citrate buffer, and
proceed as with final extn of Zn, 944. 09F.
Plot T in logarithmic scale (or A on linear scale) against
concn and draw smooth curve thru points. (Intercept of this
curve may vary slightly from day to day, depending on actual
concn of dithizone used in final extn, but slope should remain
essentially same.)
Refs.: JAOAC 27, 325(1944); 28, 271(1945).
CAS-7440-66-6 (zinc)
969.32 Zinc in Food
Atomic Absorption Spectrophotometric Method
First Action 1969
Final Action 1971
(Caution: See safety notes on A AS, wet oxidation, nitric acid,
and sulfuric acid.)
AOAC Official Methods of Analysis (1990)
Zinc
273
A. Principle
Representative sample is dry or wet ashed. Residue is taken
up in acid and dild to optimum working range. A of this soln
as detd by A A spectrophotometry at 213.8 nm is converted to
Zn concn thru calibration curve.
B. Reagents
(Use Pyrex glassware exclusively; clean thoroly before use with
hot HNO^. ^ glass beads are used to prevent bumping, clean
first with strong alkali followed by hot HN0 3 . Since Pt used
in laboratory may contain significant traces of metals, clean
Pt dishes by KHS0 4 fusion followed by 10% HC1 leach.)
(a) Zinc std solns. — (7) Stock soln. — 500 |xg/mL. Dis-
solve 0.500 g pure Zn metal in 5-10 mL HC1. Evap. almost
to dryness and dil. to 1 L with H 2 0. Soln is stable indefinitely.
(2) Working soln. — Dil. aliquots of stock soln with H 2 S0 4 (1
+ 49) or 0. 17V HC1 (depending on method of ashing) to obtain
5=5 solns within range of instrument. Prep, stds in 0-10 jxg/
mL range daily. (Do not use <2 mL pipets or <25 mL vol.
flasks.)
(b) Acids. —Reagent grade HN0 3 , HC1, and H 2 S0 4 . Test
acids for freedom from Zn by A A measurement of appropri-
ately dild sample. If contaminated, purify HN0 3 and HC1 by
distn. Further test purity of reagents and efficiency of cleaning
by conducting blank detns by appropriate ashing method.
C. Preparation of Sampie Solution
Prep, representative sample by mixing, blending, or grind-
ing.
(a) Wet ashing.— Accurately weigh, into 300 or 500 mL
Kjeldahl flask, representative sample ^10 g, estd to contain
25-100 |xg Zn. (If sample is liq., evap. to small vol.) Add ca
5 mL HNO3 and cautiously heat until first vigorous reaction
subsides. Add 2.0 mL H 2 S0 4 and continue heating, maintain-
ing oxidizing conditions by adding HNO3 in small increments
(large amts may introduce Zn) until soln is colorless. Continue
heating until dense fumes of H 2 S0 4 are evolved and all HN0 3
has been removed. Cool, dil. with ca 20 mL H 2 0, filter thru
fast paper (pre-washed) into 100 mL vol. flask, and dil. to vol.
with H 2 0. Dil. further, if necessary, with H 2 S0 4 (1 + 49) to
attain working range of spectrophtr.
(b) Dry ashing. — Accurately weigh, into clean Pt dish, rep-
resentative sample estd to contain 25-100 (xg Zn. Char under
IR lamp and ash at temp. <525° until C-free. (Raise temp, of
furnace slowly to 525° to avoid ignition.) Dissolve ash under
watch glass in min. vol. HO (1 + 1). Add ca 20 mL H 2 and
evap. to near dryness on steam bath. Add 20 mL 0.1/V HC1
and continue heating ca 5 min. Filter thru fast paper into 100
mL vol. flask. Wash dish and filter with several 5-10 mL
portions of 0AN HC1, cool, and dil. to vol. with 0.UV HC1.
Dil. further, if necessary, with 0. IN HO to attain working
range of instrument.
D. Determination
Set instrument to previously established optimum conditions
or according to manufacturer's instructions. Det. A of ashed
soln or diln, and >5 stds within optimum working range, tak-
ing >2 readings (before and after sample readings). Flush burner
with H 2 and check point between readings. Det. Zn content
from std curve obtained by plotting A against |xg Zn/mL:
ppm Zn = [(fig Zn/mL from curve)
X (diln factor, mL)]/g sample
Refs.: J AOAC 51, 1042(1968).
CAS-7440-66-6 (zinc)
10. Pesticide and industrial Chemical Residues
Associate Chapter Editors:
Leon D. Sawyer
Food and Drug Administration
Bernadette M. McfVlahon
Food and Drug Administration
W, Harvey fMewsorne
Health and Welfare Canada
Gail Abbott Parker
Florida Department of Agriculture and Consumer Services
MULTIRESIDUES
GENERAL CONSIDERATIONS
970.52 Organochlorine
and Organophosphorus Pesticide Residues
General Multiresidue Methods
A. Principle
Thoroly mixed sample is extd with CH 3 CN (high-H 2 foods)
or aq. CH 3 CN (low-H 2 or high sugar foods). Fat is extd from
fatty foods and partitioned between pet ether and CH 3 CN. Ali-
quot (nonfatty samples) or entire soln (fatty samples) of CH 3 CN
is dild with H 2 and residues are extd into pet ether. Residues
are purified by chromatgy on Florisil column, eluting with mixt.
of pet and Et ethers. Residues in coned eluates are measured
by GC and identified by combinations of gas, thin layer, or
paper chromatgy.
Analyst competence in applying method for trace residues
should be assured before analysis. Recoveries of added compds
thru method should be >80%.
Absence of interferences arising from laboratory and reagent
contamination should also be assured by regular performance
of reagent blanks. Solvs in particular, because of their concn
during methods, can contribute significant interference if not
sufficiently purified. Solvs of adequate purity are com. avail-
able from several manufacturers, but each batch must be tested
under conditions of method in which it will be used.
Other reagents and app. (rubber, plastics, glass wool, etc.)
are also potential source of interferences. See references for
recoveries obtained during collaborative and validation studies
and Table 970. 52 A for commodities approved. See 970. 52H,
introductory par., and 970.52H(c) for GC performance re-
quirements: sensitivity, sepn capability, and linearity. Behav-
ior of >200 pesticides and industrial chems in method is given
in JAOAC 61, 640(1978).
B. General Reagents
Solvs must be purified and final distn conducted in all-glass
app. (Caution: See safety notes on distillation, flammable sol-
vents, toxic solvents, acetonitrile, diethyl ether, hexane, and
petroleum ether.) See 970.52A.
Solvent purity test. — Electron capture GC requires absence
of substances causing detector response as indicated by fol-
lowing test: Place 300 mL solv. in Kuderna-Danish concen-
trator fitted with 3-ball Snyder column and calibrated collec-
tion vessel, and evap. to 5 mL. Inject 5 jxL cone, from 10 |iL
syringe into gas chromatograph, using conditions described in
970.52H(c). Cone, must not cause recorder deflection >1 mm
from baseline for 2-60 min after injection.
(a) Acetonitrile . — See solv. purity test. Purify tech. CH 3 CN
as follows: To 4 L CH 3 CN add 1 mL H 3 P0 4 , 30 g P 2 0„ and
boiling chips, and distil in all-glass app. at 81-82°. Do not
exceed 82°.
Some lots of reagent grade CH 3 CN are impure and require
distn. Generally vapors from such lots will turn moistened red
litmus paper blue when held over mouth of storage container.
Pronounced amine odor is detectable.
(b) Acetonitrile saturated with petroleum ether.— Sat.
CH 3 CN, (a), with redistd pet ether, (m).
(c) Alcohol.— TJSP, reagent grade, or JvleOH, ACS.
(d) Alcoholic alkali soln. — 2%. Dissolve 2 g KOH in al-
cohol, and dil. to 100 mL.
(e) Eluting solvent, 6%.— Dil. 60 mL Et ether, (h), to 1 L
with redistd pet ether, (m).
(f) Eluting solvent, 15%. — Prep, as in (e), using 150 mL
Et ether.
(g) Eluting solvent, 50%. — Prep, as in (e), using 500 mL
Et ether.
(h) Ethyl ether. — See solv. purity test. Redistd at 34-35°,
and stored under N. Add 2% alcohol. Must be peroxide-free
by test in Definitions of Terms and Explanatory Notes.
(1) Florisil.— 60/ \Q0 PR grade, activated at 675°C (1250°F),
available from Floridin Co., 3 Pennsylvania Center, Pitts-
burgh, PA 15235. When 675°C activated Florisil is obtained
in bulk, transfer immediately after opening to ca 500 mL (1
pt) glass jars, or bottles, with g-s or foil -lined, screw-top lids,
and store in dark. Heat >5 hr at 130° before use. Store at 130°
in g-s bottles or in air-tight desiccator at room temp, and reheat
at 130° after 2 days.
Prep, mixed pesticide std soln in hexane contg 1, 4, I, 2,
1, 2, and 4 (xg/mL, resp., of ronnel, ethion, heptachlor epox-
ide, parathion, dieldrin, endrin, and malathion.
Test each batch of activated Florisil by placing 1 mL mixed
pesticide std on prepd column and eluting as in Cleanup,
970.520. Cone, eluates from Florisil column to 10 mL. Inject
aliquot (see 970. 52H) of each eluate into gas chromatograph
and det. quant, recovery of each compd as in 970. 52R. Florisil
that quant, elutes heptachlor epoxide, ronnel, and ethion in 6%
eluate; dieldrin, endrin, and parathion in 15% eluate; and mal-
athion in 50% eluate, is satisfactory.
Adsorptivity of lots of Florisil may be tested with lauric acid
and size of column adjusted to compensate for variation in
adsorptivity (IAOAC 51, 29(1968)). Test adjusted column be-
fore use by performing elution test above.
274
AOAC Official Methods of Analysis (1990)
MULTIRESIDUES
275
Table 970.52A Compounds and Commodities to Which
General Method Applies
Compound
Official Final Action
Dieldrin (CAS-60-57-1)
Heptachlor epoxide (CAS-1 024-57
BHC (CAS-608-73-1)
DDE (CAS-72-55-9)
DDT (CAS-801 7-34-3)
TDE (CAS-72-54-8)
Lindane (CAS-58-89-9)
Methoxychlor (CAS-72-43-5)
Ethylan (CAS-72-56-0)
Aldrin (CAS-309-00-2)
Endrin (CAS-72-20-8)
Heptachlor (CAS-76-44-8)
Mirex (CAS-2385-85-5)
0,p'-DDT (CAS-801 7-34-3)0
Diazinon (CAS-333-41-5)
Ethion (CAS-563-12-2)
Malathion (CAS-1 21 -75-5)
Me parathion (CAS-298-00-0)
Parathion (CAS-56-38-2)
Ronnel (CAS-299-84-2)
PCBs (CAS-1 2767-79-2)
*)
Group I nonfatty foods, dairy prod-
ucts, fish, vegetable oils, whole
Group I nonfatty foods, dairy prod-
ucts, fish, whole eggs
Group I nonfatty foods, dairy prod-
ucts, whole eggs
Group I nonfatty foods, whole eggs
Group I nonfatty foods, dairy prod-
ucts
J
Group II nonfatty foods
Poultry fat, fish, dairy products
Group I nonfatty foods: apples*, apricots, barley*, beets, bell peppers,
broccoli*, cabbage*, cantaloupes, cauliflower*, celery, collard greens, corn
meal and silage, cucumbers*, eggplant, endive, grapes*, green beans, hay,
kale*, mustard greens*, oats*, peaches, pears, peas, plums, popcorn, po-
tatoes*, radishes, radish tops, spinach, squash*, strawberries, sugar beets,
sweet potatoes, tomatoes*, turnips*, turnip greens*, wheat*
Group II nonfatty foods: Group I nonfatty foods marked with asterisk (*)
plus carrots, green peppers, and lettuce
(j) Hexane. — See solv. purity test. Reagent grade, redistd
in all-glass app.
(k) Magnesium oxide. — Adsorptive magnesia (Fisher Sci-
entific Co. No. S-120). Treat as follows: Slurry ca 500 g with
H 2 0, heat on steam bath ca 30 min, and filter with suction.
Dry overnight at 105-130° and pulverize to pass No. 60 sieve.
Store in closed jar.
(I) Magnesia-C elite mixture. — Mix treated MgO, (k), with
Celite 545, 1 + 1 by wt. Pet ether ext of Celite should be free
of electron-capturing substances.
(m) Petroleum ether. — See solv. purity test. Reagent grade,
redistd in all-glass app. at 30-60°.
(n) Sodium sulfate. — Anhyd., granular.
C. Reagents for Thin Layer Chromatography
(a) Aluminum oxide. — Neutral A1 2 3 "G" (Type E, EM
Science No. 1090), or equiv., for TLC.
(b) Developing solvents for organochlorine pesticides. — (7)
w-Heptane, com. grade. (2) n- Heptane contg 2% reagent grade
acetone.
(c) Chromogenic agent for organochlorine pesticides. —
Dissolve 0.100 g AgN0 3 in 1 mL H 2 0, add 20 ml 2-phen-
oxyethanol (Practical, Eastman Kodak Co.), dil. to 200 mL
with acetone, add very small drop 30% H 2 2 , and mix. Store
in dark overnight and decant into spray bottle. Discard after 4
days.
(d) Developing solvents for organophosphorus pesti-
cides. — (7) Immobile. — 15 or 20% /V^-dimethylformamide
(DMF) in ether. Dil. 75 or 100 mL DMF to 500 mL with ether
and mix. (2) Mobile. — Methyleyclohexane.
(e) Chromogenic agents for organophosphorus pesti-
cides. — (7) Stock dye soln. — Dissolve 1 g tetrabromophe-
nolphthalein Et ester (Eastman No. 6810) in 100 mL acetone.
(2) Dye soln. — Dil. 10 mL stock dye soln (7) to 50 mL with
acetone. (3) Silver nitrate soln. — Dissolve 0.5 g AgN0 3 in 25
mL H 2 and dil. to 100 mL with acetone. (4) Citric acid soln. —
Dissolve 5 g granular citric acid in 50 mL FLO and dil. to 100
mL with acetone.
D. Reagents for Paper Chromatography*
—Surplus 1980
See 29.004, 13th ed.
E. General Apparatus
(a) High-speed blender. — Waring Blend or, or equiv.
(b) Chromatographic tubes. — With Teflon stopcocks and
coarse fritted plate or glass wool plug; 22 mm id x 300 mm.
(c) Chromatographic tubes without stopcocks. — 22 mm id
x 300 or 400 mm.
(d) Filter tubes. — Approx. 22 mm id x 200 mm with short
delivery tube and coarse fritted plate or glass wool plug.
(e) Kuderna-Danish concentrators. — 500 and 1000 mL with
Snyder distilling column and 5 or 10 mL plain, vol., and grad-
uated receiving flasks (Kontes Glass Co. No. K-570000, K-
621400, and K-570050, or equiv.).
(f) Separators. — 1000 and 125 mL with Teflon stopcocks.
(g) Micro-Snyder column. — 2-ball (Kontes Glass Co. No.
K-569001, or equiv).
(h) Micro-Vigreaux column. — Kontes Glass Co. No. K-
569251, or equiv.
F. Apparatus for Thin Layer Chromatography
(a) Desaga/Brinkmann standard model applicator, or equiv.
(b) Desaga/Brinkmann standard mounting board, or equiv.
(c) Desaga/Brinkmann drying rack } or equiv. — Accom-
modates ten 8 x 8" plates.
(d) Desaga/Brinkmann model 51 stainless steel desiccating
cabinet, or equiv.
(e) Window glass. — 8 X 8", double strength window glass
plates of uniform width and thickness; smooth off corners and
edges with file or other tool.
(f) Chromatographic tank and accessories. — With metal
instead of glass troughs.
(g) Dipping tank and accessories. — Stainless steel, 8V2 x
8V2 x l U- 3 /w" inside width with metal supports and close-
fitting U-shaped cover ca 9 x V2"- Capacity ca 300 mL.
(h) Spotting pipets. — 1 jxL.
(i) Spray bottle. — 8 oz (Thomas Scientific No. 2753-J10 or
Lurex Scientific No. 131-0514, 250 mL).
( j) Chromatography spray flask. — 250 mL (Microchemical
Specialties Co., 1825 Eastshore Hwy, Berkeley, CA 94710,
No. S-4530-D).
(k) Tank liner.— Cut 2 pieces, U l / 4 x 8 3 / 4 ", from desk
blotter, white or colored, and bend into L-shape to fit tank.
(!) Strong ultraviolet light source. — Such as germicidal lamps
(General Electric Co., Nela Park, Cleveland, OH 44112), either
(7) two 30 watt, 36" tubes, No. G30T8, mounted in std 30
watt reflector fixture ca 20 cm above papers; or (2) two 15
watt, 18" tubes, No. G15T8, mounted in std 15 watt desk lamp
fixture placed ca 10 cm above papers. Shield to protect eyes
and skin at all times.
G. Apparatus for Paper Chromatography*
—Surplus 1980
See 29.007, 13th ed.
H. Apparatus for Gas Chromatography
(See also JAOAC 47, 326-342(1964); 49, 8-21(1966).)
Gas chromatgc system when operated with column, (b), and
approx. conditions described in Gas Chromatography, 970. 52R,
276
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
should be capable of producing ca V2 scale deflection for 1 ng
heptachlor epoxide by electron capture detection and for 2 ng
parathion by KC1 -thermionic detection, and should resolve mixt.
of heptachlor, aldrin, heptachlor epoxide, ethion, and carbo-
phenothion into sep. peaks. Retention time for aldrin should
be ca 4.5 min. Compds of interest must not be degraded by
any part of GC system.
(a) Gas chromatograph. — Instrument consisting of on-col-
umn injection system, all-glass column in oven controlled to
±0.1°, electron capture and thermionic detectors, each with
independent power supply, electrometer, and appropriate mv
recorder.
(b) Column. — Glass, 1.85 m (6') X 4 mm id packed with
10% DC-200 (w/w) on solid support: (/) 80-100 mesh Chro-
mosorb W HP (Manville Filtration and Minerals, manufac-
turer, but available thru many GC distributors); (2) 80-100
mesh Gas-Chrom Q (Applied Science Laboratories, inc.); (3)
80-90 mesh Anakrom ABS (Analabs, Inc.). DC-200 may be
replaced by OV-101 (available from many GC distributors).
Weigh 2 g Dow Corning 200 silicone fluid (12,500 centi-
stokes) or OV-101 into beaker. Dissolve in CHC.I 3 and transfer
to 300 mL Morton-type flask, using total of ca 100 mL CHC1 3 .
Add 18 g solid support, (7), (2) or (J), to flask. Swirl, and
let stand ca 10 min. Place flask on rotary evaporator and re-
move solv. slowly with intermittent rotation, using 50° H 2
bath and slight vac. (Foaming may occur initially.) When sol-
ids appear damp, increase vac. Remove last traces of CHC1 3
without rotation or by air drying. Use only free-flowing ma-
terial to fill column. Use care at all stages of column prepn to
prevent fracturing solid support. Condition column at 250-260°
with N flow of ca 100 mL/min >48 hr or until endrin exhibits
single peak.
(c) Electron capture detector (ECD). — Concentric design,
for use with dc voltage supply and 3 H source (ca 150 mCi 3 H,
U.S. Nuclear Regulatory Commission license is required.)
Det. detector operating characteristics as follows: Apply dc
voltage to detector. After system becomes stable (overnight),
det. current-voltage relationship at various voltages between
200 and v. (Current measurements at voltages of 200, 150,
100, 75, 50, 40, 30, 25, 20, 15, 10, 8, 6, 4, 2, and 1 provide
points for smooth curve.) Slightly lower, stable, standing cur-
rent may be obtained after detector has been at operating temp,
several hr. This is probably due to loss of some easily removed
radioactive material. Det. and plot response- voltage relation-
ship at 1 X 10~ 9 amp full scale sensitivity for 1 ng injections
of heptachlor epoxide at same voltages used in obtaining cur-
rent-voltage curve. Select as operating voltage that voltage at
which heptachlor epoxide causes ca 40-50% full scale re-
corder deflection. Check linearity of system from 0.2 to 2.0
ng heptachlor epoxide.
Other electron capture detectors may be substituted for dc
voltage concentric design 3 H detector, which is no longer mar-
keted. Const current, variable frequency 63 Ni electron capture
detectors are acceptable substitutes when operated at condi-
tions to produce stable, reproducible, linear responses. Opti-
mum conditions may produce more sensitive response than from
3 H detector. To maintain same method limit of quantitation of
3 H detector, inject proportionately smaller equiv. sample wt
into 63 Ni detector system. The 63 Ni electron capture detector
may provide different relative responses for pesticides than those
obtained with 3 H electron capture detector. Use of Ar-CH 4 car-
rier gas, as recommended for most 63 Ni detectors, precludes
use of KC1TD dual detection system, (d), (h)-(j).
(d) Potassium chloride thermionic detector (KCITD). —
Hame ionization detector modified to incorporate coil with KC1
coating prepd as in (/) or (2). Detector voltage is 300 v dc.
Use in dual arrangement with electron capture detector.
All dual detector systems described are capable of compa-
rable performance. In-series, (h), arrangement is preferred be-
cause of simplicity and ease of operation.
(7) Coil with potassium chloride for in-series dual detec-
tor. — See Fig. 970. 52A (may be used with all detector ar-
rangements). Wind Pt-Ir wire (B&S gage 26) on 7 mm diam.
rod into 2 turn helix so that turns are touching. Approx. 5 mm
below helix, continue to wind wire on 3 mm rod, or rod with
same outside diam. as flame jet, making 3-turn spiral. Cut
wire so that 7 mm helix is supported 4 mm above flame jet
when 3 mm spiral is slipped over jet. Fill 30 mL tall -form Pt
crucible ca V4 full with KC1 (ACS). Heat with Meker burner
until all salt melts. Continue heating until bottom of crucible
glows red, imparting pink glow to melt. Remove heat and be-
gin dipping the 2-turn helix of coil into melt at 5 sec intervals
as melt cools. (Make sure only 2-turn helix touches melt and
do not raise coil above top of crucible.) When melt is at proper
temp., salt clings to coil. Remove coil from melt. Place probe
in center of coil while salt is molten. This causes crystn around
probe tip. Remove center of coil. Remove any rough edges on
coil coating by holding coil in burner flame 1 sec; id of prop-
erly coated coil is 5 mm. Position coil over flame jet.
(2) Coil with potassium chloride for parallel and in-series
split dual detectors.— See Fig. 970.52B. Wind Pt-Ir wire (B&S
gage 26) on 5 mm diam. rod into 5-turn helix so that turns are
close together or touching. Continue to wind wire on 3 mm
rod, or rod having same outside diam. as flame jet, making
3-turn spiral. Cut wire so that 5 mm helix is supported 2 mm
above flame jet when 3 mm spiral is slipped over jet. Grasp
formed wire by end opposite 5 mm helix with forceps. Dip 5
mm helix into satd KC1 (recrystd twice from H 2 0) soln, or
apply KC1 soln with dropper. Fuse in flame. {Caution: Use
safety glasses; spattering occurs.) Repeat application of KC.1
soln 3-4 times until helix is coated with fused KC1. Coating
should appear almost crystal clear. Position coil over flame
jet.
(e) Hydrogen. — From generator or cylinder of compressed
H gas (cylinder preferred). Equip cylinder with pressure drop
of stainless steel capillary tubing (0.020" id) to restrict H flow
to ca 30 mL/min at 20 lb delivery pressure. Place H source
close to detector and use gas lines with min. dead vol. to re-
duce outgassing time in lines. (For fine precise control of H
flow, insert Nupro Fine Metering Valve, "S" series (Swagelok
Co., 31400 Aurora Rd, Solon, OH 44138; Part Number B-1S)
between exit end of capillary tubing pressure drop and inlet of
detector H line. Caution: Do not use Nupro valve as shut-off
valve. Repeated tightening damages needle.) Use Swagelok
fittings for all connections.
& — 7mm— h
FSG. 970.52A — KCI thermionic detector coil for in-series dual
detection system
AOAC Official Methods of Analysis (1990)
MULTIRESIDUES
277
FIG. 970.52B— KCI thermionic detector coil for parallel and
in-series split dual detection systems
(f) Air. — Min. air requirement for thermionic detector is
300 mL/min. Cylinder of compressed air or aquarium air pump
is recommended.
(g) Capillary T-tube .—{See Figs. 970.52C and 970.52D.)
Prep. 1:1 stream splitter (B) for parallel and in-series split dual
detection systems. Fit two 4.5 cm lengths of stainless steel
capillary tubing, 0.010" id, Vir/ od, into 1 cm length of std
wall, Vs" stainless steel tubing. Fit 1" length of No. 16 hy-
podermic tubing at right angles in hole drilled into the piece
of Vs" tubing. Silver braze all connections. Prep, capillary T-
tube (E) for introducing purge gas to parallel system. Fit two
2.5 cm lengths of No. 16 hypodermic tubing into 1 cm length
of std wall, Vs" stainless steel tubing. Fit 1 cm length of No.
16 hypodermic tubing at right angles in hole drilled into piece
of Vs" tubing. Silver braze as above.
(h) Assembly of in-series dual detection system. — Assem-
ble as in Fig. 970.52E. Introduce column effluent (A) of 120
N120 ml/min ^ N120ml/min _B_ N 60 ml/min
» > ECO -S- CZZ) »> KC/TD
Atmosphere
FIG. 970.52C~-ln-series split dual detection system
N 60 ml/min
N60ml/mi'n
120 ml/min
^-**ECD
N 120 ml/min . , .
a M b
N 60 ml/min
-^KOTD
FIG. 970.52D — Parallel dual detection system
N 120 ml/min . Nl20ml/m/n v
-**■ ECO -*- ^KCITD
FIG. 970.52E— In-series dual detection system
mL/min directly to ECD inlet. Connect ECD outlet directly
to KC1TD inlet, using No. 16 std wall Teflon tubing.
Note: For in-series, (h), and in-series split, (i), operation,
thoroly check ECD for gas leaks, particularly at Teflon insu-
lator.
(i) Assembly of in-series split dual detection system. — As-
semble as in Fig. 970. 52C. Introduce column effluent (A) of
120 mL/min directly to ECD inlet. Connect 1:1 stream splitter
(B) between ECD outlet and KC1TD inlet so that only 60 mL
N/min enters KC1TD and remaining 60 mL N/min exits to
atm. Use No. 16 std wall Teflon tubing for all connections.
See Note in (h).
( j) Assembly of parallel dual detection system. — Assemble
as in Fig. 970.52D. Split column effluent (A) of 120 mL/min
by passing thru 1:1 stream splitter (B) so that each detector
receives 60 mL effluent/min. Increase flow to ECD by intro-
ducing 60 mL N/min from second N source (C) thru capillary
T-tube (E). Preheat N from C by passing thru stainless steel
capillary tube (D) (0.040" id) which extends 120 cm into col-
umn bath and returns to detector bath where addnl 35 cm of
tubing is coiled into small helix. Connect capillary tubes and
splitters to detectors with No. 16 std wall Teflon tubing. Mea-
sure flow at each end of splitter (B) to ensure exact 1:1 split.
(k) Potassium chloride thermionic detector operation. — Zero
recorder with zero control before detector flame is ignited (no
signal). Turn on H (ca 30 mL/min) and ignite flame. Adjust
H with flame burning to give baseline current (BLC) of 0.2-
0.8 X 10~ 8 amp. (Sensitivity to P compds is directly related
to KCI temp., which depends on H concn in flame.) Select
operational electrometer setting and adjust H concn to obtain
40-50% full scale recorder deflection for 2 ng parathion en-
tering detector. When baseline has stabilized, measure BLC
precisely, at electrometer setting of 1 x 10~ 8 amp full scale.
Return to operational electrometer setting and zero recorder
pen, using current balance control to "buck out" current gen-
erated by detector. Check linearity of system from 0.4 to 4.0
ng parathion. Monitor BLC frequently during operation. If drift
occurs, readjust H concn to maintain same BLC. For accurate
quantitation, BLC must be identical during chromatgy of sam-
ple and std.
Concentration Technics
I. Purified Extracts
(Never evap. purified exts to dryness.)
(a) To approximately 5 mL or more. — Evap. on steam bath
in Kuderna-Danish concentrator fitted with 3 -ball Snyder col-
umn and vol. flask or graduated collection tube; 20-mesh boil-
ing chip is necessary.
(b) To less than 5 mL. — Evap. to ca 5 mL as in (a). Re-
move collection tube from concentrator and fit tube with 2-
ball micro- Snyder or micro-Vigreux column. Evap. to slightly
less than desired vol., permit condensate to drain into tube,
and remove column. Min. attainable vol is 0.2-0.4 mL.
J. Extracts Containing Fats, Oils, or Plant Extractives
(a) Kuderna-Danish concentrator. — Fitted with 3-ball Sny-
der column and vol. flask or graduated collection tube. Use
on steam bath.
(b) Flash evaporator. — Keep flask in H 2 bath at room
temp.
(c) Beaker. — Evap. in beaker in H 2 bath at 35-40° under
stream of clean, dry air. Remove from heat and air stream as
soon as last of sol v. evaps. Let residual H 2 evap. sponta-
278
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
neously. Solvs may be evapd from fats on steam bath for short
periods.
Preparation of Sample and Extraction
K. Nonfatty Foods
{Caution: See safety notes on blenders, distillation, flammable
solvents, toxic solvents, acetonitrile, and petroleum ether.)
Pit soft fruits, if necessary. Chop or blend representative
sample of leafy or cole-type vegetables, pitted soft fruits, firm
fruits, and roots. Mix thoroly to obtain homogeneous sample
before taking portions for analysis. Grind dry or low moisture
products, e.g., hays, to pass No. 20 sieve and mix thoroly.
Proceed as in (a), (b), (c), or (d).
(a) High moisture (more than 75% H 2 0) products contain-
ing less than 5% sugar. — (I) Products other than eggs. — Weigh
100 g chopped or blended sample into high-speed blender jar,
add 200 mL CH 3 CN and ca 10 g Celite, and blend 2 min at
high speed. Filter with suction thru 12 cm buchner fitted with
sharkskin paper into 500 mL suction flask. Transfer filtrate to
250 mL graduate and record vol. (F). Transfer measured fil-
trate to 1 L separator, and proceed as in (e). (2) Whole eggs. —
Discard shells and blend combined yolks and whites at low
speed >5 min or until sample is homogeneous. Low-speed
blending will minimize foaming or "whipping"' of sample.
Weigh <25 g thoroly mixed yolks and whites into high-speed
blender jar, and proceed with addn of CH 3 CN as in (/).
(b) High moisture (more than 75% H2O) products contain-
ing 5-15% sugar.— Add 200 mL CH 3 CN and 50 mL H 2 to
100 g sample in blender and proceed as in (a). Transfer <250
mL filtered ext (record vol. (F)) to 1 L separator, and proceed
as in (e).
(c) High moisture (more than 75% H 2 0) products contain-
ing 15-30% sugar, e.g., grapes. — Heat mixt. of 200 mL
CH 3 CN and 50 mL H 2 to 75°, add to 100 g sample in blender,
and immediately proceed as in (a). Before filtered ext cools,
transfer <250 mL (record vol. (F)) to 1 L separator. Let cool
to room temp, and proceed as in (e).
(d) Dry or low-moisture products, e.g., hays. — Add 350
mL 35% H 2 0-CH 3 CN (350 mL H 2 dild to 1 L with CH 3 CN)
to 20-50 g ground sample in blender (if larger sample is re-
quired, add enough addn I extn mixt. to wet sample and permit
thoro blending). Blend 5 min at high speed, and proceed as
in (a), beginning "Filter with suction ..." Transfer <250
mL filtered ext (record vol. (F)) to 1 L separator, and proceed
as in (e).
(e) Transfer of residues to petroleum ether. — Carefully
measure 100 mL pet ether and pour into separator contg fil-
trate. Shake vigorously 1-2 min and add 10 mL satd NaCl
soln and 600 mL H 2 0. Hold separator in horizontal position
and mix vigorously 30-45 sec. Let sep., discard aq. layer, and
gently wash solv. layer with two 100 mL portions H 2 0. Dis-
card washings, transfer solv. layer to 100 mL g-s cylinder, and
record vol. (P). Add ca 15 g anhyd. Na 2 S0 4 and shake vig-
orously. Do not let ext remain with Na 2 S0 4 >1 hr or losses
of organochlorine pesticides by adsorption may result. Trans-
fer soln directly to Florisil column, 970.520, or cone, to 5-
10 mL in Kuderna-Danish concentrator for transfer.
(f) Calculation for fruits and vegetables. — Calc. g sample
as S x (F/T) x (P/100); where S = g sample taken; F = vol.
filtrate; T - total vol. (mL H 2 in sample + mL CH 3 CN added
- correction in mL for vol. contraction); P = mL pet ether
ext; and 100 = mL pet ether into which residues were parti-
tioned. When 50 mL H 2 is added to CH 3 CN for extn of high
sugar products, total vol., T, is increased by 45, i.e., T = 325
instead of 280 for samples contg 85% H 2 0.
Example: 100 g sample contains 85 g H 2 0; 200 mL CH 3 CN
is added; vol. contraction is 5 mL. Total vol., T, is 280 mL.
If vol. filtrate is 235 mL, vol. pet ether ext is 85 mL, and
residue is transferred to 100 mL pet ether, then 100 x (235/
280) X (85/100) - 71 g sample.
Consult refs on food composition for av. H 2 content. Water
content of most fresh fruits and vegetables may be assumed to
be 85%.
For 25 g whole eggs and 200 mL CH 3 CN, use 215 as T.
(g) Calculation for dry or low moisture products, e.g.,
hays. — Calc. g sample as in fruits and vegetables, (f), except
T = total vol. (mL H 2 in sample 4- mL 35% H 2 0-CH 3 CN
added - correction in mL for vol. contraction). If H 2 content
of sample is <10%, disregard and use vol. of extg mixt.
as 7\
L Fat-Containing Foods
(After isolation of fat, proceed with CH 3 CN partitioning,
970.52H.)
(a) Animal and vegetable fats and oils. — If solid, warm un-
til liq. and filter thru dry filter.
(b) Butter. — Warm at ca 50° until fat seps and decant fat
thru dry filter.
(c) Milk. — (Caution: See safety notes on distillation, flam-
mable solvents, diethyl ether, and petroleum ether.) To 100
mL fluid milk (dil. evapd milk 1 + 1 with H 2 0) in 500 mL
centrf. bottle, add 100 mL alcohol or MeOH and ca 1 g Na
or K oxalate, and mix. Add 50 mL ether and shake vigorously
1 min; then add 50 mL pet ether and shake vigorously I min.
Centrf. ca 5 min at ca 1500 rpm. Blow off solv. layer with
wash bottle device, Notes, into 1 L separator contg 500- 600
mL H 2 and 30 mL satd NaCl soln. Re-ext aq. residue twice,
shaking vigorously with 50 mL portions ether-pet ether (1 +
1); centrf. and blow off solv. layer into separator after each
extn. Mix combined exts and H 2 cautiously. Drain and dis-
card H 2 0. Rewash solv. layer twice with 100 mL portions H 2 0,
discarding H 2 each time. (If emulsions form, add ca 5 mL
satd NaCl soln to solv. layer or include with H 2 wash.) Pass
ether soln thru column of anhyd. Na 2 S0 4 , 50 x 25 mm od,
and collect eluate in 400 mL beaker. Wash column with small
portions pet ether and evap. solv. from combined exts at steam
bath temp, under air current to obtain fat.
(d) Cheese. — Place 25-100 g (to provide 3 g fat) diced
sample, ca 2 g Na or K oxalate, and 100 mL alcohol or MeOH
in high-speed blender and blend 2-3 min. (If experience with
product indicates emulsions will not be broken by centrfg, add
1 mL H 2 0/2 g sample before blending.) Pour into 500 mL
centrf. bottle, add 50 mL ether, and shake vigorously 1 min;
then add 50 mL pet ether and shake vigorously 1 min (or di-
vide between two 250 mL bottles and ext each by shaking
vigorously 1 min with 25 mL each ether). Proceed as in (c),
beginning "Centrf. ca 5 min at ca 1500 rpm."
(e) Fish. — (Caution: See safety notes on blenders, distil-
lation, flammable solvents, and petroleum ether.)
Weigh 25-50 g thoroly ground and mixed sample into high-
speed blender. (If fat content is known or can be estd, adjust
sample size so that max. of ca 3 g fat will be extd.) Add 100
g anhyd. Na 2 S0 4 to combine with H 2 present and disintegrate
sample. Alternately blend and mix with spatula until sample
and Na 2 S0 4 are well mixed. Scrape down sides of blender jar
and break up caked material with spatula. Add 150 mL pet
ether and blend at high speed 2 min. Decant supernate pet
ether thru 12 cm buchner, fitted with 2 sharkskin papers, into
500 mL suction flask. Scrape down sides of blender jar and
break up caked material with spatula. Re-ext residue in blender
jar with two 100 mL portions pet ether and blend 2 min each
time. (After blending 1 min, stop blender, scrape down sides
of blender jar, and break up caked material with spatula; con-
AOAC Official Methods of Analysis (1990)
MULTIRESIDUES
279
tinue blending 1 min.) Scrape down sides of blender jar and
break up caked material between extns. Decant supernate pet
ether from repeat blend ings thru buchner and combine with
first ext. After last blending, transfer residue from blender jar
to buchner, and rinse blender jar and material in buchner with
three 25-50 mL portions pet ether. Immediately after last rinse,
press residue in buchner with bottom of beaker to force out
remaining pet ether. Pour combined exts thru 40 X 25 mm od
column of anhyd. Na 2 S0 4 and collect eluate in 500 or 1000
mL Kuderna-Danish concentrator with plain tube. Wash flask
and column with small portions pet ether and evap. most of
pet ether from combined exts and rinses in Kuderna-Danish
concentrator. Transfer fat so In to tared beaker, using small amts
pet ether. Evap. pet ether at steam bath temp, under current
of dry air to obtain fat. When pet ether is completely removed,
weigh and record wt of fat extd.
Record wt of fat taken for cleanup. ((Wt fat for cleanup/wt
fat extd) X wt original sample = wt sample analyzed.) If it is
known that <3 g fat will be extd from particular sample, do
not isolate and weigh fat before CH 3 CN partitioning. Detn is
then on basis of wt of original sample.
Notes: To siphon off ether, use tube similar to delivery tube
of ordinary wash bottle but with intake end bent up into U
shape in opposite direction to outlet end, with opening 6-12
mm higher than bottom of U, cut off horizontally. (Avoid ex-
cessive constriction when bending.) Set delivery tube loosely
enough in stopper that it can be raised or lowered. In oper-
ating, adjust opening of U bend to ca 3 mm above surface of
aq. layer and blow ether layer off by gently blowing thru
mouthpiece tube inserted in adjacent hole in stopper.
M. Soil
(Official final action (1976) for aldrin, p,//-DDE, o,//-DDT,
/?,//- DDT, dieldrin, endrin, heptachlor, heptachlor epoxide,
lindane, and p.p'-TDE (DDD))
Weigh 10.0 g undried soil, sieved thru 2 mm sieve and mixed
thoroly, into 250 mL erlenmeyer. Add 7 mL 0.2M NH 4 Cl soln
(10.7 g/L) and let stand 15 min. Add 100 mL hexane-acetone
(1 + 1), stopper tightly, and shake overnight (>12 hr) on re-
ciprocal or wrist-action shaker at 180 rpm.
Carefully pour supernate, avoiding aq.-clay phase, thru 2-
3 cm column (22 mm id) of Florisil, 970.52B(i), and collect
eluate in 1 L separator. Rinse flask and soil with two 25 mL
portions hexane-acetone and decant thru column. Rinse col-
umn with 10 mL hexane-acetone.
Add 200 mL H 2 to separator and shake gently ca 30 sec.
Drain aq. phase into second separator and ext with 50 mL hex-
ane. Combine hexane layers in first separator and wash with
100 mL H 2 0. Drain and discard H 2 0. Pour hexane thru 2 cm
column (22 mm id) Na 2 S0 4 , cone, to 100 mL, and make pre-
liminary injection of 5-10 |ulL into gas chromatograph. If peaks
are present at retention times of DDE or dieldrin, cone, to 10
mL in Kuderna-Danish concentrator, 970.521(a), and sep. DDE
or dieldrin as in 970.520. (This cleanup may also be necessary
with exts from high org. matter soils.) Proceed as in 970. 52Q,
using ECD, (b). To calc. to dry basis, dry sep. sample of 10
g ca 16 hr at 105° to obtain % solids.
Refs.: JAOAC 51, 403, 472(1968); 56, 728(1973); 57,
604(1974).
Cleanup Technics
N. Acetonitrile Partitioning
(Caution: See safety notes on distillation, flammable solvents,
and petroleum ether. Different fats and oils may show varying
tendencies to emulsion formation.)
Weigh <3 g fat into 125 mL separator, and add pet ether
so that total vol. of fat and pet ether is 15 mL. Add 30 mL
CH 3 CN satd with pet ether, 970.52B(b), shake vigorously 1
min, let layers sep., and drain CH 3 CN into 1 L separator contg
650 mL H 2 0, 40 mL satd NaCl soln, and 100 mL pet ether.
Ext pet ether soln in 125 mL separator with 3 addnl 30 mL
portions CH 3 CN satd with pet ether, shaking vigorously 1 min
each time. Combine all exts in the 1 L separator.
(If experience with particular sample (e.g., fish) indicates
that cleanup may not be sufficient, perform partitioning as fol-
lows: Drain CH 3 CN phase from first partitioning into second
125 mL separator contg 15 mL pet ether, shake vigorously 1
min, let layers sep., and drain CH 3 CN into 1 L separator contg
650 mL H 2 0, 40 mL satd NaCl soln, and 100 mL pet ether.
Pass CH 3 CN phase from each of 3 addnl partitionings thru
same 15 mL pet ether in 125 mL separator. Shake vigorously
each time and combine CH 3 CN exts in the 1 L separator.)
Hold separator in horizontal position and mix thoroly 30™
45 sec. Let layers sep. and drain aq. layer into second 1 L
separator. Add 100 mL pet ether to second separator, shake
vigorously 15 sec, and let layers sep. Discard aq. layer, com-
bine pet ether with that in original separator, and wash with
two 100 mL portions H z O. Discard washings and draw off pet
ether layer thru 50 x 25 mm od column of anhyd. Na 2 S0 4
into 500 mL Kuderna-Danish concentrator. Rinse separator and
then column with three ca 10 mL portions pet ether. Evap.
combined ext and rinses to ca 10 mL in Kuderna-Danish con-
centrator for transfer to Florisil column.
O. Florisil Cleanup
(Caution: See safety notes on distillation, flammable solvents,
toxic solvents, diethyl ether, and petroleum ether.)
Prep. 22 mm id Florisil column, 970.52E(b), contg 10 cm,
after settling (or amt detd by lauric acid test, 970. 52B(i)), of
activated Florisil topped with ca 1 cm anhyd. Na 2 S0 4 . Pre wet
column with 40-50 mL pet ether. Place Kuderna-Danish con-
centrator with vol. flask or graduated collection flask under
column to receive eluate. Transfer pet ether ext or cone, to
column, letting it pass thru at <5 mL/min. Rinse containers
and Na 2 S0 4 , if present, with two ca 5 mL portions pet ether,
pour rinsings onto column, rinse walls of tube with addnl small
portions pet ether, and elute at ca 5 mL/min with 200 mL 6%
eluting solv., 970.528(e). Change receivers and elute with 200
mL 15% eluting solv., 970.528(f), at ca 5 mL/min. Change
receivers and elute with 200 mL 50% eluting solv., 970.528(g),
at ca 5 mL/min.
Cone, each eluate to suitable definite vol. in Kuderna-Dan-
ish concentrator. When vol. <5 mL is needed, use 2-ball mi-
cro-Snyder or micro-Vigreux column.
First eluate (6%) contains or gano chlorine pesticides (aldrin,
BHC, DDE, DDD (TDE), o<p'- and p t p'-DDT, heptachlor,
heptachlor epoxide, lindane, methoxychlor, mirex, and ethy-
lan), industrial chems (polychlorinated biphenyls (PCB)), and
organophosphorus pesticides (ethion and ronnel) and is usu-
ally suitable for GC directly. If further cleanup is necessary,
repeat Florisil cleanup, using new column. Second eluate (15%)
contains organochlorine pesticides (dieldrin and endrin) and
organophosphorus pesticides (diazinon, Me parathion, and
parathion). If further cleanup is necessary, det. organophos-
phorus pesticides by GC and TLC; then proceed with Mag-
nesia Cleanup, 970. 52P, and/or Saponification, 970. 52Q,
which are applicable only to organochlorine pesticides in 15%
eluate (organophosphorus pesticides are degraded). Third eluate
(50%) contains organophosphorus pesticide malathion.
P. Magnesia Cleanup
(Applicable only to organochlorine pesticides in 15% eluate
when addnl cleanup is necessary)
280
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
Transfer ca 10 g MgO-Celite mixt., 970.52B(!), to chro-
matgc tube without stopcock, 970.52E(c), using vac. to pack.
Prewash with ca 40 mL pet ether, discard prewash, and place
Kudema-Danish concentrator under column. Transfer 15%
Florisil eluate, coned to ca 5 mL, to column, rinsing with small
portions pet ether. Force pet ether into column with slight vac.
or pressure. Then elute with 100 mL pet ether. Cone, eluate
to suitable vol. Proceed with detn, or saponification, if re-
quired.
Q. Saponification-First Action
(Applicable only to those chems stable to hot alkali treatment.
Use as supplemental cleanup if 15% eluate or MgO-Celite
eluate is not substantially free from oily materials.)
Cone. pet. ether-ether (85 + 15) fraction under current of
air to 2 ml, add 1 mL 2% ale. KOH, attach micro-Snyder
column, and carefully reduce to <1 mL on steam bath. Reflux
sample 15 min, remove, and cool. Add 2 mL alcohol~H 2 (1
+ 1) and 5 mL hexane, and shake 1 min. Centrf. to sep. lay-
ers. Transfer as much hexane layer as possible to second tube,
using disposable Pasteur pipet, and repeat extn with 5 mL hex-
ane. Cone, combined hexane to appropriate vol. for GC anal-
ysis.
Detection Methods
R. Gas Chromatography— Tentative Identification and
Quantitative Measurement
(Applicable to organochlorine pesticides, organophosphorus
pesticides, and polychlorinated biphenyls (PCB). Method is
applicable to PCB residues when present alone in sample. If
pesticidal or other compds are detected in chromatogram of
the PCB residue, other chemical or physical operations must
be applied to eliminate or minimize their interference before
PCB quantitation.)
Inject suitable aliquot (3-8 jjlL) of coned eluate from Florisil
or MgO-Celite column contg amt of compd within linear range
into gas chromatograph, 970. 52H, using 10 jjlL syringe. Ten-
tatively identify residue peaks on basis of retention times.
Measure area or ht of residue peak(s) and det. residue amt by
comparison to peak area or ht obtained from known amt of
appropriate ref. material(s). To ensure valid measurement of
residue amt, size of peaks from residue and ref. std should be
within ±25%. Chromatograph ref. material(s) immediately af-
ter sample.
Measure PCB residues by comparing total area or ht of res-
idue peaks to total area or ht of peaks from appropriate Aro-
clor(s) (Analabs, Inc.) ref. materials. Measure total area or ht
response from common baseline under all peaks. Use only those
peaks from sample that can be attributed to chlorobiphenyls.
These peaks must also be present in chromatogram of ref. ma-
terial. Mixt. of Aroclors may be required to provide best match
of GC patterns of sample and ref.
Alternatively, det. PCB residues by individual peak area
comparisons using Aroclor ref. material wt factors in Table
970. 52B. Calc. each PCB peak against appropriate individual
ref. peak with exactly same absolute retention. Sum individual
peak values to obtain total ppm PCB. (This method is rec-
ommended for PCB residues with chromatgc patterns which
are altered extensively from that of any Aroclor ref.)
(a) Recommended operating conditions for 10% DC-200 or
OV-J01 column. — Glass column, 1.8 m (6') x 4 mm id. Temps
(°): injector, 225; column 200; 3 H electron capture detector,
210 max.; carrier gas flow, 120 mL N/min.
(b) Electron capture detection (ECD). — (Use for detn of
Table 970.52B Weight % Factors for Individual Gas
Chromatographic Peaks in Aroclor Reference
Materials
(Peaks are identified by their retention time
relative to p,p'-DDE=100 at conditions
consistent with 970.52R(a) and (b))
AROCLOR
1016
1242
1248
1254
1260
Rdde(100x)
(77-029) a
(71-696) 3
{71-697) a
(71-698) a
(71-699) a
11
0.2
16
3.8
3.4
0.3
21
8.1
10.3
1.1
24
1.2
1.1
0.2
28
16.8
15.8
6.0
32
7.6
7.3
2.6
37
18.5
17.0
8.7
40
14.6
13.0
7.4
47
11.6
9.9
15.7
7.1
54
7.7
7.1
9.3
2.7
58
6.4
4.4
8.3
1.2
70
3.4
8.7
18.2
14.7
2.4
78
1.9
6.4
84
4.6
18.6
3.6
98
3.4
8.3 1
104
3.3
14.1 j
2.8
112
1.0
117
4.4
125
2.3
15.6
11.0
146
1.2
9.0
13.3
160
5.5
174
7.4
10.0
203
1.3
10.9
232-244
11.2
280
12.5
332
4.2
360-372
5.4
448
0.8
528
2.0
a Food and Drug Administration Lot Nos. (Wt factors are valid only for these
FDA Lot Nos.) Aroclor ref. materials are available from Food and Drug
Administration, Division of Contaminants Chemistry, HFF-420, 200 C St SW,
Washington, DC 20204.
organochlorine pesticides in fruits, vegetables, and food contg
fats and for detn of PCB in foods and paperboard.) Select for
3 H electron capture detector operating voltage that voltage (ca
50 v dc) at which 1 ng heptachlor epoxide produces 40-50%
full scale recorder deflection at 1 or 3 x 10~ 9 amp full scale
sensitivity.
Operate 63 Ni electron capture detector to produce stable, re-
producible, linear response, and adjust amt of injected sample
to accommodate differences in instrument sensitivity.
(c) Potassium chloride thermionic and electron capture dual
detection. — (Use one of the 3 dual detection systems specified
in 970.52H(h), (i), (j), for detn of organophosphorus and or-
ganochlorine pesticides and PCB. In-series system, (h), is pre-
ferred because of simplicity and ease of operation.) (/) In-
series dual detection. — Operate ECD as in (b). For KC1TD,
adjust H flow producing 0.2-0.8 X 10~ 8 amp baseline current
and select electrometer setting at which 2 ng parathion pro-
duces 40- 50% full scale recorder deflection. (2) In-series split
dual detection. — Same as (3), Parallel, except ECD receives
entire injection and KC1TD receives V2 amt injected into col-
umn. (3) Parallel dual detection. — Same as (7), In-series dual,
except column effluent is split; therefore, inject twice as much
sample to obtain desired limit of quantitation.
Thin Layer Chromatography — Confirmation of Identity
Method I
(Applicable to organochlorine and organophosphorus pesti-
cides except where indicated)
AOAC Official Methods of Analysis (1990)
MULTIRESIDUES
281
S. Preparation of Adsorbent Layer
Before coating, wash plates in hot soapy water and thoroly
rinse with distilled H 2 0. Press plates snugly into position on
mounting board that has retaining ledge on one side and one
end. Plastic board is mounted so that long side with raised
ledge faces operator while short side with ledge is to right of
operator. Before coating, wipe plates with few mL alcohol.
Position applicator, trough open, with left edge 6 mm in from
edge of first plate to be coated.
To coat 5 plates, weigh 30 g A1 2 3 G, 970.52C(a), into 250
mL J erlenmeyer. Add 50 mL H 2 0, stopper, and shake mod-
erately 45 sec. Violent shaking produces bubbles, resulting in
"pock-marked" layer.
Suspensions that contain adsorbents with binders set rap-
idly, and entire operation from prepn of slurry to final coating
must be completed within 2 min.
After shaking, immediately pour slurry into applicator
chamber. Rotate chamber by turning large lever handle thru
1 80°. After few sec, slurry begins to flow out of exit slit. Grasp
applicator with both hands and pull it manually with steady
motion across series of plates. Approx. 5 sec is required for
actual coating operation. Immediately after application, tap edge
of mounting board or shake entire board gently to smooth out
slight ripples or imperfections in wet coating.
Let coated plates dry in position on mounting board 15 min.
Then dry plates in forced-draft oven 30 min at 80° . Remove
plates and cool.
Examine plates carefully in transmitted and reflected light
for imperfections or irregularities in coating. Discard any plates
showing extensive rippling or mottling of layer.
Prep. 5 more plates while first set is drying. Be sure appli-
cator is thoroly cleaned and dried before reusing. The 10 coated
and dried plates may be pre washed immediately.
T. Prewashing of Adsorbent Layer
Scrape 1 cm of adsorbent off edge of plate with razor blade.
Pour 15 mL 50% aq. acetone into metal trough inside chro-
matgc tank. Cut out 2 x 20 cm strip of Whatman No. 1 filter
paper, wet with solv., and place over scraped off portion with
6 mm overlapping adsorbent layer. Place plate in chromatgc
tank, seal tank with masking tape, and develop with 50% aq.
acetone to within 4 cm from top of plate (75-90 min). Remove
plate from tank, remove filter paper wick, invert plate, and
dry in hood 5 min. Dry plate 45 min at 80°. Remove plate
from oven, cool, and store in desiccator. Use prepd plates within
1 week after prepn.
U. Sample Spotting
Make pencil mark 4 cm from bottom of plate at both sides.
Imaginary line between the two points indicates sample spot-
ting or origin "line." Draw line (which removes coating) com-
pletely across plate 14 cm from bottom edge; this line repre-
sents solv. front after development. On lower edge of adsorbent
starting 2 cm in from left edge of plate, make 18 marks with
pencil at 1 cm intervals. (Fewer marks with longer intervals
may be used, if desired. Marks serve as horizontal guides to
sample application. Identity of samples and stds may be etched
directly into adsorbent layer above these marks above solv.
front line.)
Imaginary spotting "line" is actually shadow line cast by
strong light source from wooden ruler supported 2 cm above
plate. Align ruler shadow on the two 4 cm marks on either
edge of plate. Shadow line and 18 marks, resp., serve as ver-
tical and horizontal guides for sample application.
For optimum semiquant. detn, spot aliquot of sample as fol-
lows:
(a) Organochlorine pesticides. — Adjust aliquot to give res-
idue spot within range 0.005-0.1 u.g. Spot stds and std mixts
at 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, and 0.2 u,g. Sample
spots >0,2 jxg are difficult to det. quant, and < 0.005 fxg may
be difficult to distinguish. Spot all 6% Florisil eluates on one
plate and 15% Florisil eluates on another plate.
(b) Organophosphorus pesticides. — Adjust aliquots of
sample and stds to give spot within range 0.1-0.5 u.g. Spot
6, 15, and 50% Florisil eluates on same plate. Ronnel and
ethion are not resolved; spot each std sep. Spot diazinon, Me
parathion, and malathion sep. or as mixt.
Vol. of sample ext spotted should be <10 jxL, if possible,
and spotting should be done repeatedly with 1, 2, or 3 julL
Kontes spotting pipet. Spot std and sample solns with same
pipet. For best results, keep size of spotted samples as small
as possible.
V. Development
(a) Organochlorine pesticides. — Place liners and metal
trough in tank, 970.52F(f). Presat. liner by pouring 75 mL
developing solv., 970.52C(b), into bottom of tank >30 min
before developing plate. Presatn decreases development time
and improves uniformity of R f values.
For plates spotted with 6% Florisil eluates, pour 50 mL n-
heptane into trough. Place lower edge of plate in metal trough
with top of plate leaning against side of tank. Place glass cover
plate on tank and seal with masking tape.
For plates spotted with 15% Florisil eluates, use acetone-n-
heptane (2 + 98) as developing solv.
(b) Organophosphorus pesticides. — Prep, chromatgc tank,
970.52F(f ), after samples and stds have been spotted on plate.
Place liners and metal trough in tank. Pour 50 mL methyl-
cyclohexane, 970. 52C (d)(2), into trough, and 75 mL into bot-
tom of tank. Quickly fill dipping tank, 970.52F(g), to within
4-5 cm from top with immobile solv., 970. 52C(d)(7). Invert
plate and dip with uncoated side touching back wall of tank
to prevent front wall from scraping the adsorbent layer during
dipping operation. Dip plate just to spotting line, remove, and
immediately place in metal trough, with top portion of plate
leaning against side of tank. Place glass cover plate on tank
and seal with masking tape.
When solv. front in (a) or (b) just reaches pencil line 10 cm
above spotting "line," remove plate and dry in hood 5 min.
W. Spraying
(Caution: See safety notes on spraying chromatograms.)
(a) Organochlorine pesticides. — Support plate on one side
and spray fairly heavily with chromogenic agent, 970.52C(c),
using lateral motions of spray bottle perpendicular to direction
of solv. flow. Spray until plate appears translucent or soaked
with reagent. Underspraying will result in poor sensitivity. Af-
ter spraying, dry plate in hood 15 min; then immediately place
under UV light source and proceed as in 970. 52X.
(b) Organophosphorus pesticides. — Immediately spray plate
moderately heavily and uniformly with dye soln, 970.52C(e)(2),
using lateral motions of spray flask, 970.52H(j), perpendic-
ular to direction of solv. flow. Plate should be vivid blue after
spraying. Using spray bottle, 970.52H(i), overspray plate lightly
and uniformly with AgN0 3 soln, 970.52C(e)(J) (at this point
plate should be bluish purple and spots should be discernible).
After 2 min, overspray plate moderately and uniformly with
citric acid soln, 970.52C(e)(4), using spray bottle, 970.52H(i).
After spraying, thiophosphate pesticides should immediately
appear as vivid blue or purple spots against yellow back-
ground. Color of spots reaches max. intensity ca 5-10 min
after citric acid spraying. After ca 10 min, background begins
to change from yellow to greenish blue, masking spots. At this
point, respraying plate with citric acid soln changes back-
ground back to yellow and makes spots stand out as well as
282
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
or better than originally. Evaluate chromatogram <10 rnin af-
ter respraying. Blue spots fade completely and irreversibly af-
ter 30-40 min from time of original citric acid spraying.
X. Exposure
(Caution: See safety notes on hazardous radiations.)
Expose plate to UV light until spot for std of lowest concn
appears; 5 ng of most organochlorine pesticides should be vis-
ible after 15-20 min exposure with equipment described under
970.52H(1). Exposure times >30 min will not harm plates.
For best results, place plates 8 cm from bottom edge of lamps.
Method II
(Applicable only to organochlorine pesticides)
Y. Preparation of Adsorbent Layer
Weigh 40 g AU0 3 G, 970.52C(a), into 500 mL centrf. bot-
tle. Add 80 mL 0.2% HN0 3 , shake well, and centrf. at ca
1200 rpm 1-2 min. Decant supernate into 100 mL graduate,
and record vol. (35-40 mL should be recovered). Add 80 mL
H 2 0, breaking up material on bottom of centrf. bottle with
glass rod, if necessary. Shake well and centrf. as before. De-
cant and record vol. supernate recovered (60-70 mL). Add 2
addn.1 80 mL portions H 2 0, shake well, centrf., and decant.
Weigh the A1 2 3 and H 2 that has been retained. (Wt should
be ca 100 g.) Add .10 mL 1% AgND 3 soln and enough H 2
to make total wt 120-130 g. Shake well, place in applicator,
and prep, plates as in 970. 52S. Let plates air dry in position
on mounting board 15 min. Place in metal drying rack, in ver-
tical position, 30 min at 100°.
Z. Sample Spotting
Spot as in 970. 52U. Draw line across plate 4 cm from top
(which removes coating). Next, scrape 6 mm of coating from
each side of plate. (Irregularities in thickness of coating on
these outer edges cause uneven flow of mobile solv.) Make
pencil mark at each side of layer 2.5 cm from bottom of plate;
imaginary line between these 2 points indicates sample spot-
ting line. Spot samples and stds at .1 cm intervals.
AA. Development and Exposure of Plates
(Caution: See safety notes on hazardous radiations.)
Develop plates as in 970.52V, except use only 25-30 mL
mobile solv. in trough, since spotting line has been lowered
to 2.5 cm. Use w-heptane to develop 6% Florisil eluates, and
acetone-yi-heptane (2 + 98) for 15% Florisil eluates.
Plates may be exposed to UV light after short drying period
(ca 5 min) after removal from tank. Spots of aldrin, DDE, and
isomers of DDT will appear within 5-10 min after exposure;
lindane, endrin, dieldrin, and all others will require more time.
Plates may be exposed 1 .5-2 hr without appreciable darkening
of background.
BB. Paper Chromatography*
See 29.028, 13th ed.
Refs.: JAOAC 42, 734(1959); 44, 171(1961); 46, 186(1963);
48, 668(1965); 49, 460, 463, 468(1966); 50, 430, 623,
1205(1967); 51, 311, 666, 892(1968); 52, 1280(1969);
53, 152, 355, 1300(1970); 54, 325, 525(1971); 55,
284(1972); 56, 721, 1015(1973); 59, 169(1976); 61,
282(1978); 63, 277(1980).
985.22 Organochlorine
and Organophosphorus Pesticide Residues
Gas Chromatographic Method
First Action 1985
Final Action 1986
(Applicable to residues of acephate, ct-BHC, chlorpyrifos,
dieldrin, monocrotophos, and omethoate in lettuce, strawber-
ries, and tomatoes)
A. Principle
Nonfatty sample is blended with acetone and filtered; pes-
ticides are transferred from aq. filtrate to org. phase by shaking
with pet ether and CH 2 C1 2 ; after drying, org. phase is coned
in presence of pet ether and then acetone to remove CH 2 C1 2 ;
aliquot of coned org. phase is injected into various GC systems
for detn of wide variety of pesticide residues.
Absence of cleanup steps permits examination for residues
of many chem. types, including many that would not be re-
covered thru methods requiring Florisil or charcoal column
chromatgc step.
B. Reagents and Apparatus
(a) Solvents. — Acetone, CH 2 C1 2 , pet. ether, distd in glass
(Burdick & Jackson Laboratories), or equiv.
(b) Sodium sulfate. — Anhyd., granular.
(c) Glass wool. — Rinse with acetone and alcohol several
times and dry. Washed glass wool will be somewhat brittle.
(d) High-speed blender. — Waring Blendor, or equiv.
(e) Kuderna-Danish concentrator. — 500 mL with Snyder
column and fitted with vol. flask or graduated receiving tube.
Calibrate receiving tube with acetone delivered from a buret.
Use buret-corrected vol. for sample wt calcn.
(f) Separatory funnels . — 1 L, with Teflon stopcocks.
(g) Gas chromato graph. — (/) For organochlorine resi-
dues. — Instrument contg any suitable methyl silicone column,
such as 2% OV-101, on 80-100 mesh Chromosorb W (HP),
6 ft X 2 mm id glass, and Hall 700 A HECD halogen -specific
detector. Column, 200°; He carrier cas, 60 mL/min; detector
900°, H reaction gas 60-100 mL/min; n-propanol solv. 0.35
mL/min; electrometer range 10 in OPR/FLT mode; attenua-
tion 5. (2) For organophosphorus residues. — Instrument with
column contg 2% stabilized DECS on 80-100 mesh Chro-
mosorb W (HP), 4 ft X 2 mm id silanized glass, and P-specific
flame photometric detector (526 nm filter). Column 180°; de-
tector 200°; He carrier gas, 60 mL/min. Condition column
(disconnect detector) by passing carrier gas thru column 0.5 h
at <80°. Program temp, at l-2°/min to 230° and hold over-
night. Establish stable flame at electrometer setting that will
produce 50% full scale deflection for 1.5 ng chlorpyrifos and
6 ng monocrotofos. If necessary, increase air/O until >:50%
response. Baseline noise should be <2%.
(h) Reference std materials. — Acephate, BHC, chlorpyri-
fos, p,p'-DDT, dieldrin, methamidophos, monocrotophos, and
omethoate (U.S. Environmental Protection Agency, Pesticides
and Industrial Chemical Respository (MD-8), Research Tri-
angle Park, NC 27709). Prep, all stds in acetone. Mixed stds. —
For Hall system, std soln should contain at least chlorpyrifos,
dieldrin, and p,p ( -DDT. For flame photometric detector, std
soln should contain at least methamidophos and chlorpyrifos.
Do not use mixed std solns for quant, of unknowns.
(i) Std solns. — Prep, all stock solns and dilns in glass -distd
acetone. Prep. GC std solns so 4 u,L injection causes 30-70%
full scale deflection in properly functioning system. Suggested
conens are given below. Check responses before beginning
analysis. Store all std solns in tightly stoppered containers in
refrigerator. Let equilibrate 1 h at room temp, before using.
AOAC Official Methods of Analysis (1990)
Organochlorines
283
Compd
ng/pL
acephate
0.5
a-BHC
0.1
chlorpyrifos
0.5
dieldrin
0.2
AP'-DDT
0.5
methamidophos
0.2
monocrotophos
2.0
omethoate
2.0
C. Preparation of Sample
Chop or blend fruits and vegetables and mix thoroly. Weigh
100 g chopped or blended sample into high-speed blender jar,
add 200 mL acetone, and blend 2 min at high speed. Do not
add Celite. Filter with suction thru 12 cm Buchner funnel fitted
with sharkskin paper. (Note: Rinse filter paper with acetone
before filtration of sample to remove artifacts that can interfere
with analysis.) Collect ext in 500 mL suction flask. Filtration
is normally complete in <1 min. Continuation of vac. for ex-
cessive period can reduce vol. of ext and cause error in calcn.
Place 80 mL sample ext in 1 L sep. funnel, and add 100
mL pet ether and 100 mL CH 2 C1 2 . Shake vigorously 1 min.
Transfer lower aq. layer to second 1 L sep. funnel. Dry upper
org. layer in first sep. funnel by passing thru ca IV2 in. Na 2 S0 4
supported on washed glass wool in 4 in. funnel, collecting in
500 mL Kuderna-Danish concentrator fitted with vol. flask or
calibrated receiving tube. To sep. funnel with aq. phase, add
7 g NaCl and shake vigorously 30 s until most NaCl is dis-
solved. Add 100 mL CH 2 C1 2 , shake 1 min, and dry lower org.
phase thru same Na 2 S0 4 . Ext aq. phase with addnl 100 mL
CH 2 C1 2 , and dry as above. Rinse Na 2 S0 4 with ca 50 mL CH 2 C1 2 .
Attach Snyder column on Kuderna-Danish concentrator (boil-
ing chips may be added) and start evapn slowly by placing
only receiver tube into steam. After 100-150 mL has evapd,
concentrator may be exposed to more steam. When liq. level
in hot concentrator tube is ca 2 mL, add 100 mL pet ether thru
Snyder column and reconc. to ca 2 mL. Add 50 mL pet ether
and repeat concn step. Add 20 mL acetone and reconc. to ca
2 mL. Do not let soln go to dryness during any concn step.
Adjust vol. of ext to suitable definite vol. with acetone.
Calculation of equivalent sample weight. — Calc. equiv.
sample wt in final soln as follows:
mg sample equiv. 80 1
(ulL final ext
100 x
x
200 + W - 10 mL final vol.
where 200 = mL acetone blended with 100 g sample; W =
amt (mL) FLO present in sample; and 10 = adjustment for
water-acetone vol. contraction. Thus, when sample contains
85% H 2 (85 mL/100 g) and final ext vol. is 7 mL, each |xL
contains:
80 1
100 x x -
200 + 85-10 7
— 4. 15 mg sample equiv. /|jlL final ext
D. Determination
Check that both GC systems are working properly by in-
jecting mixed std soln into each. Inject ca 12 mg sample equiv.
into each system. Tentatively identify any GC responses on
basis of retention times. Quantitate residue peak(s) by area
comparison with that obtained from known amt of ref. mate-
rials). To ensure valid measurement of residue amt, area of
peaks from residue and ref. std should be within ±25%. Cau-
tion: Repeated injection of sample exts which have had min.
cleanup can be detrimental to GC columns. Replace packing
material at front of GC columns as needed to maintain chro-
matgc quality and prolong column life.
Refs.: JAOAC 68, 64(1985); 70, 329(1987).
CAS-30560-19-1 (acephate)
CAS-319-84-6(a-BHC)
CAS-2921-88-2 (chlorpyrifos)
CAS-60-57-1 (dieldrin)
CAS-50-29-3 (DDT)
CAS- 1 1 1 3-02-6 (omethoate)
CAS- 10265-92-6 (methamidophos)
CAS-6923-22-4 (monocrotophos)
ORGANOCHLORBNE RESIDUES
983.21 Organochlorine Pesticide
and Polychlorinated Biphenyl Residues in Fish
Gas Chromatographic Method
First Action 1983
Final Action 1985
A. Principle
Chlorinated pesticides and polychlorinated biphenyls (PCBs)
are extd from prepd fish sample with pet. ether, cleaned up
on Florisil column, and detd by GC against ref. stds.
See 970. 52B, E, H-J for general app., reagents, and tech-
nics.
B. Apparatus
(a) Gas chromato graph. — With on-column injection sys-
tem, 6 ft glass column (4 mm id), packed with 10% DC-200
on 80-100 mesh Chromosorb WHP, and electron capture de-
tector. Other liq. phases such as 5% OV-101 on suitable sup-
ports may be substituted if known to give adequate resolution
for compds present in samples.
Linearized 63 Ni detector capable of producing V2 scale de-
flection for I ng heptachlor epoxide is suggested; however,
other equiv. electron capture detectors may be used. Operate
GC in accordance with manuf. directions, adjusting to provide
necessary response and resolution.
(b) Chromatographic tube. — 10 mm id x 300 mm column
with Teflon stopcock, coarse fritted disk, I" 24/40 top joint
(Kontes Glass Co. K -420550, or equiv.).
(c) Kuderna-Danish concentrators . — Snyder distg column
(Kontes K-503000-0121); 125 mL K-D flask (Kontes K-570001-
9010) (special item) 1 19/22 lower joint; 10 mL concentrator
tube (Kontes K-570050-1025).
(d) Micro Snyder column. — Kontes K-569251, ¥ 19/22.
C. Reagents
(a) FhrisiL— PR grade, 60-80 mesh (Floridin Co.). Must
meet 970.52B(i) specifications.
(b) Solvents. — Pet. ether, ethyl ether, hexane, and alcohol,
known to be suitable for pesticide residue detn.
(c) Glass wool (Pyrex). — Must be free of interference with
electron capture detection.
(d) Sodium sulfate . — Anhyd., granular, reagent grade, free
of interference with electron capture detection.
D. Extraction
Weigh 20 g thoroly ground and mixed sample into metal
blender cup. Moisten 40 g granular Na 2 S0 4 with pet. ether and
add to sample. Mix sample, using stirring rod, let stand 20
min, and mix again. Add 100 mL pet. ether to sample and
blend 1-2 min. (Lourdes blender in series with rheostat set at
40-60%, or equiv., may be used.) Centrf. balanced sample
cup 1-2 min at ca 2000 rpm to obtain clear pet. ether ext.
Place glass wool plug in funnel, overlay with 20 g granular
Na 2 S0 4 , and place funnel in 250 mL vol. flask. Decant pet.
284
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
ether ext thru Na 2 S0 4 into vol. flask. Mix sample again with
stirring rod, add 100 mL pet. ether, and ext as before. Repeat
using 70 mL pet. ether. Oil. to vol. with pet. ether.
Transfer 25 mL aliquot to tared 100 mL flat bottom extn
flask. Place flask on steam bath to evap. solv., leave addnl 30
min on steam bath, remove, and cool. Weigh flask and det.
% fat in fish.
For fish contg <10% fat, transfer 25 mL aliquot to 125 mL
K-D concentrator. For fish contg >10% fat, take aliquot contg
not >200 mg fat. Add several granules of 20-30 mesh car-
borundum and cone, to ca 3 mL on steam bath. Let cool and
remove Snyder column. Rinse concentrator with two 1 mL
portions of pet. ether and, using only current of air, cone, sam-
ple to 3 mL for transfer to Florisil column.
E. Florisil Cleanup
Use 4 g Florisil adjusted for lauric acid value (J AOAC 51,
29(1968)). Add Florisil to 300 x 10 mm id chromatgc tube
and add Na 2 S0 4 to ht 2 cm above Florisil. Completely open
stopcock, tap tube to settle adsorbent, and mark tube 1 cm
above Na 2 S04 layer.
Add 20-25 mL pet. ether wash to Florisil column; as solv.
level reaches mark, place 125 mL K-D flask under column.
Using disposable Pasteur pipet, transfer 3 mL sample to col-
umn, and wash tube with 1 mL pet. ether and add wash to
column. Solv. level must not go below mark. Temporarily close
stopcock if necessary. Add 35 mL pet. ether-ether mixt. (94
+ 6) and elute PCBs and DDT and its analogs. When solv.
level reaches mark, change K-D flask, and add 35 mL pet.
ether-ether (85 + 15) to elute compds such as dieldrin and
endrin. Add several granules of carborundum to first concen-
trator, attach Snyder column, and carefully cone, on steam
bath. Let concentrator cool, remove Snyder column and evap.
solv. under air to appropriate vol. for GC detn. Fractions contg
mixt. of PCBs and chlorinated compounds such as DDE may
require addnl sepn technics.
F. Additional Cleanup
Often addnl cleanup is required for second fraction (85 +
15) to prevent deterioration of GC column. Use 970. 52Q.
G. Gas Chromatography
See 970.52R.
Ref.: J AOAC 66,
1983).
984.21 Organochlorine Pesticide
Residues in Animal Fats
Gel Permeation Chromatographic Method
First Action 1984
Final Action 1985
(Applicable to beef, poultry, and swine fats)
A. Principle
Liq. fats are dissolved in ChLC^-cyclohexane (1 + 1). Res-
idues are sepd from lipid by gel permeation chromatgy (GPC),
and identified and measured in coned eluates by GC-EC de-
tection .
0. Reagents and Apparatus
(a) Solvents. — CH 2 C1 2 , cyclohexane (C 6 H ]2 ), isooctane. Must
meet criteria in 970. 52B.
(b) Gel permeation chromatographic system (GPC). —
AutoPrep gel permeation chromatograph (Model 1002B, An-
alytical Bio-Chemistry Laboratories, Inc., PO Box 1097, Co-
lumbia, MO 65205) or equiv. with 60 g BioBeads SX-3 resin,
200-400 mesh, in 60 X 2.5 cm id chromatgc tube, ca 48 cm
bed length, elution solv. CH 2 C1 2 -C 6 H 12 (1 + 1). Flow rate cal-
ibrated to 5.0 mL/min, operating pressure 7-10 psig.
(c) Flash evaporator. — Rotary evapn system with 30° H 2
bath.
(d) Gas chromatograph. —EC detector ( 63 Ni) operated as in
970.52H. 1.85 m x 4 mm id column packed with 1.5%
SP2250/1.95% S-2401 on 100-120 mesh Supelcoport (Su-
pelco. Inc.). Operating conditions: injector 250°, column 200°,
detector 350°, N flow 60-80 mL/min. Condition column 2
days at 250°.
C. GPC Calibration Procedure
Chromatographic system will fractionate effluent from col-
umn into 23 equal fractions for elution calibration. (It is nec-
essary to det. correct "dump" and "collect" times for desired
residues, as function of pump flow rate.) Before fractionating,
check flow rate with SX-3 gel column connected and adjust
flow to 5.0 ± 0.2 mL/min (start pump >5 min before mea-
suring flow to let flow equilibrate and improve accuracy).
Fractionate vol. of eluant from 150 to 320 mL to ensure res-
idue collection. Evap. fractions, re suspend in isooctane, and
analyze to det. collection vol. for samples (fractionation pro-
cedure is described in instrument manual). Check calibration
for quant, recovery with 2.0 g corn oil fortified with relevant
compds .
D. Preparation of Sample
Place ca 40 g representative fat sample in glass funnel (8.0
cm) with glass wool plug. Place funnel in flint glass bottle or
250 mL beaker on hot plate at < 1 10° until fat ceases to drip.
Mix thoroly.
E. Cleanup
Weigh 2.0 g liq. fat into 10 mL vol. flask. (Fortifications
of corn oil to check recoveries may be made here with stds
dild in CH 2 C1 2 -C 6 H 12 (1 + 1).) Dil. to 10 mL with CH 2 C1 2 -
C 6 H 12 (1 + 1) and mix thoroly. Centrf. or filter if particulate
matter is visible. Use ca 7 mL sample to load sample loops
on precalibrated GPC (5 mL aliquot (1.0 g equiv. of sample)
is accepted into sample loop). Process thru GPC system using
dump/collect times from fractionation procedure and collect
eluate in 250 mL boiling flask. Rotary-evap. to just dry at
<30°. Transfer quant, with 10 mL isooctane or equiv. GC-EC
compatible solvent into a precalibrated culture tube. Adjust vol.
under gentle, dry N stream to 5.0 mL.
F. Gas Chromatography
Inject 3-6 \xL aliquots into a gas chromatograph operated
as in 970.52H with 63 Ni ECD. Measure peaks (ht or area). If
necessary, dil. sample to give residue concn approx. that of
std soln. Inject aliquot of pesticide std soln (in same solv. as
sample) and again measure peaks.
Each residue, ppm — concn std (fxg/mL) x (peak size sam-
ple/peak size std) X (jxL std/|xL sample) x (diln vol/1.0 g
sample).
(Note: Since only 5 mL of original 10 mL vol. contg 2.0 g
fat is injected into GPC sample loop, only 1.0 g fat is ana-
lyzed.)
Refs.: JAOAC 67, 284(1984); 68, 267(1985).
976.23 Endosulfan, Endosuifan Sulfate,
Tetradifon, and Tetrasul Pesticide Residues
Gas Chromatographic Method
First Action 1976
Final Action 1977
(Applicable to apples and cucumbers)
AOAC Official Methods of Analysis (1990)
POLYCHLORINATED BlPHENYLS
285
A. Principle
Pesticides are extd with CH 3 CN, partitioned with pet ether,
eluted thru Florisil column with mixts of CH 2 C1 2 , hexane, and
CH 3 CN, and detd by gas chromatgy. Method is variation of
970.52A-R, as it applies to nonfatty foods. Pesticides are eluted
from Florisil column with different eluants to improve cleanup
for these compds.
B. Apparatus
See 970.52E(a)-(h) and 970.52H(a)-(c).
C. Reagents
(a) Florisil. —See 970.52B(i).
(b) Solvents. — Hexane, CH 2 C1 2 , and CH 3 CN, all distd in
glass and free from electron capturing substances (see 970.52B).
(c) Eluant mixtures. — (7) Eluant L — 20% CF^CJ^-hexane.
DiL 200 mL CH 2 C1 2 with hexane. Let mixt. reach room temp.
and adjust vol. to 1 L with hexane. (2) Eluant IL~~5Q% CH 2 C1 2 -
0.35% CH 3 CN-49.65% hexane. Pipet 3.5 mL CH 3 CN into 500
mL CH 2 C1 2 , and dil. with hexane. Let mixt. reach room temp.;
dil. to I L with hexane.
D. Preparation of Sample and Extraction
See 970.52K(a), (b), (e)-(g).
E. Column Chromatography
(Caution: See safety notes on distillation, acetonitrile, and
hexane.)
Add wt activated Florisil detd from lauric acid absorption
value, 970.52B(i), to 22 mm id chromatgc tube, 970.52E(b).
Gently tap chromatgc column to settle Florisil. Top column
with ca 12 mm anhyd. granular Na 2 S0 4 . Wet column with 40-
50 mL hexane. Use Kuderna-Danish concentrator with volu-
metric or graduated tube to collect eluate. Transfer pet ether
or hexane soln of sample ext to column, and let it elute at ca
5 mL/min. Rinse container (and Na 2 S0 4 , if present) with 2
ca 5 mL portions hexane, transfer rinsings to column, and rinse
walls of chromatgc tube with addnl small portions of hexane.
Elute tetrasul at ca 5 mL/min with 200 mL eluant I. Change
receivers and elute endosulfan I and II, endosulfan sulfate, and
tetradifon at ca 5 mL/min with 200 mL eluant II. Cone, each
eluate to suitable definite vol. in Kuderna-Danish concentra-
tor. For evapn <5 mL, use 2- ball micro Snyder or Vigreux
column.
F. Determination
See 970.52H(a)-(c).
Using the 10% DC-200 column, retention times relative to
aldrin are ca 1.6 for endosulfan I, 2.2 for endosulfan II, 2.5
for tetrasul, 2.7 for endosulfan sulfate, and 5.4 for tetradifon.
Ref.: J AOAC 59, 209(1976).
CAS- 115-29-7 (endosulfan)
CAS- 103 1-07-8 (endosulfan sulfate)
CAS-1 16-29-0 (tetradifon)
CAS-2227-13-6 (tetrasul)
974.21 Polychlorinated Biphenyls
in Paper and Paperboard
Gas Chromatographic Method
First Action 1974
Final Action 1982
A. Apparatus
(a) Gas chromato graph. — Equipped with electron capture
detector and 1 .85 m (6') x 4 mm id glass column contg either
(/) 10% DC-200 or (2) 1 + 1 mixt. of 15% QF-1 + 10% DC-
200 on 80-100 mesh Chromosorb W(HP). Operating condi-
tions: temps (°) — column and detector 200, injector 225; flow
rate, 120 mL N/min; concentric design electron capture de-
tector operated at dc voltage to cause V2 full scale recorder
deflection for 1 ng heptachlor epoxide when full scale deflec-
tion is 1 X 10" 9 amp (see 970.52H(c)).
(b) Chromatographic tubes. — See 970.52E(b).
(c) Filter tube.— See 970.52E(d).
(d) Kuderna-Danish concentrator, — See 970.52E(e), and
(g).
(e) West condenser. — 400 mm jacket length with I inner
drip joint to fit 250 and 500 mL erlenmeyers.
B. Reagents
(a) Florisil.— See 970.52B(i).
(b) Alcoholic potassium hydroxide soln. — 2% KOH in al-
cohol or redistd MeOH.
(c) Petroleum ether.— See 970.52B(m).
(d) Polychlorinated biphenyls . — Com. mixts (Aroclors) for
ref. in GC detn (Analabs, Inc.).
C. Extraction
(Caution: See safety notes on flammable solvents, and petro-
leum ether.)
Cut paper sample representative of lot into pieces ca 6 x 6
mm and mix thoroly.
Weigh 10 g sample into 250 mL erlenmeyer. Do not pack
tightly. (See note below if vol. of 10 g sample is >50 mL.)
Add 60 mL 2% ale. KOH, and fit flask with West condenser
cooled with circulating cold tap H 2 0. Reflux gently on steam
bath 30 min. Rinse inside of condenser with small amt of al-
cohol. Transfer soln thru glass wool plug in small funnel, to
250 mL separator, avoiding transfer of any paper material. Rinse
paper and flask with three 40 mL portions pet ether, combining
rinses in separator. Add 60 mL H. 2 to separator and shake
vigorously 30 sec. Drain lower aq. layer into second 250 mL
separator. Add 60 mL pet ether to second separator and shake
vigorously 30 sec. Discard aq. layer and combine pet ether
layers in first separator. Rinse second separator with several
small portions pet ether, collecting rinses in first separator.
Wash pet ether with three 40 mL portions FLO, discarding
each wash. Dry pet ether thru 50 mm column, (c), of anhyd.
Na 2 S0 4 , collecting eluate in Kuderna-Danish concentrator. Rinse
separator and then column with 3 ca 20 mL portions pet ether,
collecting rinses. Cone, combined pet ether ext and rinses on
steam bath to ca 5 mL. Ext is ready for cleanup on Florisil
column, 974.21D. If experience with particular sample types
indicates that Florisil column cleanup is not required, proceed
to GC detn, 970.52R.
Note: Adequate extn of low density paper such as newspaper
or tissue paper will require adjustment of either amt of sample
to <10 g or vol. of reflux soln to >60 mL. Preferably, reduce
sample to wt that is completely covered and wetted by 60 mL
KOH soln. Increase in vol. of reflux soln >60 mL must be
accompanied by proportional increases in vols of pet ether rin-
ses of sample, H 2 diluent added to ale. reagent in separator,
and size of erlenmeyers and separators.
Refs.: JAOAC 56, 957(1973); 57, 518(1974).
D. Florisil Cleanup
Proceed as in 970.520, pars 1 and 2, except prep. 10 g
column, pre-wet column with 20 mL pet ether, and elute at ca
5 mL/min with 150 mL pet ether. Coned eluate is suitable for
analysis by GC with electron capture detection, 970. 52R.
Note: Waxes, if present in ext, can be removed before Flor-
286 Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
isil chromatgy by partitioning between pet ether and CH 3 CN,
970.52N
ORGANOPHOSPHORUS RESIDUES
974.22 Organophosphorus
Pesticide Residues
Carbon Column Cleanup Method
First Action 1974
Final Action 1976
(CH 3 CN extn and charcoal cleanup column using KC1 therm-
ionic or flame photometric detector for residues of parathion,
paraoxon, carbophenothion and its O analog, and EPN on ap-
ples and green beans)
A. Reagents
(a) Solvents. — Redistd from glass (see 970.52A): EtOAc,
CH 3 Cl 2 , benzene, hexane, CH 3 CN, and isopropanol.
(b) Acid-treated charcoal. — Slurry 200 g Norit SG Extra
(no longer marketed) or 100 g Nuchar C-190N (no longer mar-
keted) with 500 mL HC1, cover with watch glass, and stir mag.
while boiling l hr. Add 500 mL H 2 0, stir, and boil addnl 30
min. Collect charcoal in buchner and wash with H 2 until
washings are neut. to universal indicator paper. Dry at 130°
in forced-draft oven.
(c) Magnesium oxide. — See 970.52B(k).
(d) Adsorbent mixture. — Mix I part acid-treated charcoal,
2 parts hydrated MgO, and 4 parts Celite 545, acid washed.
Keep sealed.
(e) Pesticide std solns. — Prep, solns contg 1 jxg/mL EtOAc
of each of following: parathion, paraoxon, carbophenothion,
carbophenothion O analog, and EPN.
(f) Eluting soln.— CH 3 CN-benzene (1 + 1).
Purity test. — Reagents must be free of substances causing
KCJ thermionic or flame photometric detector response, as in-
dicated by following test: Carry reagents thru entire method,
and inject 5 jjlL from final cone, into gas chromatograph , using
conditions described in 968. 24F. Cone, must not cause re-
corder deflection >1 mm from baseline for 2-60 min after
injection.
B. Apparatus
See also 970.52E and 968.24B(a).
(a) Vacuum adapter. — Kontes Glass Co., No. K-954002,
or equiv.
(b) Gas chromatograph. — With potassium chloride therm-
ionic detector (See 968.24B(i) and (k).) or flame photometric
detector (See 974.226(e) and (i).).
(c) Column.— See 968.24B(j).
(d) Potassium chloride thermionic detector (KC1TD). — See
970.52H(d)C0 or (2), (e), (f), and (k). Also check linearity
of GC system to paraoxon and carbophenothion O analog.
(e) Flame photometric detector (FPD). — With P selective
optical filter for 526 nm wavelength (Tracor Instruments, Inc.).
Equiv. to KC.1TD for detn of organophosphorus pesticides in
fruits and vegetables. (Note: Older commercial models of FPD
may give rise to adsorption and /or degradation of O analogs
of organophosphorus pesticides within detector's gas mixing
chamber. Design changes of detectors manufactured after mid-
1973 have generally corrected this problem. Flameout in FPD,
on injection of sample, can be avoided by letting H enter de-
tector (lower part) so that H and GC column effluent mix be-
fore burner area. Air-O enters detector thru upper part. This
arrangement reverses that recommended by manufacturer.
Specifications for physical modification of FPD to correct above
problems are available from Division of Chemistry and Phys-
ics, Food and Drug Administration, Washington, DC 20204.)
Use highly stabilized 0-750 v dc variable power supply ca-
pable of 10 ma output (Keithley Instruments, Inc., 28775 Au-
rora Rd, Cleveland, OH 44139, or equiv.), 6.3 v ac ignitor
power supply, electrometer with bucking capability of I X 10~ 6
amp (Tracor Instruments, or equiv.), and variable transformer
capable of delivering 150 watts to control temp, of flame hous-
ing. Strip chart recorder should be compatible with electrom-
eter.
(f) Hydrogen. — From cylinder of compressed H gas. Equip
cylinder with regulator, delivery line, and variable flow con-
troller capable of 200 mL/min delivery. Metering shut-off valve
is required sep. from controller.
(g) Air. — Cylinder of compressed air equipped as in (f) to
deliver up to 100 mL/min. Sep. shut-off valve is not needed.
(h) Oxygen. — Cylinder of compressed O gas equipped as
in (f) to deliver up to 50 mL/min. Combine with air using std
Swagelok tee before detector inlet.
(i) Flame photometric detector operation. — Adjust temp,
of burner housing to ca 170-180° before igniting flame. Temp,
will rise 20-30° after ignition. Do not allow detector to exceed
220°. Adjust gas flows at controllers to ca 150-300 mL/min
H, 50-100 mL/min air, and 5-20 mL/min O. Adjust column
effluent flow, 970.52R(a), to 1 20 mL N/min. Turn off H flow
with metering shut-off valve (f). (Caution: Before attempting
ignition, make certain H has been purged from detector with
other gases. One min interval between ignition attempts is ad-
equate.) Apply ca 750 v to photomultiplier tube from power
supply. Zero recorder with electrometer set at appropriate sen-
sitivity (ca 1 x 10~* to 1 x 10~ 9 amp full scale). Push ignitor
button and then slowly open H metering shut-off valve. Re-
corder pen will not return to zero baseline if flame ignites. If
ignition is not effected, shut off H valve, increase O flow, and
repeat ignition procedure. Establish proper baseline with buck-
out control after flame is lit. Operate at sensitivity that pro-
duces j /2 full scale recorder deflection for 2 ng parathion. Re-
duce photomultiplier voltage to reduce sensitivity. Alternatively,
use electrometer sensitivity and attenuator controls to achieve
proper response. Check linearity of GC system to paraoxon
and carbophenothion O analog.
C. Preparation of Sample
Blend and filter sample as in 970.52K(a), or (b). Transfer
aliquot of CH 3 CN ext (30-35 mL) equiv. to ca 10 g sample
from suction flask to 125 mL separator, add equal vol. CH 2 C1 2 ,
shake vigorously 30 sec, and set aside 10-15 min to sep. Calc.
g sample in aliquot as g sample x [mL aliquot/(mL H 2 in
sample + mL extg sol v. added — correction in mL for vol.
contraction)].
D. Charcoal Cleanup
Fit 1-hole No. 5 rubber stopper onto tip of chromatgc tube,
970.52E(b), add side-arm vac. adapter and ¥ 24/40 receiving
flask, open stopcock, and connect app. to open vac. line. Place
I g Celite 545 in tube, tamp, add 6 g adsorbent mixt., and
tamp again. Add 2 cm glass wool plug on top of adsorbent.
Prewash column with 100 mL eluting soln. Close stopcock
when eluting soln is ca 2 cm above glass wool and maintain
this head to ensure clean column. Disconnect vac, replace
flask with 500 mL Kuderna-Danish flask equipped with 10 mL
tube, 970.52E(e) (check calibration at 1 mL), and reconnect
to open vac. line.
Drain lower CH 2 C1 2 layer in separator onto column, retain-
ing H 2 layer (upper phase) in separator. Open column stop-
cock to vac. and adjust flow to ca 5 mL/min. Re-ext H 2
layer cautiously (do not shake vigorously) with two 10 mL
AOAC Official Methods of Analysis (1990)
Organophosphates
287
portions CH 2 CJ 2 and add exts to column. Discard H 2 phase.
Elute column with 120 m'L eluting soln (column may be taken
to dryness). Disconnect app. and rinse column tip and vac.
adapter with several mL EtOAc. Collect all rinses in same Ku-
derna-Danish concentrator with tube attached. Add 1 or 2 small
boiling chips, attach Snyder column, and cone, cautiously over
steam bath to ca 1 mL. (Caution: Begin heating very gently
due to differences in densities and bps of individual solvs.)
When cool, disconnect evaporative app. from Mills tube. Sub-
stitute column, 970.52E(h), on Mills tube, add boiling chips
again, and cone, sol v. to <1 mL. While app. is still immersed
in steam bath, add 3-4 mL isopropanol (to remove CH 3 CN
azeotropically) and distil under gentle reflux. Repeat isopro-
panol addn and cone, to ca 0.5 mL. Remove from heat, cool,
remove column, and adjust vol. to 1.0 mL with EtOAc.
B. Gas Chromatography
Proceed as in 968. 24F, using recommended operating con-
ditions / specified for column, 968.24B(j)(7). (See 974.22B(e)-
(i) if using flame photometric detector.
Refs.: JAOAC 54, 513(1971); 57, 930(1974).
CAS-786-19-6 (carbophenothion)
CAS-2 104-64-5 (EPN)
CAS-56-38-2 (parathion)
CAS-7 173-84-4 (carbophenothion O analog)
CAS-31 1-45-5 (paraoxon)
968.24 Organophosphorus
Pesticide Residues
Sweep Codistillafion Method
First Action 1968
Final Action 1977
(Sweep codisti Nation cleanup for parent organophosphorus
residues of carbophenothion, diazinon, ethion, malathion, Me
parathion, and parathion in kale, endive, carrots, lettuce, ap-
ples, potatoes, and strawberries (fresh or non-sugared frozen);
this cleanup is not adequate for electron capture gas chromatgc
detector. Use only with KG thermionic or flame photometric
detector.)
A. Reagents
(a) Ethyl acetate. — Redistd from glass. Check suitability of
reagent by concg 100 mL to 2 mL. Inject 5 jjlL into GC (KCl
thermionic detector) with operating conditions specified in
968.24B(i). Chromatogram should show no peaks to 20 min
with chart speed of l"/2 min.
(b) Pesticide std soln. — Prep. EtOAc soln contg 1 p,g/mL
of each of following: carbophenothion, diazinon, ethion, mal-
athion, Me parathion, and parathion.
B. Materials and Apparatus
(a) Glass wool. — Si lane-treated (available from Applied
Science).
(b) Anakrom ABS. — 80-90 mesh. Remove fines by stirring
with EtOAc, decanting several times, and drying.
(c) Teflon tubing. — AWG No. 16, std, natural.
(d) Disposable glass capillary pipets. — 145 mm long, 6 mm
id, with capillary stem (Thomas Scientific No. 7760-B30, or
equiv.).
(e) Syringes. — I mL Tuberculin Luer-Lok and 2 mL Luer-
Lok with Luer-Lok 2" No. 25G needles.
(f) High-speed blender. — 400 mL capacity. Omni mixer
(available from DuPont Instruments Co, Sorvall Operations,
Peck's Ln, Newtown, CT 06470), or equiv.
(g) Sweep codist illation apparatus. — See Fig. 968.24. Fol-
lowing tubes are required: (/) S tor her r tube. —24.5 cm long,
6 mm id (Kontes Glass Co., No. K-898600, or equiv.). (2)
Concentrating tube. — \Q mL calibrated to 0.5 mL. (3) Adapter
for extension of concentration tube. — 7 cm long, ¥ 19/22 (Kontes
No. K-570100 (K-500750 part 355), or equiv.).
(h) Kuderna-Danish concentrators . — 500 mL with Snyder
distilling column and 5 mL vol. and 10 mL graduated (Mills
tube) receiving flasks, J 19/22 (Kontes Glass Co., No. K-
570000, K-621400, and K-570050, or equiv.),
(i) Gas chromato graph with potassium chloride thermionic
detector. — See 970.52H(a). Only thermionic detector,
970.52H(d), is required.
Following conditions are important in operation of GC and
KCl thermionic detector:
(/) Every day before starting work change silane-treated glass
wool plug insert in injection port of GC column. Remove and
replace only that portion affected by syringe.
(2) Every week before starting work reheat KCl spiral over
gas burner and reinsert into detector. Detector must then equil-
ibrate ca 2 hr before use.
(j) Column. — 10% DC 200 or OV-101 on 80-100 mesh
Chromosorb W HP in glass column 1 .85 m (6') x 4 mm id;
see 970.52H(b). Adjust column temp to give retention time
for parathion of ca 5 min.
(/) Recommended operating conditions: I. — Temps (°): in-
jection 225, column 200, detector 210; N flow 120 mL/min.
Split column effluent with 1:1 stream splitter so that only 60
mL N/min enters KCITD. (2) Recommended operating con-
ditions 11. — Temps (°): column 220, injector and detector 240;
N flow 60 mL/min.
(k) Potassium chloride thermionic detector (KCITD). — See
970.52H(d)(7) or (2), (e), (f), and (k).
C. Preparation of Apparatus
App. is constructed in 3 parts: removable Storherr tube, per-
manent heating coil, and distillate collector (see Fig. 968.24),
(a) Preparation of removable Storherr tube. — Pack Stor-
herr tube with silane-treated glass wool. Use silane-treated glass
wool as received. Do not pack glass wool too tightly; other-
wise removal for cleaning is difficult. Only 13-15 cm portion
from injection end requires packing. Insert injection septum
and two 1-hole septums. Tube is now ready for use. Use clean
tube for each sample. Clean tubes thoroly with soap and H 2
after use, rinse with acetone, and dry. Soak tubes difficult to
clean in chromic acid cleaning soln before cleaning with soap
and H 2 0.
(b) Preparation of permanent heating coil. — Attach bi-
metallic wires of calibrated pyrometer directly to outside mid-
dle area of Cu tube (length 20 cm (8") x 11 mm Cfw) id).
(Thermometer with stem covered with A I foil may also be used
for temp, measurement.) Wrap heating tape (60 X 1.3 cm)
uniformly around outside of Cu tubing and over bimetal lie wires
or thermometer, and secure ends. Cover heating tape with as-
bestos tape and secure with glass tape or glass thread. Cover
asbestos with several layers of Al foil and secure with tape.
Place heating assembly on ring stand, using asbestos-cov-
ered 3-prong clamp. Orient and use heating coil in near hor-
izontal position. Attach heating tape leads to variable trans-
former. Adjust transformer so pyrometer reads 180-185°. Use
this setting or temp, for all crop cleanup.
Add N flow, 600 mL/min, to sidearm of Storherr tube. (For
N pressure gage to give meaningful readings, add stainless steel
capillary tube to reduce gas flow. Connect capillary tube di-
rectly to 1-hole septum in sidearm of tube with short length
Teflon tubing.) Measure N flow with gas flow gage, and cal-
ibrate pressure regulator gage by this means.
288 Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
COPPER r
Tu8E ^C
^INSULATION
^<^ASBESTOS
g|j=jj=^lE ATING TAPE
=r> wmmmmMamawm
\^
\To PYROMETER
WATER
AlCE
BATH
-GLASS WOOL
(Silamzmd)
-4 cm ANAKROM
-ADAPTER
-GLASS WOOL
(Stlaniz«d)
J 19/22
STORHERR TUBE
CONTAINING 5-6"
SlLANlZED GLASS WOOL
n w
lim N ? INLET
variable
Transformer
CONCENTRATION
TUBE
BEAKER
Of
H 2
FIG. 968.24 — Sweep codistillation apparatus
(c) Sample distillate collector. — Construct in 3 parts: cool-
ing coil, scrubber tube, and concn tube with extended adapter.
(/) Cooling coil. — Cut 120 cm length of Teflon tubing. Form
this tubing into three 7 cm diam. loops having 2 arms of ca
20 cm lengths. Attach Teflon cooling coil and 1-hole septum
directly to Storherr tube. Place coils in 250 mL beaker contg
ice and H 2 0. Place 250 mL beaker inside 400 mL beaker for
insulation.
(2) Scrubber tube. — Insert silane-treated glass wool plug in
constricted end of disposable pipet. On outside of pipet place
marks 4 and 6 cm above top of glass wool plug. Add Anakrom
ABS to 6 cm mark and pack Anakrom to 4 cm mark by com-
pressing with 3 mm rod. Place silane-treated glass wool plug
on top of packed Anakrom and 1-hole septum in pipet top.
Connect exit arm of Teflon cooling coil directly into 1-hole
septum in scrubber tube and extending ca 2 cm below septum.
Secure scrubber tube on sep. ring stand with 3-prong clamp.
Scrubber tube must be lower than cooling bath, especially in
rinsing step.
(3) Concentration tubes. — Use 10 mL calibrated tubes,
968.24B(h). Adapter, (g)(3), is needed for insertion into concn
tube to prevent splash during sweep and rinsing steps. Place
tip of scrubber thru adapter and into concn tube. If possible,
place scrubber tip against wall of concn tube. Hold tube in
place with clamp.
Adjust heat to 180-185° and N flow to 600 mL/min (mea-
sure before entering Storherr tube). Flush several 0.5 mL EtOAc
injections thru entire system, using 2 mL syringe (used for all
rinsings). Replace concn tube with clean tube and insert adapter;
assembly is ready for use.
D. Extraction
Ext all crops with EtOAc in exact order as follows: To high-
speed blender, add 25 g chopped sample, 125 mL EtOAc from
pipet, and 25 g anhyd. granular Na 2 S0 4 . Blend 5 min at slow
speed; then 5 min at high speed with mixer cup immersed in
ice-H 2 bath. Decant liq. thru 2.5 cm silane-treated glass wool
plug contained in short glass chromatgc tube. (Do not add sol-
ids to glass wool plug.) Collect EtOAc ext (ca 100 mL) in 125
mL flask or bottle. Remove 50 mL aliquot (equiv. to 10 g
original sample) and place in Kuderna-Danish concentrator with
Snyder column, calibrated Mills tube, or 5 mL vol. receiving
flask, and cone, to ca 5 mL. Adjust vol. to exactly 5.0 mL,
using air jet or adding EtOAc. Use 1 mL aliquots (2 g sample)
for sweep codistn cleanup.
Store all stds and crop solns at <0° when not in use. Warm
to room temp, ca 1 hr before use.
E. Sweep Codistillation Cleanup
Assemble app. as in Fig. 968.24, except position Storherr
tube and heating unit so exit end of Storherr tube is ca 10°
below horizontal to avoid backup of sample into N inlet arm.
If sample backs up, discard detn. Check temp. (180-185°), N
flow (600 mL/min), and receiver tube. Inject 1 mL (2 g) sam-
ple, using 1 mL Luer-Lok tuberculin syringe. Immediately fol-
low sample with injection of 0.5 mL EtOAc sweeping sol v.
and repeat 0.5 mL EtOAc injection every 3 min for 21 min.
After last injection wait 1 min until solv. has cleared cooling
coil and scrubber tube; then disconnect cooling coil arm with
septum from Storherr tube. Disconnect septum with attached
cooling coil arm from scrubber tube and rinse 2 cm Teflon
AOAC Official Methods of Analysis (1990)
Organophosphates
289
projection, collecting rinse in scrubber tube (still in position
in concn tube). With septums in place on disconnected cooling
coil arms, reverse coil arms and place that end formerly in
Storherr tube into scrubber tube. Make certain that Teflon tub-
ing in this arm extends 2 cm into scrubber tube below inserted
septum (similar to position when cleaning up sample). Slowly
inject 1 mL EtOAc rinse from 2 mL syringe directly into open
end of cooling coil arm formerly in scrubber tube. Gently force
rinse, using N flow from disconnected Storherr tube, thru cooling
coil into scrubber tube and into concn tube. Repeat 1 mL EtOAc
rinse 1-2 addnl times. Rinse scrubber tip end and inside of
adapter, remove scrubber, disconnect adapter, and rinse J joint.
Collect all rinses in concn tube. Rinse down sides of concn
tube and cone, to 1 mL, using N or air jet. Prevent H 2 con-
densation inside tubes by placing tube in room temp. H 2 bath
during this step. If cleaned up soln is too coned for GC detn
(>2 u.g/mL), dil. soln to 5 or 10 mL with EtOAc. If calibra-
tions of Mills tube are incorrect (most usually are except for
1 mL mark), quant, transfer the coned soln to 5 or 10 mL vol.
flask, using disposable pipet with attached rubber bulb. Rinse
inside of tube with EtOAc and transfer rinse in same pipet.
Repeat this rinse of tube and pipet several times; then rinse
inside of pipet into flask, using EtOAc. Dil. to vol. Further
diln with EtOAc or concn may be necessary to bring concn
within measurement range.
Anakrom scrubber tube is used repeatedly without change.
Final EtOAc rinses after each run keep it clean. However, if
Anakrom becomes discolored, prep, new tube.
F. Determination by Gas Chromatography
Operate chromatograph under conditions specified for col-
umn, 968.24B(j). Inject 3-8 |ulL aliquot coned, cleaned-up
soln contg amt of pesticide within linear range of gas chro-
matgc system, (i), using 10 )ulL syringe. Tentatively identify
residue peaks on basis of retention times. Det. amt of pesticide
by comparing area under peak with that from known amt of
appropriate std pesticide. For accurate detn, baseline current
of sample and std must be identical during chromatgy.
Injections <3 p,L are difficult to reproduce; injections >8
p,L may cause flame blow-out. Sample wt is not critical — use
injections equiv. to <1 mg or several hundred mg. Inject ap-
propriate std immediately after every sample. Peak ht also may
be used for detn, but only if ht of ref. std is ca same ht as
sample unknown (width of base should then be same).
Refs.: JAOAC 51, 662(1968); 59, 472(1976).
CAS-786-19-6 (carbophenothion)
CAS-333-41-5 (diazinon)
CAS-563-12-2 (ethion)
CAS- 121 -75-5 (malathion)
CAS-298-00-0 (methyl parathion)
CAS-56-38-2 (parathion)
970.53 Organophosphorus
Pesticide Residues
Single Sweep Oscillographic
Polarographic Confirmatory Method
First Action 1970
Final Action 1974
(Applicable to diazinon, malathion, Me parathion, and
parathion)
A. Apparatus
(Wash all glassware with hot HN0 3 (1 + 1) and rinse with
H 2 0.)
(a) Polarograph. — Any voltammetric or polarographic in-
strument capable of linear sweep voltammetry at 10 ng pes-
ticide/mL cell soln (equiv. to 0.01 ppm based on 1 g sample
in 1 mL cell soln).
(b) Silver wire electrode. — Deposit very thin coating of AgCl
on No. 20 or 22 gage Ag wire as follows: Dip wire in 10%
HN0 3 , rinse in H 2 0, and then let stand 10 min in IN HC1.
B. Reagents
(See statement regarding solvs, 970. 52A.)
(a) Acetonitrile . — Distd in glass at 82 ± 1°.
(b) Acetone. — Distil at 56.5° with 0.25 g KMn0 4 /L. Distn
must be performed as directed.
(c) Ethyl acetate. — Distd in glass at 77 ± 1°.
(d) Petroleum ether.— Distd. in glass at 30-60°.
(e) Nitrogen. — Prepurified, H 2 0-pumped.
(f ) Tetramethyl ammonium bromide. — Eastman Kodak No.
670, or equiv.
(g) Electrolyte solns. — (/') For diazinon. — Dissolve 7.7 g
Me 4 NBr in 300 mL H 2 0. Add 115 mL HOAc and dil. to 500
mL with LLO. (2) For malathion. — Dissolve 15.4 g Me 4 NBr
in 300 mL H 2 0. Add 0.2 g LiCl and 4.1 mL HC1, and dil. to
500 mL with H 2 0. (3) For methyl parathion and parathion. —
Dissolve 2.2 g NaOAc .3H 2 and 1.17 g NaCl in 100 mL
H 2 and adjust to pH 4.8 with HOAc, using pH meter.
(h) Pesticide std solns. —(I) Stock solns. — Prep, individual
solns contg 1 .00 mg pesticide/mL EtOAc. Store at 0°. (2) In-
termediate solns. — 0.2 mg/mL. Transfer 5 mL stock soln to
25 mL vol. flask and dil. to vol. with pet ether for diazinon,
MeOH for malathion, and acetone for Me parathion and par-
athion .
C. Preparation of Standard Curves
(a) Diazinon. — Transfer 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0
mL intermediate std soln of diazinon to individual 100 mL vol.
flasks and dil. to vol. with pet ether. Transfer 1.0 mL of each
soln to sep. 50 mL erlenmeyers and evap. to incipient dryness
under gentle jet of dry air. Evap. remaining solv. with warmth
of hand. Dissolve residue in 5.0 mL electrolyte soln, (g)(7).
Transfer soln to polarographic cell, adjust to 25 ± 1°, and
bubble N thru soln 5 min. Polarograph between -0.70 and
— 1.2 v against either Hg pool or Ag wire ref. electrode.
Peak potential for diazinon at 25° is -0.90 ± 0.05 v against
either electrode. Plot fxg diazinon /mL cell soln against peak
ht in units X instrument sensitivity setting.
(b) Ma lathion.— Transfer 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0
mL intermediate std soln of malathion to individual 25 mL vol.
flasks and dil. to vol. with MeOH. Transfer 2.0 mL of each
soln to sep. 50 mL erlenmeyers and add 1 .0 mL 0. 17V KOH.
After 3 min, add 2.0 mL electrolyte soln, (g)(2), mix well,
and let stand 5 min. Transfer to polarographic cell, adjust to
25 ± 1°, and bubble N thru soln 5 min. Polarograph between
—0.5 and — 1.0 v against either Hg pool or Ag wire electrode.
Peak potential for malathion at 25° is -0.82 ± 0.05 v against
Ag wire and -0.85 ± 0.05 v against Hg pool ref. electrodes.
Plot |mg malathion/mL cell soln against peak ht in units x
instrument sensitivity setting. (Note: Compd actually polaro-
graphed is fumaric acid resulting from basic hydrolysis of mal-
athion.)
(c) Parathion and methyl parathion. — Transfer 0.0, 1.0,
2.0, 3.0, 4.0, and 5.0 mL intermediate parathion (or Me par-
athion) std soln to individual 100 mL vol. flasks and dil. to
vol. with acetone. Transfer 5.0 mL aliquots of each soln to
sep. 50 mL erlenmeyers, add 5.0 mL electrolyte soln. (g)(3),
mix well, and transfer ca 5 mL to polarographic cell. Adjust
to 25 ± 1°, bubble N thru soln 5 min, and polarograph be-
290
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
tween —0.4 and —0.9 v against either Hg pool or Ag wire ref.
electrode.
Peak potential for parathion and Me parathion at 25° is —0.68
± 0.05 v against Hg pool and -0.70 ± 0.05 v against Ag
wire ref. electrodes. Plot |xg pesticide/mL cell soln (10 mL)
against peak ht in units X instrument sensitivity setting. Cell
soln vol. = 5 mL sample soln + 5 mL electrolyte soln.
D. Preparation of Sample Solution
Prep., ext, and clean up samples as in 970, 52K— O. Cone.
15% and 50% eluates from Florisil column to suitable definite
vol. in Kuderna-Danish concentrator. All eluting solvs must
be peroxide-free by test in Definitions and Explanatory Terms.
E. Determination
(a) Parathion and/ or methyl parathion. — Transfer aliquot
of coned 15% Florisil eluate, equiv. to 5 g crop, to 50 mL
erlenmeyer. Carefully evap. to dryness under gentle jet of air
at room temp. Dissolve residue in 3.0 mL acetone. (Note: Since
good polarograms can be obtained by using as little as 0.5 mL
soln in cell, min. of 0.25 mL acetone can be used to dissolve
residue.) Add 3.0 mL electrolyte soln, (g)(5), mix well, trans-
fer to polaro graphic cell, and adjust to 25 ± 1°. Bubble N thru
soln 5 min and polarograph as in 970.53C(c). Measure ht of
wave whose peak potential corresponds to that of parathion,
and det. concn from freshly prepd std curve or by comparing
wave hts of sample soln with those of std soln polarographed
immediately before or after sample. (Latter method is rec-
ommended for greater accuracy.)
Calc. |xg/mL as follows:
Qamplc = l.(WH sampll .) X (IS sample )
x (C« d )]/[(WH std ) x (lS std )]
where C = |xg pesticide/mL cell soln; WH — wave ht; IS =
instrument sensitivity setting.
ppm = (C sam pie x mL sample soln)/g sample
Limit of quant, detn is 0.01 ppm based on 1 g crop in 1 mL
cell soln.
Me parathion, parathion, and paraoxon polarograph at ca
same peak potential . If any one of these pesticides is present
as indicated by multiple residue methods, it should be polar-
ographed against that std. If these pesticides are present to-
gether, use mixed std contg ratio of pesticides as estd from
analysis by multiple residue method. (Paraoxon will not be
recovered by cleanup specified.)
Other pesticides known to give polarographic peak poten-
tials similar to parathion are pentachloronitrobenzene (PCNB),
l,2,4,5-tetrachloro-3-nitrobenzene (TCNB, tecnazene), and O-
ethyl 0-/?-nitrophenyl phenylphosphonothioate (EPN). PCNB
and TCNB are recovered in 6% Florisil eluate and will not
interfere. Verify presence or absence of EPN by GC or TLC.
(b) Diazinon. — Transfer aliquot of coned 15% Florisil eluate,
equiv. to 5 g crop, to 50 mL erlenmeyer. Carefully evap. just
to dryness, using gentle jet of dry air at room temp. Dissolve
residue in 5.0 mL electrolyte soln, (g)(/). Transfer soln to po-
larographic cell and adjust to 25 ± 1°. Bubble N thru soln 5
min and polarograph as in 970.53C(a). Calc. amt of diazinon
present as in (a).
Limit of quant, detn is 0.2 ppm based on 1 g crop sample
in 1 mL cell soln.
(c) Malathion. — Transfer aliquot of coned eluate from 50%
Florisil eluate, equiv. to 5 g crop, to 50 mL erlenmeyer. Care-
fully evap. just to dryness under gentle jet of dry air at room
temp. Dissolve residue in 2.0 mL MeOH, add 1.0 mL 0.1 N
KOH, and let stand 3 min. Add 2.0 mL electrolyte soln, (g)(2),
mix well, and let stand 5 min. Transfer to polarographic cell,
adjust to 25 ± 1°, bubble N thru soln 5 min, and polarograph
as in 970.53C(fo). Calc. amt of malathion present as in (a).
Limit of quant, detn is 0.3 ppm based on 1 g crop in 1.0
mL cell soln.
Note 1 : If polarogram cannot be obtained because of high
residual currents, check coned eluate for peroxides. If per-
oxides are present, transfer 5 mL coned eluate to small sep-
arator contg 25 mL 3% FeS0 4 soln; shake well and discard
aq. layer. Transfer 1.0 mL ether layer to 50 mL erlenmeyer
and proceed as in (a), (b), or (c).
Note 2: All glassware used for polarographic detns should
be thoroly washed with hot HN0 3 (1 + 1) and rinsed with
distd H 2 0.
Ref.: JAOAC 52, 811(1969).
CAS-333-4 1-5 (diazinon)
CAS-121-75-5 (malathion)
CAS-298-00-0 (methyl parathion)
CAS-56-38-2 (parathion)
964.1 7* Organophosphorus
Pesticide Residues
Cholinesterase Inhibition Method
First Action 1964
Surplus 1974
See 29.049-29.055, 12th ed.
FUMIGANT RESIDUES
977.18 Volatile Fumigants in Grain
Gas Chromatographic Method
First Action 1977
Final Action 1981
(Applicable to CC1 4 , CHC1 3 , BrH 2 CCH 2 Br, and CLCCC1H in
wheat and corn grain)
A. Apparatus and Reagents
(a) Column. — 4 m X 2.2 (id) mm stainless steel packed
with 15% polypropylene glycol (LB 550X, Ucon fluid) on 60-
80 mesh Chromosorb W.
(b) Gas chromatography — Isothermal with source-heated
electron capture detector and glass-lined heated injection block.
(100-200 mCi 3 H with Ar as p -ionization detector is more
useful for multiresidue detns than 63 Ni and N.) Use 1 mv re-
corder with max. response time of 1 sec and chart speed of
0.5 cm /min. Operate electron capture detector with N at 25
psi (173 kPa) at 95° with polypropylene glycol column for CCJ 4
(retention time, ca 6 min). Use 120° for CHC1 3 (3 min),
C1 2 CCC1H (4 min), and BrH 2 CCH 2 Br (8 min).
(c) Acetone. — Check for interfering peaks by gas chro-
matgy before use.
B. Determination
(Caution: See safety notes on acetone.)
Store sample at ^5°. Quickly weigh 50 g and immerse in
150 mL acetone-H 2 (5 + 1) in 250 mL g-s flask, and stopper.
Let stand 48 hr in dark at 20-25°, swirling at 24 hr. Decant
10 mL supernate into 25 mL g-s graduate, add 2 g NaCl, stop-
per, and shake vigorously 2 min. Let stand until layers sep.
Pour 5 mL clear upper layer into 10 mL g-s graduate, add 1
g anhyd. CaCL, stopper, and shake 2 min. Let stand 30 min
with occasional shaking.
AOAC Official Methods of Analysis (1990)
Carbamates
291
Withdraw 0.5 jjlL aliquots from upper layer into 1 |xL sy-
ringe. Inject into gas chromatograph. Dil. 10 x or 100x with
dry acetone, if necessary to avoid overloading detector. Inject
all solns in triplicate and average results.
Construct calibration curve daily of peak hts against ng fu-
migant/125 mL acetone for suitable range.
Refs.: Analyst 99, 570(1974). JAOAC 60, 368, 405(1977).
CAS-56-23-5 (carbon tetrachloride)
CAS-67-66-3 (chloroform)
CAS- 106-93-4 (ethylene dibromide)
CAS-79-01-6 (trichloroethylene)
CARBAMATE RESIDUES
975.40 A/-Methylcarbamate
Insecticide Residues
Gas Chromatographic Method
First Action 1975
Final Action 1976
(Carbanolate, Carbaryl, Carbofuran, and Propoxur)
(Applicable to apples, cabbage, collards, corn kernels, green
beans, kale, and turnip tops. Rinse all glassware with acetone
and then distd H 2 before use.)
A. Principle
Residue is extd from crop with CH 3 CN, and ext is purified
by partitioning with pet ether and coagulating with H 3 P0 4 -NH 4 CI
soln. Phenolic impurities are largely eliminated by partitioning
CH 2 C1 2 ext with KOH soln. Carbamate residues are treated
with l-fluoro-2,4-dinitrobenzene to form ether derivative.
Residues may be detd at levels ^0.05 ppm. Recoveries range
from 90 to 110%.
B. Reagents
(a) Borax. — 5% aq. soln.
(b) Diatomaceous earth. — Wash thoroly with acetone and
dry 2 hr at 110°.
(c) Coagulating soln . — ( 1 ) Stock soln . — Di ssol ve 20 g N H 4 C1
and 40 mL H3PO4 in 360 mL H 2 0. (2) Working soln.—T)i\.
100 mL stock soln to 1 L for coagulation.
(d) 1 -Fluoro-2 ,4-dinitrobenzene soln, — (Eastman Kodak
Co.) Redistil at 128° and 1 mm pressure. Dissolve 1.5 mL in
25 mL acetone.
(e) Pesticides. — Best quality obtainable from manufac-
turer; anal, grades when available.
(f) Potassium hydroxide soln. — 0.5N aq. soln.
(g) Sodium chloride soln. — 30% aq. soln.
(h) Solvents. — Acetone, CH 2 C1 2 , isooctane, CH 3 CN, and
pet ether (distd in glass; see statement regarding solvs, 970.52A);
acetophenone and MeOH (anal, grade).
C. Gas Chromatographic Apparatus
Gas chromatograph equipped with 3 H electron capture de-
tector and 46 X 0.64 (od) cm (18 X l / 4 ") glass column contg
10% DC-200 (12,500 est) on 60-70 mesh Anakrom ABS (An-
alabs, Inc.). Porous Teflon end plugs for '// od glass tubing
(Chemical Research, PO Box 888, Addison, 1L 60101) are
preferable, but glass wool can be used at outlet and omitted
at inlet if necessary. (Glass wool at inlet tends to adsorb de-
rivatives gradually and to release them later, giving rise to "ghost
images" of compds.)
Equilibrate column 2 days at 250° and 2 weeks at 212°. Op-
erating conditions: temps (°) — column 212, detector 218,
standby temps 190 and 200, resp.; N carrier gas 60 mL/min;
sensitivity 1- X 10~ 9 amp full scale; and detector potential either
25 or 50 V, depending on response level needed (V3 to 2 / 3 full
scale peak ht with injections of 4 ng carbamate).
Alternatively, use instrument with 63 Ni detector and 1.8 m
(6') x 4 mm id glass column contg 10% DC-200 on 60-70
mesh Anakrom ABS. Do not use glass wool at beginning of
column. Operating conditions: temps (°) — column 232, detec-
tor 250, N carrier gas 80 mL/min, sensitivity 1 x 10~ 9 amp
full scale, and detector potential 50 or 75 V.
D. Extraction of Pesticides
(Caution: See safety notes on blenders, acetonitrile, and pe-
troleum ether)
Place 100 g sample and 200 mL CH 3 CN (add 50 mL H 2
with fruit or other samples contg 5-15% sugar) in sq screw-
top jar, and macerate in blender operated 2 min at moderate
speed. Filter with suction into 500 mL r-b flask thru rapid pa-
per in 1 1 cm buchner. Transfer aliquot equiv. to 40 g crop
(mL aliquot = (mL H 2 in sample + mL CH 3 CN added +
mL H 2 added - 5 mL vol. contraction) x 40/100) to 250
mL separator. Shake 10 sec with 25 mL NaCl soln. Drain and
discard aq. phase. Repeat with fresh NaCl soln. Add 100 mL
pet ether, and shake 30 sec. Drain CH 3 CN into 1 L separator.
Strip pet ether by shaking 20 sec with 50 and 10 mL portions
CH 3 CN, draining each into the 1 L separator. Add 300 mL
H 2 0, 25 mL NaCl soln, and 50 mL MeOH. Ext mixt. with
100 mL and two 25 mL portions CH 2 C1 2 , shaking each 20 sec,
and drain lower layer into 500 mL r-b flask. Add 2 drops ace-
tophenone, and evap. in rotary evaporator connected to aspi-
rator pump. During evapn, keep H 2 bath within 40-50° range
and remove flask from H 2 bath when ext vol. has been re-
duced to few mL, so that final evapn to dryness takes place
at low temp.
Add 5 mL acetone, and swirl flask to dissolve residue. Add
50 mL coagulating soln, swirl to mix, add 1-2 g diat. earth,
and swirl again to mix. Pour soln into 150 mL suction filter
of medium porosity packed with 6 mm (V/) diat. earth, and
collect filtrate in 500 mL r-b flask. Break vac. immediately
after liq. is drawn into diat. earth layer. Rinse sides of flask
with 5 mL acetone, swirl, and repeat coagulation. Rinse flask
with 20 mL coagulating soln, and add rinse to filter just after
liq. of second coagulation is drawn into diat. earth layer. After
filtration is complete (ca 5 min), transfer filtrate to 250 mL
separator. Ext carbamates by shaking 20 sec with three 25 mL
portions CH 2 CI 2 , rinsing filter flask with each portion before
adding to separator. Drain CH 2 C1 2 (lower) ext into another 250
mL separator. Soln may be held overnight at this point. Add
40 mL H 2 and 10 mL 0.5N KOH, mix briefly by gentle
swirling, and shake 20 sec. Drain CH 2 C1 2 thru granular anhyd.
Na 2 S0 4 supported by glass wool in filter funnel, and collect
filtrate in 250 mL erlenmeyer. Add 10 mL CH 2 C1 2 to sepa-
rator, swirl gently, and drain org. phase. Repeat once. Rinse
filter with two 10 mL portions CH 2 C1 2 . Add 2 drops aceto-
phenone, and evap. with same technic used in first evapn.
E. Determination
Add 100 mL H 2 0, 2 mL 0.5^ KOH, and 1 mL l-fluoro-
2,4-dinitrobenzene soln. Stopper, and mix 20 min at high speed
on mech. agitator. Add 10 mL 5% borax, swirl to mix, and
heat on steam bath 20 min. Cool to room temp, by placing
flasks in shallow H 2 bath 10 min. Add 5 mL isooctane, stop-
per, shake 3 min at high speed, and pour into 250 mL sepa-
rator. Drain aq. phase, and rinse twice with H 2 0. Drain isooc-
tane soln thru funnel contg 6 mm glass wool plug into g-s test
tube. Soln may be held overnight at this point. Inject 10 jxL
sample into gas chromatograph. If necessary to dil. sample,
292
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
transfer 1 mL of isooctane ext to another test tube, dil. to exact
vol. with isooctane, and shake to mix. Chromatograph std and
sample solns at approx. same level of response.
Methylcarbamates, ppm
= concn std x (peak ht sample/peak ht std)
X (jjlL std/u,L sample) X (diln vol. /aliquot vol.) x 5/40
F. Preparation of Standard Curves
Dissolve 50 mg each carbamate in 1 00 mL benzene and store
in brown bottles. Dil. 5 mL aliquots from these solns to 50
mL with benzene, and store in brown bottles. Transfer 50 |xL
to 250 mL erlenmeyer, and derivatize as in 975. 40E. After
extn of derivatives, solns will contain equiv. of 0.5 ng each
carbamate/uX. Chromatograph 4, 6, 8, and 10 julL and plot
mm response against ng carbamate. If response is nonlinear,
adjust GC parameters and/or prep, more dil. ref. soln, e.g.,
equiv. of 0.25 jag carbamate /mL, and establish suitable linear
working range.
Refs.: JAOAC 56, 713(1973); 58, 562(1975).
CAS-67 1-04-5 (carbanolate)
CAS-63-25-2 (carbaryl)
CAS- 1563-66-2 (carbofuran)
CAS- 1.14-26-1 (propoxur)
985.23 AMVIethylcarbamate Insecticide
and Metabolite Residues
Liquid Chromatograhic Method
First Action 1985
Final Action 1986
(Applicable to residues of aldicarb, bufencarb, carbaryl, car-
bofuran , methiocarb, methomyl, oxamyl, and metabolites
aldicarb sulfone and 3-hydroxycarbofuran in grapes and
potatoes)
A Principle
Sample is extd with MeOH and cleaned up by liq.-liq. par-
titioning and Nuchar-Celite column chromatgy. Residues are
sepd by reverse phase liq. chromatgy and detected by in-line
post-column fluorometric technic.
B. Reagents
(a) Solvents.— CH 3 CN, MeOH, CH 2 C1 2 , pet ether, and tol-
uene. Distd-in-glass grade (Burdick & Jackson Laboratories,
Inc.).
(b) LC acetonitrile . — UV grade distd-in-glass (Burdick &
Jackson Laboratories, Inc.). Before use, degas CH 3 CN in glass
bottles by applying vac. and slowly stirring solv. with mag.
stirrer 5 min.
(c) Ultrapure water. — Prep, using Milli-Q H 2 purification
system (Millipore Corp.). For LC use, degas H 2 as described
above for LC CH 3 CN.
(d) NaOH soln.— 0.05N. Pipet 27 mL clear supernate NaOH
in H 2 (1 + 1), 936.16B(b), into 100 mL vol. flask. Dil. to
vol. with H 2 and mix (5N NaOH). Pipet 10 mL 5N NaOH
into 1 L vol. flask. Dil. to 1 L with degassed ultrapure H 2 0,
and mix well but gently to minimize reincorporation of air into
soln.
(e) Na 2 S0 4 . — Anhyd., granular. Heat at 600° overnight and
then cool in desiccator.
(f) Sodium tetraborate soln. — 0.05M. Add 19. 1 g ACS grade
sodium tetraborate decahydrate (Na 2 B 4 O 7 .10H 2 O) and ca 500
mL degassed ultrapure H 2 to 1 L vol. flask. Heat flask in
steam bath to dissolve sodium borate, cool to room temp., and
dil. to vol. with degassed ultrapure H 2 0. Mix well but gently
to minimize reincorporation of air into soln.
(g) Reaction soln. — Weigh 500 mg o-phthal aldehyde
(Fluoropa, Dionex Corp., 1228 Titan Way, Sunnyvale, CA
94088), transfer to 1 L vol. flask, add 10 mL MeOH, and swirl
flask to dissolve o-phthalaldehyde. Add ca 500 mL 0.05M so-
dium tetraborate soln and 1.0 mL 2-mercaptoethanol (Aldrich
Chemical Co., Inc., nos. 22, 173-0 and M370-1, resp.) and
dil. to vol. with 0.05M sodium tetraborate soln. Mix well but
gently to minimize reincorporation of air into soln.
(h) Silanized Celite 545.—§\mvy 150 g Celite 545 (Man-
ville Filtration and Minerals) with 1 L HCI-H 2 (1 + 1) in 2
L beaker, cover with watch glass, and stir mag. while boiling
10 min. Cool slurry, filter, and wash with distd or ultrapure
H 2 until filtrate is neut. Wash Celite with 500 mL MeOH
followed by 500 mL CH 2 C1 2 and then air-dry Celite in hood
on watch glass to remove solv. Transfer Celite to 1 L erlen-
meyer with g-s joint. Heat unstoppered flask in 120° oven
overnight and then cool flask in desiccator. Place flask in hood
and carefully pipet 3 mL dichlorodimethylsilane (Pierce
Chemical Co.) onto Celite. Stopper flask, mix well, and let
flask remain at room temp. 4 h. Add 500 mL MeOH to flask,
mix, and let stand 15 min. Filter silanized Celite and wash
with isopropanol until neut. Air-dry silanized Celite in hood
to remove isopropanol. Dry silanized Celite in 105° oven 2 h,
and cool in desiccator. Store silanized Celite in g-s container.
Test Celite for total silanization by placing ca 1 g in 50 mL
H 2 and placing ca 1 g in 20 mL toluene saturated with methyl
red. Silanized Celite should float on H 2 0, and appear yellow
with methyl red -toluene soln. Repeat silanization of Celite with
dichlorodimethylsilane if particles of Celite are dispersed in
H 2 0, and/or appear pink with methyl red-toluene soln, indi-
cating active sites.
(i) Nuchar S-N.— Slurry 100 g Nuchar S-N (Eastman Ko-
dak no. 1180454) with 700 mL HC1, cover with watch glass,
and stir mag. while boiling 1 h. Add 700 mL H 2 0, stir, and
boil addnl 30 min. Cool slurry, filter, and wash with distd or
ultrapure H 2 until neut. Then wash Nuchar S-N with 500 mL
MeOH followed by 500 mL CH 2 C1 2 , and air-dry Nuchar S-N
in hood to remove solv. Dry Nuchar S-N in 120° oven 4 h.
Cool in desiccator. Store Nuchar S-N in g-s container.
( j) Nuchar S-N-silanized Celite 545 chromatographic mix-
ture. — Mix well 1 part Nuchar S-N with 4 parts silanized Cel-
ite 545 (w/w). Test each Nuchar S-N with mixed carbamate
soln (carbaryl, methiocarb, methiocarb sulfoxide, methomyl).
Note: Use freshly prepd mixed std soln because methiocarb
sulfoxide degrades in soln. Prep, mixed carbamate soln in MeOH
at concn of 5 fxg each/mL. Pipet 5 mL of this soln into 250
mL r-b flask and 5 mL into 25 mL actinic vol. flask. Dil. soln
in vol. flask to 25 mL with MeOH and use as LC ref. std.
Evap. std soln in r-b flask just to dryness with vac. rotary
evaporator as described in Extraction. After last trace of MeOH
has evapd, remove r-b flask from evaporator and dissolve car-
bamate residue in 10 mL CH 2 C1 2 . Transfer CH 2 C1 2 soln in
r-b flask to prepd adsorbent column and elute as described in
Coextractive Removal — Chromatographic . After evapn of eluate
in r-b flask, dissolve residue in 25 mL MeOH. Filter 5-8 mL
of this soln thru Swinny filter holder as described in Coex-
tractive Removal — Chromatographic . Quantitate recovery of
carbamates using LC detn. Nuchar S-N is considered satisfac-
tory if av. recovery of carbamates is >95%, with no one compd
<90% rec.
(k) Carbamate LC std solns. — Dissolve carbamate std(s)
(EPA /FDA Reference Standards, Environmental Protection
Agency, Pesticides and Industrial Chemicals Repository (MD-
8), Research Triangle Park, NC 27709) in MeOH to give 1
|uig/mL concn or as needed. Store soln(s) in actinic glassware,
AOAC Official Methods of Analysis (1990)
Carbamates 293
and when not in use store in refrigerator. Most carbamate stds
stored in this manner are stable for several months. However,
methiocarb sulfone and sulfoxide degrade within hours and days,
respectively, even with storage precautions.
C. General Apparatus
(a) Homogenizer.—Polytron Model PT 10-35, with PT 35K
generator contg knives (Brinkmann Instruments, Inc.).
(b) Homogenizer jar. — Four side glass qt jar (Tropicana
Products, Inc., Bradenton, FL 33506).
(c) Vacuum rotary evaporator. — Model RE rotavapor
(Brinkmann Instruments, Inc.). Maintain soln in condensing
coils and around receiving flask at —15°. (Refrigerated H 2 0-
antifreeze soln works well.) Use vac. pump fitted with ma-
nometer and needle valve to control vac. in evaporator.
(d) Chromatographic tubes. — Chromatlex 30 cm x 22 mm
id column (size 233) with coarse porosity frit with Varibor
stopcock (size 2) (No. K-420540-9042, Kontes).
(e) Swinny filter holder. — 1 3 mm filter size (No.
XX3001200, Miliipore Corp.).
(f) Milt ex filters. — 5 fjtm, 13 mm diam., white, plain (No.
LSWP 01300, Miliipore Corp.).
D. LC Apparatus
LC app. (Fig. 985. 23 A) must be capable of performing as
described in LC Operating Parameters. Following specific in-
dividual items of app. have been found to meet operating pa-
rameters and are listed as guide for analyst:
(a) Mobile phase delivery system. — Model 322 MP pro-
grammable gradient system (replacement Model System Gold
Protein II, Beckman Instruments, Inc., 2350 Camino Ramon,
PO Box 510.1 , San Ramon, CA 94583-0701).
(b) Injector. — Model 16 AS-7000 automatic sampler with
10 uX injection loop.
(c) Guard column. — 7 cm X 2.1 mm id column pellicular
ODS (no. 4390-413, Whatman Inc.)
(d) Analytical column. — 25 cm x 4.6 mm id column contg
6 fxm Zorbax C-8 spherical particles (DuPont Co.). Equiv. col-
umn should contain 5 or 6 |xm spherical silica particles that
have been bonded with a monofunctional octyl silane reagent
to form a monomolecular bond.
(e) Column oven. — Custom-built forced draft oven (66 x
13 x 11 cm).
(f) Carbamate hydrolysis chamber. — Column bath (18 X
18 X 13 cm) from Model 5360 Barber-Coleman gas chro-
matograph with Model 700-115 proportional temp, controller
(replacement Model 70A, Dowty Electronics Co., Prospect St,
PO Box 250, Brandon, VT 05733-0250) contg 3 m X 0.48
mm id No. 321 stainless steel tubing (Tubesales, 175 Tubeway
St, Forest Park, GA 30051).
-INJECTOR
- ODS GUARD COL.
~C-8 ANAL. COL.
o-PHTHALALDEHYDE
2-MERCAPTOETHANOL
HYDROLYSIS CHAMBER
FLUOR. DET.
hL
RECORDE R
10%
FSD
FIG. 985.23A — LC system with in-line post-column fiuorome-
tric detector
5 10 15 20 25 30
Retention [mini
FIG. 985.23B — Typical chromatogram of separation of carba-
mates and carbamate metabolites: 1, aldicarb sulfoxide; 2, al-
dicarb sulfone; 3, oxamyl; 4, methomyl; 5, 3-hydroxy carbof-
uran; 6, methiocarb sulfoxide; 7, aldicarb; 8, carbofuran; 9,
carbaryl; 10, methiocarb; 11, bufencarb (1 and 6 not included
in official method).
(g) Fluorescence detector. —Model 650-lOLC, with 20 liL
cell (Perkin-Elmer Corp.).
(h) Recorder. — Model 4000 microprocessor/printer plotter
(Spectra Physics, 3333 N First St, San Jose, CA 95134).
(i) NaOH and reaction soln reservoirs. — 60 cm X 25 mm
id glass columns with Teflon fittings (No. 125029, Spectrum
Medical Industries, Inc., 60916 Terminal Annex, Los Ange-
les, CA 90060). Pressurize reservoirs with N. Connect 6 m X
0.5 mm id Teflon restriction coil from reservoir to \5 cm x
0.18 mm id stainless steel tubing. Connect stainless steel tub-
ing to 0.74 mm id stainless steel reaction tee (No. ZVT-062,
Valco Instruments Co., Inc., PO Box 55603, Houston TX
77255).
(j) Connecting tubing. — Use No. 304 stainless steel tubing
(1.6 mm od x 0.18 mm id) to connect injector, columns, and
first tee.
E. LC Operating Parameters
Adjust mobile phase flow rate to 1.50 ± 0.02 mL/min at
50% CH 3 CN in H 2 0. Equilibrate system at 12% CH 3 CN in
H 2 for 10 min, inject sample, and begin 30 min linear gra-
dient to 70% CH 3 CN in H 2 0. Adjust flow rate of 0.05N NaOH
and reaction soln to 0.50 ± 0.02 mL/min each. Operate col-
umn oven at 35° and hydrolysis chamber at 100°. Set fluores-
cence detector excitation and emission wavelengths to 340 and
455 nm, resp.; slit widths 15 and 12 nm, resp. Set detector
PM gain to low and time const to 1 s. Adjust sensitivity so
that 10 ng carbofuran produces 50 ± 5% full scale response
on recorder. Baseline noise should be <2%. Carbamates are
to elute as shown in chromatogram (Fig. 985. 23B). Note: If
system will not be used for several days, replace H 2 mobile
phase with MeOH and pump thru system, drain NaOH and
reaction solns from reservoirs, and wash reservoirs and asso-
ciated tubing first with H 2 and then MeOH. When starting
up system, change mobile phase to H 2 0, and wash reaction
reservoirs and associated tubing with H 2 before adding re-
action solns.
294
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
F. Extraction
High moisture (more than 75% water) products. — Add 150
g chopped sample and 300 mL MeOH to homogenizer jar.
Homogenize sample with Polytron for 30 s at ca half speed
(setting of 7) and then 60 s at full speed. Vac. -filter homog-
enate through 12 cm perforated buchner contg sharkskin or
597 S&S filter, collecting filtrate in 500 mL filter flask. (Note:
Reduce vac. during filtration if filtrate begins to boil.) Transfer
portion of filtrate equiv. to 100 g sample to 2 L I 24/40 r-b
flask. (Note: Vol. 100 g sample = mL H 2 in 100 g sample
+ 200 mL MeOH - 10 mL contraction factor.) Add distd or
ultrapure H 2 to r-b flask to give total of 100 mL H 2 0. Also
add small star mag. stirrer to r-b flask.
Place 250 mL I 24/40 trap on 2 L r-b flask and attach to
vac. rotary evaporator. Apply vac. slowly to minimize froth-
ing. After full vac. is supplied, slowly place flask in 35° H 2
bath. Cone, ext to 75 mL.
G. Coextractive Removal — Partitioning
Transfer coned ext from r-b flask to 500 mL sep. funnel
contg 15 g NaCl. Shake sep. funnel until NaCl is dissolved.
Wash r-b flask with three 25 mL portions of CH 3 CN, trans-
ferring each to 500 mL sep. funnel, shake 30 s, and let layers
sep. 5 min. Drain aq. phase into 250 mL sep. funnel contg 50
mL CH 3 CN, shake 20 s, let layers sep., and discard aq. layer.
Add 25 mL 20% aq. NaCl soln to CH 3 CN in 500 mL sep.
funnel, shake 20 s, let layers sep., and transfer aq. soln to 250
mL sep. funnel. Shake 250 mL sep. funnel 20 s, let layers
sep., and discard aq. layer.
Add 100 mL pet ether to 500 mL sep. funnel, shake 20 s,
let layers sep. and drain CH 3 CN layer into second 500 mL sep.
funnel. Transfer CH 3 CN in 250 mL sep. funnel to first 500
mL sep. funnel which contains pet ether, shake 20 s, let layers
sep., and transfer CH 3 CN to second 500 mL sep. funnel. Add
10 mL CH 3 CN to first 500 mL sep. funnel, shake, let layers
sep., and transfer CH 3 CN to second 500 mL sep. funnel. Dis-
card pet ether layer.
Add 50 mL 2% aq. NaCl soln to CH 3 CN in second 500 mL
sep. funnel. Ext mixt. successively with 100, 25, and 25 mL
portions of CH 2 C1 2 , shaking each 20 s (shake 25 mL portions
gently). Drain lower CH 2 C1 2 -CH 3 CN layers thru 22 mm id col-
umn contg ca 5 cm anhyd. granular Na 2 S0 4 . Collect eluate in
1LI 24/40 r-b flask. Evap. soln to dryness with rotary evap-
orator as described earlier. Remove r-b flask from evaporator
immediately after last traces of soln have evapd and then add
10 mLCH 2 Cl 2 to r-b flask.
H. Coextractive Removal — Chromatographic
Fit 1-hole No. 5 rubber stopper onto tip of chromatgc tube
with Varibor stopcock, add J 24/40 side arm vac. adaptor and
500 mL ¥ 24/40 r-b flask, open stopcock, and connect app.
to vac. line. Place 0.5 g silanized Celite 545 in chromatgc
tube, tamp, add 5 g Nuchar S-N-silanized Celite 545 (1+4)
mixt., and tamp again. Add 1-2 cm glass wool plug (Corning)
on top of adsorbent. Prewash column with 50 mL toluene-
CH 3 CN (1+3) eluting soln. Close stopcock when prewash
soln is ca 0.5 cm from top of glass wool. Disconnect vac,
discard eluting soln in r-b flask, and reconnect flask to app.
Transfer sample in 10 mL CH 2 C1 2 from r-b flask to column
and elute column at 5 mL/min. Wash 1 L r-b flask with 10
mL CH 2 C1 2 and then with 25 mL eluting soln. Transfer each
sep. to column and elute each to top of glass wool before add-
ing next soln. Next, add 100 mL eluting soln and elute column
at 5 mL/min. Turn off stopcock when top of eluting soln reaches
top of glass wool. Evap. soln in 500 mL r-b flask just to dry-
ness using vac. evaporator as before. Remove flask from evap-
orator immediately after all soln has evapd. Immediately pipet
5 mL MeOH into 500 mL r-b flask to dissolve residue. Pour
MeOH sample soln into 10 mL glass syringe contg Swinny
filter holder with 5 |xm filter. Push MeOH soln thru filter with
syringe plunger, collecting filtrate in 10 mL centrf. tube or
other suitable container. Note: Approximately 4.5 mL filtrate
will be collected. Vol. of filtrate collected is not critical be-
cause g sample /mL MeOH is known. If soln needs to be dild,
pipet aliquot into another container and dil. to vol. as needed.
/. Determination
Inject 10 |mL MeOH sample soln onto LC column using
chromatgc app. and parameters as described. Tentatively iden-
tify residue peaks on basis of retention times. Measure peak
area or peak ht and det. residue amt by comparison to peak
area or peak ht obtained from known amt of appropriate ref.
material(s). To ensure valid measurement of residue amt, size
of peaks from sample residue and ref. std should match within
±25%. Chromatograph ref. material (s) immediately after sam-
ple.
CAS- 116-06-3 (aldicarb)
CAS- 1646-88-4 (aldicarb sulfone)
CAS-8065-36-9 (bufencarb)
CAS-63-25-2 (carbaryl)
CAS- 1563-66-2 (carbofuran)
CAS- 16655-82-6 (3-hydroxycarbofuran)
CAS-2032-65-7 (methiocarb)
CAS- 16752-77-5 (methomyl)
CAS-23 135-22-0 (oxamyl)
Ref.: JAOAC 68, 726(1985).
INDIVIDUAL RESIDUES
961 .1 2* Azinphos-Methyl
Pesticide Residues
Spectrophotometric Method
First Action 1961
Surplus 1974
(Applicable to cole-type crops and to apples, plums, peaches,
grapes, apricots, and cherries.)
See 29.102-29.107, 12th ed.
956.05* Benzene Hexachloride
Pesticide Residues
Colorimetric Method
First Action 1956
Final Action 1960
Surplus 1970
See 24.101-24.105, 10th ed.
958.07* Lindane and Technical BHC
Pesticide Residues
Distinguishing Method
First Action
Surplus 1970
See 24.106-24.110, 10th ed.
AOAC Official Methods of Analysis (1990)
Biphenyl
295
968.25 BiphenyS Pesticide Residues
in Citrus Fruits
Thin Layer Chromatographic-Spectrophotometric Method
First Action 1968
Final Action 1969
A. Principle
Biphenyl is extd from blended pee J or pulp by steam- 1 iq.-
liq. extn. Ext is subjected to TLC and biphenyl zone is com-
pletely scraped from developed plate. Biphenyl is eluted from
adsorbent with alcohol for spectrophtric detn.
0. Reagents
(a) Silica gel. — GF-254 (Brinkmann Instruments, Inc. No.
7730).
(b) Biphenyl std solns. — (7) Stock soln. — Approx. 0.5 mg/
mL. Dissolve ca 50 mg accurately weighed biphenyl in n- hep-
tane and dil. to 100 mL with n-heptane. (2) Limit soln. — Ap-
prox. 0.01 mg/mL. Dil. 5 mL stock std to 250 mL with n-
heptane.
Use stock std soln for spectrophtric quantitation after TLC
step. Limit std soln aids in locating biphenyl zone and in estg
small amts.
C. Apparatus
(a) Applicator. — For depositing thin layer on glass plates.
(b) Glass plates. — 8 X 8" or 2 x 8"; of uniform thickness.
(c) Plastic board. — 22 x 1 13 cm, with retaining edges 1.8
cm wide along short and long sides.
(d) Developing jars or tanks. — Use equipment, 970.52G(a),
for 8 x 8" glass plates and glass cylinders for small plates.
Cylinders can be covered with plastic caps.
(e) Spotting pipet. — 100 fxL.
(f) Tank liner. — Whatman 3MM paper cut to fit tank.
(g) Moisture test apparatus. — Similar to lighter-than-H 2
volatile oil trap, 962.17A(a), Fig. 962.17, with cold finger
condenser (Lurex Scientific, No. JM-8590, or equiv.).
D. Preparation of TLC Plates
Mix 40 g silica gel with 80 mL H 2 0, shaking vigorously
few sec, and finally swirling ca 30 sec to eliminate air bubbles.
Spread slurry 0.3 mm thick over 5 plates. Let plates air dry
in place ca 1 hr. Put plates in drying rack and place in 100°
oven 2 min. Remove plates and store in desiccator over silica
gel or CaCl 2 until used. Plates may be stored up to 30 days.
E. Preparation of Sample
Sort out and discard rotten units. Completely peel >6 whole
fruits (include all white material under peel in peel portion).
Weigh peelings and peeled fruit, and calc. wt ratio of peelings
to peeled fruit.
(a) Peel. — Grind combined peel in food grinder. Blend 200
g ground peel with 400 g H 2 at high speed 5 min (or in five
1 min increments if blender becomes very warm), using high-
speed blender. (Larger batches may be blended with large
blender as long as peel-H 2 ratio is same.)
(b) Peeled fruit. — Cut peeled fruit into small pieces and blend
at high speed 5 min (or in five 1 min increments if blender
becomes very warm).
F. Extraction
Accurately weigh ca 300 g recently blended peel slurry or
ca 100 g recently blended peeled fruit, and transfer to I L r-
b ¥29/42 flask with enough FLO to yield total vol. of ca 500
mL; add few boiling chips (6 mesh granular SiC is conve-
nient). Connect extn unit of moisture test app. to flask and fill
side arm with FLO to overflowing. Place ca 3 mL rc-heptane
on top of H 2 layer and insert cold finger cooled with very
rapid flow of cold FLO. Gradually heat flask with mantle (con-
trolled by variable transformer) until even boiling is obtained,
then intensely enough to maintain vigorous boiling. Continue
extn 3 hr from time mixt. starts boiling. (Wrap exposed por-
tion of flask and connector arm between flask and extn unit
with Al foil.) Initial carry-over of froth does not interfere. Af-
ter 3 hr, discontinue heat and drain entire contents of extractor
into 125 mL separator. Discard lower layer and drain heptane
ext thru 2.5 cm column of granular anhyd. Na 2 S0 4 (8-10 mm
id column) into 10 mL vol. flask. Rinse separator with 1 mL
H-heptane and add rinse to column. Rinse cold finger and extn
unit with five 2 mL portions alcohol, collecting successive
rinses in separator. Add 5 mL w-heptane to separator and shake
vigorously few sec; add 50-75 mL H 2 and shake moderately
few sec. Let layers sep. (lower layer may remain slightly cloudy)
and discard lower layer. Pass heptane layer thru same Na 2 S0 4
column into vol. flask. Rinse separator and column with enough
n-heptane to dil. to vol.
G. Thin Layer Chromatography
Pre-sat. tank contg liner with n-heptane >l hr before use.
Establish imaginary spotting line 3 cm from bottom edge of
plate. For each intended spot, use tip of 100 \xL pipet to scratch
mark in adsorbent layer just size of pipet tip. (Space spots
evenly with max. of 7 spots including blank.) Spot 100 (xL
each stock and limit std solns on extreme spots (one on far
right and one on far left of plate). Spot 100 \iL //-heptane as
blank and 100 pL sample between std spots. Use same pipet
for all spots, rinsing thoroly with rc-heptane between appli-
cations. Keep size of spots uniform at 1.5-2 cm diam. by us-
ing following technic: Fill 100 fxL pipet past mark with soln
to be spotted. Carefully drain excess into absorbent towel until
soln is at exact vol. mark. Press pipet tip against exposed glass
in center of spotting mark on plate (hold pipet in vertical po-
sition at all times). Regulate size of spot by holding finger over
top of pipet and pressing tip tightly against plate. Blow across
spot (orally) only when necessary to regulate size of spot and
never lift pipet from place once spotting is begun.
Pour 10-1.5 mL n-heptane in tank trough, insert plate, and
seal tank. Develop until sol v. is within 2.5 cm from top of
plate (ca 30 min). Remove plate, air dry few min, and view
under UV light. Biphenyl appears as bright blue spot on yel-
low background.
If no biphenyl appears in sample, end analysis at this point.
If biphenyl is found, remove spots from plate without delay.
Score upper and lower extremes of biphenyl zone horizontally
across plate. Score vertical lines in adsorbent between bi-
phenyl spots to include approx. equal area in each rectangle,
scribing same area for ref. spot. Use razor blade to scrape off,
and discard all adsorbent below biphenyl zone and outside ex-
treme vertical lines. Use absorbent tissue and alcohol to clean
exposed glass thoroly. Carefully scrape adsorbent from one
extreme rectangular zone onto glazed paper and transfer to funnel
inserted in 10 mL vol. flask; do not use solv. to rinse paper.
Rinse off razor blade into funnel with small portion of alcohol.
Tip plate at angle to facilitate rinsing of scraped area into fun-
nel and rinse with several small portions alcohol. Rinse funnel
and finally dil. to vol. with alcohol. Shake mixt. vigorously
and let stand 5 min, shaking occasionally. Remove each bi-
phenyl spot same way, working inward from each side of plate
and cleaning and drying each previously removed zone. Filter
each mixt. thru Whatman No. 44 paper, or equiv., and store
filtrate in stoppered vessel for spectrophtric detn.
H. Spectrophotometry
Det. A of each soln at 248 and 300 nm in 1 cm cell with
alcohol as ref.
296
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
ppm Biphenyl = (AA 248 sample/ A A 24H std)
x (|xg std spotted/g sample spotted)
where AA 248 = A 2 4$ - [A 300 x (A 24 s blank /A 30o blank)].
Ref.: JAOAC 50, 934(1967).
CAS-92-52-4 (biphenyl)
959.10* 2-(p-ferf-Butylphenoxy)-
1-Methylethyl 2-Chloroethyl Sulfite
(Aramite) Pesticide Residues
Colorimetric Method
Final Action 1965
Surplus 1975
See 29.067-29.071, 12th ed.
957.14 Captan Pesticide Residues
Spectrophotometric Method
Final Action
(Applicable to firm fruits such as apples, pears, peaches, and
plums and to green vegetables)
A. Principle
Captan is extd from crop with benzene; H 2 0, color, and
appreciable amts of waxes are removed, and red color is de-
veloped by fusion of captan with resorcinol at 135°; color
changes to yellow on addn of HOAc.
B. Reagents
(a) Resorcinol. — Must be free of discoloration and pass fol-
lowing tests: Fuse 0.5 g and dissolve in 25 raL HOAc. A at
425 nm is <0.015, against HOAc. 1.00 g should not lose >2
mg in 4 hr over H 2 S0 4 ; if more is lost, dry over H 2 S0 4 until
test is satisfactory.
(b) Cleanup mix. — 10 parts Nuchar, 5 parts Hyflo Super-
Cel, and 5 parts anhyd. Na 2 S0 4 .
(c) Captan std solns. — (I) Stock soln. — 3 mg/mL. Trans-
fer 150 mg pure captan (available from Chevron Chemical Co.,
PO Box 4010, Richmond, CA 94804) to 50 mL vol. flask and
dil. to vol. with benzene. (2) Intermediate soln. — 300 |xg/
mL. Pipet 10 rnL stock soln into 100 mL vol. flask and dil.
to vol. with benzene. (3) Working soln. — 30 (xg/mL. Pipet
10 mL intermediate std soln into 100 mL vol. flask and dil.
to vol. with benzene.
C. Preparation of Sample
(Caution: See safety notes on flammable solvents, toxic sol-
vents, and benzene.)
(a) Fruits. — Accurately weigh ca 500 g sample into clean,
dry jar with screw cap faced with sheet cork gasket covered
with wet filter paper, or other sol v. -tight lid, and add 500 mL
benzene. Multiples of sample-to-benzene ratio can be used.
Agitate 15 min, drain benzene into container, and transfer to
separator. (Transfer to separator may be omitted where there
is no separable aq. layer.)
Transfer ca 100 mL sepd benzene layer to 250 mL g-s flask,
and decolorize and dehydrate with 3-4 g cleanup mix, (b), by
shaking vigorously ca 5 min. Filter thru folded paper, rejecting
first 10-15 mL.
(b) Green vegetables. — Chop sample in food chopper such
as Hobart Food Cutter, mix, and transfer 100 g to explosion-
proof blender. Add 200 mL benzene and blend 2 min; add 20
g anhyd. Na 2 S0 4 and blend 2 min more. Pour mixt. into 500
mL centrf. bottle, stopper with cork, and centrf. at ca 1400
rpm 5-10 min. Decant benzene layer into 250 mL g-s erlen-
meyer, add ca 6 g cleanup mix, (b)/100 mL benzene, and
shake vigorously ca 5 min. Filter thru folded paper, discarding
first 10 mL. If water- white soln does not result, repeat cleanup
treatment. Pipet 50 mL into 100 mL vol. flask and dil. to vol.
with benzene.
D. Determination
(Caution: See safety notes on pipets, toxic solvents, pesti-
cides, and benzene.)
Pipet 5 mL filtrate, 957.14C(a), or aliquot, (b), into 25 X
200 mm test tube and add 0.5 ± 0.1 g resorcinol. Heat 20
min in oil bath at 135 ± 5°, cautiously at first to evap. ben-
zene; then immerse reaction tubes to depth of ca 5 cm but do
not let them touch bottom of bath. Remove, and immediately
add 10-15 mL HOAc, followed by rapid immersion in H 2
at room temp. Transfer quant, to 25 mL vol. flask, using HOAc,
dil. to vol. with HOAc, and mix.
Det. A at 425 nm in 1 cm cell against HOAc within 1 hr.
Calc. ppm from std curve.
B. Preparation of Standard Curve
Prep, std curve simultaneously with samples. Pipet 0, 2, 4,
and 5 mL aliquots of working std soln into 25 x 200 mm test
tubes and add benzene to make total vol. of 5 mL in each tube.
Add 0.5 ±0.1 g resorcinol and continue as in detn, beginning
"Heat 20 min in oil bath . . ."
Note: One drop H 2 in reaction tube will cause apparent
loss of ca 20% captan. Do not leave benzene aliquots in un-
stoppered reaction tubes where condensation of moisture will
take place.
Refs.: JAOAC 40, 219(1957); 46, 143, 241(1963).
CAS- 133-06-2 (captan)
964.18 Carbaryl Pesticide Residues
Colorimetric Method
First Action 1964
Final Action 1965
A. Reagents
(a) Acetone. — Redistd.
(b) Coagulating soln. — Dissolve 0.5 g NH 4 C1 in 400 mL
H 2 contg 1 mL H 3 P0 4 .
(c) Color reagent. — Dissolve 25 mg p-nitrobenzenediazo-
nium fluoborate in 5 mL MeOH and add 20 mL HOAc. Prep,
just before use.
(d) Methylene chloride. — Redistd CH 2 C1 2 .
(e) Alcoholic potassium hydroxide soln. — 0. IN in MeOH.
(f) Polyethylene glycol soln. — Dil. 1 mL polyethylene gly-
col to 100 mL with CH 2 C1 2 .
(g) Carbaryl std solns. — Ref. std material is available from
Rhone-Poulenc Ag Co., 2 T.W. Alexander Dr, PO Box 12014,
Research Triangle Park, NC 27709. (/) Stock soln.— 0.5 mg/
mL. Place 50.0 mg in 100 mL vol. flask and dil. to vol. with
CH 2 C1 2 . (2) Intermediate soln. — 50 u.g/mL. Transfer 10 mL
stock soln to 100 mL vol. flask and dil. to vol. with CH 2 C1 2 .
(3) Working soln. — 5.0 |xg/mL. Transfer 10 mL intermediate
soln to 100 mL vol. flask and dil. to vol. with CH 2 C1 2 .
B. Apparatus
Evaporative concentrator. — See Fig. 964.18. Vac. mani-
fold connected thru stopcock to antisurge column, 250 x 19
mm od, contg glass marble, or Snyder column, attached to
AOAC Official Methods of Analysis (1990)
Carbaryl
297
FIG. 964.18— Evaporative concentrator. A, glass manifold. B,
pressure tubing. C, stopcock. D, adapter, ¥ 24/40. E, antisurge
column, 25 cm x 19 mm od. F, glass marble. G, erlenmeyer,
¥ 24/40, 250 ml
¥ 24/40 erlenmeyer. Use surgical tubing wherever contact with
sample is likely.
C. Preparation of Sample Solution
Transfer 50 g sample to high-speed blender and add 150 mL
CH 2 C1 2 and 100 g powd anhyd. Na 2 S0 4 . Blend at high speed
2 min and let settle 1 min. Decant solv. into 9 cm buchner
fitted with Whatman No. 1 , or equiv., paper covered with thin
coat of Hyflo Super-Cel, or equiv., filter aid. Cautiously apply
vac. until all solv. has filtered. Repeat extn with two 100 mL
portions CH 2 C1 2 . Treat combined filtrates as in (a) or (b):
(a) Transfer combined filtrates to 500 mL ¥ erlenmeyer and
add 1 mL polyethylene glycol soln. Connect to evaporative
concentrator, place flask in H 2 bath at 25-30°, and carefully
reduce pressure to ca 20 mm (2.7 kPa). After solv. evaps,
immediately disconnect antisurge column from manifold. Rinse
down walls of column and flask with 5 mL acetone from pipet,
swirl flask, and warm gently under hot H 2 tap 30 sec. Add
50 mL coagulating soln thru column, and swirl. Remove col-
umn, let mixt. stand 30 min, and filter with vac. thru 3 mm
layer of Super-Cel in No. 1 buchner. Wash flask and pad with
two 15 mL portions coagulating soln.
Transfer filtrate to 125 mL separator, add 25 mL CH 2 C1 2 ,
shake well, and let sep. completely. Drain lower layer into
¥ 250 mL erlenmeyer. Repeat extn of aq. layer with 25 mL
CH 2 C1 2 , adding ext to same 250 mL erlenmeyer. If combined
exts are cloudy, add 5-10 g granular anhyd. Na 2 S0 4 , and shake.
Decant solv. into clean 250 mL ¥ erlenmeyer, rinsing with
small portion CH 2 C1 2 . (If residue is expected to be >2 ppm,
dil. exts to vol. in 100 mL vol. flask, and use appropriate
aliquot.)
(b) Add 1 mL polyethylene glycol. Stopper, carefully re-
duce pressure to ca 150 mm, and warm on steam bath. When
vol. is ca 5 mL, remove from steam bath and swirl until dry.
Release vac, remove stopper, and let cool. Continue as in (a),
beginning "Rinse down walls ..." except column is not pres-
ent.
D. Determination
To soln in erlenmeyer add 1 mL polyethylene glycol soln
and connect to column and evaporator. Evap. solv. as before,
immediately disconnect, and remove column. Rinse down walls
of flask with 2 mL 0. IN KOH in MeOH from pipet, rotating
to ensure complete contact. Let stand 5 min, add exactly 17
mL HOAc, and with swirling add 1 mL color reagent. Let
stand exactly 1 min and det. A in 1 cm cell at 475 nm against
reagent blank processed along with sample as ref. Det. [xg
from std curve.
ppm Carbaryl
~ (|xg/g sample) x (diln factor if aliquot was used)
E. Preparation of Standard Curve
{Caution: See safety notes on pipets and pesticides.)
Pipet 0, 1,3, 5, and 10 mL aliquots working std soln to
500 mL ¥erlenmeyers, add 300 mL CH 2 C1 2 to each, and pro-
ceed as in 964. 18C, beginning "Treat combined filtrates as in
(a) or (b):"
Plot A against (xg carbaryl to obtain std curve.
Refs.: JAOAC 47, 283(1964); 48, 676(1965).
CAS-63-25-2 (carbaryl)
968.26 Carbaryl Pesticide Residues
Qualitative and Semiquantitative Method
Final Action 1976
(Applicable to apples and spinach)
A Reagents
(a) Adsorbent. — A1 2 3 G (contains 10% CaS0 4 ). See
970.52C(a).
(b) Coagulating soln. — See 964.18A(b).
(c) Chromogenic spray soln. — Sat. diethylene glycol-al-
cohol soln (1+9) with p-nitrobenzene-diazonium fluoborate
(practical grade, ca 25 mg/100 mL) by stirring ca 2 min. Fil-
ter, keep cold during use, and store in refrigerator. Do not use
after 3 days.
(d) Diethylene glycol soln. — Dil. 10 mL diethylene glycol
to 100 mL with redistd CH 2 C1 2 .
(e) Carbaryl std.— Up 141-142°. See 964.18A(g). Re-
crystallize from alcohol and H 2 0, if necessary.
B. Apparatus
(a) TLC apparatus. — App. suitable for 8 x 8" plates. See
970.52F.
(b) Evaporative concentrator. — Two chamber, ¥ 24/25,
micro-Snyder column (Kontes Glass Co. K-569001); with 10
mL Mills tube, graduated (Kontes K-570050).
C. Extraction and Cleanup of Sample
Transfer 25 g sample to blender. Add 150 mL CH 2 C1 2 and
100 g powd (150 g granular) anhyd. Na 2 S0 4 . Blend 2 min at
low speed and let settle. Attach 9 cm buchner contg rapid pa-
per to 500 mL filter flask. Cover paper with thin coat of Hyflo
Super-Cel prepd as slurry in CH 2 C1 2 . Decant ext into buchner
and cautiously apply vac. Rinse blender with 50 mL CH 2 C1 2
and filter. Return residue to blender. (Complete sepn of resi-
due from Super-Cel is unimportant.) Add 150 mL CH 2 C1 2 , re-
ext, filter, and rinse again with 50 mL CH 2 C1 2 . Add 1 mL
diethylene glycol soln to filter flask. Place flask with buchner
contg original filter pad attached on steam bath and apply vac.
When vol. in flask is ca 5 mL, remove flask from steam bath
and swirl until dry. Release vac, remove buchner, and let flask
cool.
Rinse down side of flask with 3 mL acetone from pipet and
swirl to dissolve residue. While gently swirling flask, add 15
mL coagulating soln and let stand >10 min with occasional
298
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
swirling. Filter, using vac, thru small fritted glass funnel, me-
dium porosity, contg ca 6 mm layer of Hyflo Super-Cel and
receive filtrate in 30 mL test tube. Wash ppt with three 2 mL
portions acetone-H 2 soln (1 + 9), letting each washing re-
main in contact with ppt ca 15 sec before applying vac. Trans-
fer filtrate and washings to 25 mL vol. flask, dil. to vol. with
acetone-H 2 soln (1 + 9), and mix.
D. Determination
{Caution: See safety notes on spraying chromatograms.)
Transfer 10 mL sample soln to 125 mL separator. Ext soln
with two 5 mL portions CH 2 C1 2 , shaking 5-10 sec each time.
Combine exts in Mills tube, add small SiC chip (<0.01 mL
vol.), fit with micro-Snyder column, and evap. to 0.1 mL on
steam bath. (Caution: Samples may be lost by vigorous ebul-
lition.)
Prepare 8 x 8" TLC plates coated with 250 (xm layer A1 2 3
adsorbent. Dry plates in forced-draft oven 30 min at 80°. Store
in desiccator cabinet. Using 1 |jlL pipet, spot aliquots equiv.
to 2 g sample and carbaryl stds (in CH 2 CI 2 ) to cover expected
range.
Place trough in chromatgc tank lined with blotting paper.
Add ca 50 mL acetone- benzene soln (1 + 4) to bottom of tank
to sat. atm., and then add 50 mL same soln to trough. Place
plate in trough and seal tank with masking tape. Develop plate
until solv. front just reaches line drawn 10 cm from origin.
Dry plate ca 15 min in hood. Spray moderately with 1 .ON ale.
KOH soln. Then spray moist plate with chromogenic soln. Blue
spot with Ri value same as std carbaryl spot indicates carbaryl
(/? r range, 0.52-0.60). Compare size and intensity of sample
and std spots for semiquant. estn of amt of pesticide. It is
possible to distinguish, for example, between 0.2 and 0.4 jxg,
but not between 0.3 and 0.4 \xg. Optimum range for quant.
estn is ca 0. 1-0.4 \xg. For amts >0.4 jxg, spot smaller aliquot
of remaining 80 |xL soln. Then spot same vol. of std soln for
valid comparison.
Ref.: JAOAC 51, 679(1968).
CAS-63-25-2 (carbaryl)
958.08* p-Chlorophenyl Phenyl
Sulfone Pesticide Residues
Spectrophotometric Method
Final Action 1965
Surplus 1969
See 29.075-29.081, 11th ed.
inated to its olefin. Ethyl an olefin is extd from reaction mixt.
into hexane and portion of hexane is injected into gas chro-
matograph with electron capture detector. Ethylan olefin pro-
duces 10-fold increase in electron capture detector response
over that of parent compd.
B. Reagents
(a) Hexane.— See 970.52B(j).
(b) Carborundum chips. — SiC, ca 20 mesh.
(c) Alcoholic potassium hydroxide soln.— Dissolve 2 g KOH
in 100 mL alcohol.
(d) Ethylan std soln. — 50 |mg/mL hexane.
(e) Ethylan olefin std soln. — 5 |ULg/mL hexane.
C. Apparatus
(a) Graduated centrifuge tube. — With No. 13 glass stopper
(Corning Glass Works, No. 8084, or equiv.).
(b) Oil bath. — 100 mL beaker contg 80 mL paraffin oil.
Heat on mag. stirrer hot plate calibrated to maintain oil temp,
at 100 ± 5°.
D. Determination
After electron capture GC of ethylan and other organochlo-
rine and organophosphorus pesticides, 970.52A-R, pipet en-
tire 6% eluate, 970.520, or aliquot contg <30 |xg ethylan into
reaction tube. Carefully evap. to dryness under gentle air cur-
rent. Add 2 mL ale. KOH soln and 2-5 SiC chips. Place re-
action tube in 100° oil bath to depth of ca L0 mL graduation
and let soln reflux 15 min. (Conduct reaction in hood. Air flow
thru hood will cool upper part of tube, which serves as con-
denser. Hood also removes odors escaping from hot oil.) Re-
move tube from oil bath, cool to room temp., and add 3 mL
H 2 0. Pipet vol. hexane (but 5:1 mL) into tube to give concn
ca 5 |xg ethylan olefin /mL. Shake vigorously ca 30 sec and
let layers sep. Det. ethylan olefin in hexane layer as in 970. 52R.
ppm Ethylan = (R/R') x (W F /W) x (307.25/270.78)
where R and /?' - responses to ethylan olefin in sample and
std, resp.; W — ng std injected; W = mg equiv. sample in-
jected; and 307.25 and 270.78 = MW ethylan and ethylan
olefin, resp.
Refs.: JAOAC 55, 1042(1972); 56, 721(1973).
CAS-72-56-0 (ethylan)
960.41 * DDT Pesticide Residues
Colorimetric Method
First Action 1960
Final Action 1965
Surplus 1980
973.39 Ethylan (Perthane)
Pesticide Residues
Gas Chromatographic Method
First Action 1973
Final Action 1974
(For low levels (less than ca 50 ng/GC injection) and for con-
firmatory quantitation of higher levels of ethylan previously
detd by 970.52A-R)
4. Principle
Method is extension of general method for multiple resi-
dues, 970.52A-R. After electron capture GC detn of ethylan
and other organochJorine and organophosphorus pesticides,
ethylan in 6% mixed ether eluate, 970.520, is dehydrochlor-
See 29.097-29.102, 13th ed.
965.36 Dichfone Pesticide Residues
Spectrophotometric Method
First Action 1965
Final Action 1966
(Applicable to fresh fruits and vegetables)
A. Reagents
(a) Dichlone std soln. — 0.2 mg/mL. Dissolve and dil. 40
mg dichlone (Eastman Kodak Co. No. 3836, or equiv.) to 200
mL with benzene.
(b) Dimethylamine. — 25% aq. soln (Eastman Kodak Co.
P601 or equiv.).
AOAC Official Methods of Analysis (1990)
Dodine
299
(c) FlorisiL— 60/100 mesh, PR Grade, activated at 1250°F
(Floridin Co.)- Heat ^4 hr at 130° and store in stoppered flasks
in desiccator prior to use.
B. Preparation of Standard Curve
Place 0, 1.00, 2.00, 3.00, 4.00, and 5.00 mL dichlone std
soln in 25 mL g-s graduates and dil. each to 10 mL with ben-
zene. To each graduate add isopropanol to 20 mL mark and
mix. Add 1 mL 25% Me 2 NH soln, dil. to 25 mL with iso-
propanol, and mix.
Read A of stds against blank in covered 1 cm cells at 495
nm, and plot A against mg dichlone (0-1.0 mg range). Color
is stable >1 hr.
C. Preparation of Cotumn
Fill 15 x 300 mm chromatgc tube, fitted with fritted glass
disk or glass wool plug, with Florisil to ca ] /3 its length. (No
stopcock is required.) Prewet Florisil with 30 mL benzene.
D. Determination
{Caution: See safety notes on distillation, flammable solvents,
toxic solvents, and benzene.)
Strip weighed sample (ca 1 kg) with 500 mL benzene by
gently turning or tumbling 10 min in suitable container (ca 4
L; 1 gal.). (Avoid breaking plant tissue.) Drain benzene into
1 L flask thru folded paper (ca 32 cm) contg ca 50 g anhyd.
Na 2 S0 4 .
Add 200 mL dried benzene strip soln to prepd chromatgc
column. Discard benzene eluate. Elute dichlone from column
with 100 mL acetone-benzene eluting mixt. (1 -f 99). Collect
eluate in beaker and evap. to ca 15 mL. (Do not let sample
overheat or go to dryness.) Rinse sample into graduate and dil.
to 20 mL with benzene. Develop color in 10 mL of this soln
as in 965. 36A.
mg Dichlone from std curve X 5
= ppm dichlone (for 1 kg sample)
If visible color is present in benzene eluate, simultaneously
develop color in remaining 10 mL aliquot, omitting Me 2 NH
and adding 1 mL H 2 0. Subtract this blank A from that of de-
veloped sample to correct for sample blank.
Ref.: J AOAC 48, 759(1965).
CAS- 117-80-6 (dichlone)
964.19 Dodine Pesticide Residues
Spectrophotometric Method
First Action 1964
Final Action 1965
(Applicable to apples, peaches, pears, pecans, and strawber-
ries)
A. Reagents
(a) Bromocresol purple soln. — Recrystallize indicator-grade
bromocresol purple from boiling toluene (ca 2 g/100 mL).
Dissolve 0.4 g recrystd material in 75 mL O.QXN NaOH; if
necessary, add addnl 0.01N NaOH to bring pH to 6.0-6.1.
Filter, if necessary, and dil. to 500 mL with C0 2 -free H 2 0.
Store in brown bottle.
(b) Buffer soln.—vM 5.5. Dissolve 15.2 g Na 2 HP0 4 .7H 2
and 74.0 g NaH 2 P0 4 .H 2 in C0 2 -free H 2 and dil. to 1 L.
(c) Dodine (DDGA) std solns.-(l) Stock soln. — 130 jxg/
mL. Dissolve 32.5 mg Ref. Std (available from American Cy-
anamid Co.) in MeOH and dil. to 250 mL with MeOH. (2)
Working soln. — 13 (xg/mL. Dil. 25 mL aliquot stock soln to
250 mL with MeOH.
B. Preparation of Sample
Grind sample in high-speed blender with MeOH-CHCl 3 (2
+ 1) in ratio of 400 mL solv./lOO g sample. Filter with suc-
tion thru 2 Whatman No. 1, or equiv., papers in buchner, and
wash pulp with MeOH-CHCL, (2 + 1), using 100 mL/lOO g
sample. Det. vol. of ext and transfer portion equiv. to 50 g
sample to 400 mL beaker.
C. Determination
Add several glass beads and 1 mL HC1 to beaker, and evap.
to 50 mL on steam bath. Add 30 mL 30% NaCl soln and 100
mL MeOH. Cool, transfer to 500 mL separator, and ext gently
with 50 mL CCJ 4 by inverting separator 6-8 times. Let phases
sep. and discard CC1 4 layer. Repeat with 50 mL CCI 4 , in-
verting separator ca twice as many times. Discard CC1 4 ; then
ext with 50 mL CC1 4 , shaking gently 30 sec. Finally, ext with
50 mL CC1 4 , shaking vigorously 1 min, and again discard CC1 4 .
Adjust pH of soln to ca 5.5 with 47V NaOH (pH meter), and
add 20 mL pH 5.5 buffer and 20 mL bromocresol purple soln.
Re-adjust pH to 5.5 and ext complex with two 50 mL portions
CHC1 3 , shaking 2 min each time. Shake combined ext 30 sec
with 25 mL pH 5.5 buffer, and transfer CHC1 3 layer to another
separator. Shake 1 min with 25 mL pH 5.5 buffer, let stand
10 min, and transfer CHCI3 to another separator. Shake with
20 mL 0.05N NaOH to remove all combined indicator and any
org. acids which may persist. Recomplex dodecylguanidine (in
CHCI3 as free base) by shaking 3 min with 5 mL bromocresol
purple soln and 20 mL pH 5.5 buffer. Wash CHC1 3 with three
15 mL portions pH 5.5 buffer, shaking 1 min each time. Transfer
CHC1 3 to dry 250 mL separator and shake 2 min with 20 mL
0.057V NaOH, measured by pipet. Read A of indicator in aq.
soln at 590 nm, using Beckman spectrophtr, or equiv. Obtain
jxg DDGA from std curve.
D. Preparation of Standard Curve
Add 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL std soln to series
of separators contg 100 mL MeOH, 50 mL H 2 0, 30 mL 30%
NaCl soln, 20 mL bromocresol purple soln, and 20 mL pH
5.5 buffer. Adjust pH of each soln to 5.5 and continue as in
964. 19C, par. 2, beginning "... and ext complex with two
50 mL portions CHC1 3 , shaking 2 min each time." Read A of
each aq. soln at 590 nm and plot against jxg DDGA. No blank
correction is necessary for stds.
ppm DDGA = jxg DDGA in aliquot/g sample in aliquot
Ref.: J AOAC 47, 300(1964).
CAS-2439-10-3 (dodine)
986.20 Ethylene Dibromide in Grains
and Grain Products
Gas Chromatographic Method
First Action 1986
Final Action 1987
A. Principle
Whole grains and intermediate grain-based products are extd
by soaking in acetone-H 2 0; ready-to-eat products are extd by
hexane co-distn. Portions of exts are dried and analyzed by
gas chromatgy with electron capture detection.
B. Reagents
(a) Solvents. — 2,2,4-Trimethylpentane (isooctane), ace-
tone, and hexane (all pesticide quality). Check for interfer-
300
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
ences by injecting 5 |xL into GC system operated as described
under Apparatus .
(b) Calcium chloride. — Analyzed reagent grade, anhyd., 8
mesh.
(c) Sodium sulfate. — Analyzed reagent grade, anhyd.,
granular.
(d) Sulfuric acid (coned). — Analyzed reagent grade.
(e) Std solns. — (7) Stock soln. — Prep, in 50 mL vol. flask
equipped with Teflon-lined screw cap. Add ca 40 mL isooc-
tane to flask and weigh flask + isooctane to nearest 0.1 mg.
Introduce 20 |xL pure ref. std (EPA Ref. Std P480) into isooc-
tane and re- weigh to det. wt of EDB. Dil. to vol. with isooc-
tane and calc. concn in [xg/mL. Store in freezer. (2) Working
std soln. — Prep, in hexane by serial diln of stock soln to final
concn of ca 4 pg/|xL. Store in glass container with Teflon-
lined screw cap. Store in refrigerator or freezer when not in
use.
C. Apparatus
(a) Volumetric flasks . — 50 mL with Teflon-lined screw caps
(Thomas Scientific Co., No. 0319-037).
(b) Soaking vessels. — 250 mL erlenmeyers with Teflon-lined
screw caps (Thomas Scientific Co., No. 4903-K23) or 250 mL
media bottles with Teflon-lined screw caps.
(c) Teflon liners. — For erlenmeyers (Thomas Scientific Co.,
No. 2390-H82).
(d) Test tubes. — 15 mL with Teflon-lined screw caps
(Thomas Scientific Co., No. 9212-K42).
(e) Centrifuge. — For use with test tubes.
(f) Distilling trap. — Barrett, 20 mL graduated, 24/40
(Thomas Scientific Co., No. 7133-K44).
(g) Condenser. — Friedrich, 24/40 (Fisher Scientific Co.,
No. 07-744-5).
(h) Heating mantle.— To fit 500 mL r-b flask.
(i) Variable electric transformer. — 0-120 V (Powerstat
Model 1 168, or equiv.)
(j) Magnetic stirrer. — Thermolyne Model (7200 Barn-
stead/Thermolyne Corp, 2555 Kerper Blvd, Dubuque, I A
52001) or equiv., with mag. stirring bar, 1 X 5 / ]6 in.
(k) Gas chromatograph. — Equipped with 1.8 m X 4 mm
glass column packed with 10% SP-1000 on 80-100 mesh Su-
pelcoport (Supelco, inc., No. 1-1872), and const current 63 Ni
electron capture detector (Hewlett-Packard 5730 or equiv.) op-
erated under following conditions: temps — injector 200°, oven
1 15°, detector 350°, CH 4 -Ar (5 4- 95) carrier gas 40 mL/min.
Adjust attenuator to give V2 FSD for 20 pg EDB. Retention
time of EDB is ca 4 min. Condition new GC column by hold-
ing at 60° for 2 h with 40 mL/min carrier gas flow. Slowly
increase temp, to 200° and hold overnight. Cool to 115°, let
equilibrate, and check EDB response. If proper sensitivity is
not achieved, longer conditioning at 200° may be required.
D. Extraction and Cleanup
Store all samples in freezer until just before analysis.
(a) Whole grain and intermediate processed grain prod-
ucts. —Weigh 50 g into appropriate soaking vessel. Add 150
mL acetone-H 2 (5 + 1) and seal with Teflon-lined screw cap.
Swirl; let whole grains soak 48 h at 20-25°, with intermittent
swirling. Follow same procedure for intermediate products,
except soak 16 h. Using disposable pipet, transfer ca 10 mL
supernate into 15 mL test tube, add 1-2 g anhyd. 8-mesh CaCL,
secure Teflon-lined screw cap, and shake 2 min. If all CaCl 2
dissolves, add more and shake again. Let stand 30 min or centrf.
Proceed with Determination.
(b) Ready-to-eat products. — Weigh 20 g into 500 mL r-b
flask. Add 150 mL H 2 and stirring bar. While cooling flask
under stream of cold tap H 2 or swirling in ice-H 2 bath,
slowly add 25 mL coned H 2 S0 4 . Stopper flask to prevent loss
of EDB. Pipet 10.0 mL hexane into flask immediately before
co-distn. Connect r-b flask to Barrett trap and Friedrich con-
denser. Place flask in heating mantle on top of stirrer and turn
on stirrer. Co-distil hexane and EDB into Barrett trap, using
heating mantle with transformer set at 75% full power. Con-
tinue distn until 1-2 mL H 2 is collected. Remove flask from
heating mantle to prevent further H 2 distn. Drain lower H 2
layer and discard. Drain hexane into 15 mL test tube, add 2—
3 g anhyd. Na 2 S0 4 , secure Teflon-lined screw cap, and shake
vigorously 1 min. Let stand 30 min or centrf. Proceed with
Determination. When flask is cool to touch, pipet second 10.0
mL hexane portion into flask and distil as before. Repeat distn
third time.
Calculation of equivalent sample weight. — (/) Whole grains
and intermediate processed grain products.
mg sample equiv. /(xL final ext = 50/125 = 0.4
where 50 and 125 == g sample weighed and mL acetone added
(H 2 is removed), resp.
(2) Ready-to-eat products:
mg sample equiv. /uX final ext = 20/10 = 2
where 20 and 10 = g sample weighed and mL hexane added
(not recovered), resp. Each distn uses same calcn.
E. Determination
Inject 5 (jlL dried ext into gas chromatograph operated as
specified under Apparatus. Quantitate EDB by comparison of
peak hts or integrator counts from sample and appropriate std.
If EDB response is >100% FDS, quant, dil. sample with hex-
ane to achieve appropriate on- scale response. Sum EDB amts
found in each distn of ready-to-eat products to obtain total.
Limits of quantitation are 2 ppb EDB for whole grains and
intermediate products, 0.4 ppb EDB for ready-to-eat products.
Ref.: JAOAC 69, 847(1986).
CAS-106-93-4 (ethylene dibromide)
978.1 6 Ethylenethiourea
Pesticide Residues
Gas Chromatographic Method
First Action 1978
Final Action 1980
(Applicable to potatoes, spinach, applesauce, and milk. Cau-
tion: See safety notes on chloroform and methanol.)
A. Apparatus
(a) Chromatographic tube. — Glass, 300 X 22 (id) mm, with
coarse fritted disk and Teflon stopcock.
(b) Filter paper. — Sharkskin (Thomas Scientific, or equiv.).
(c) Gas chromatograph. — With flame photometric detector
(Meloy Laboratories, Inc., 6715 Electronic Dr, Springfield,
VA 22151, or equiv.) contg S filter and 1.8 m X 4 (id) mm
coiled glass column packed with 5% Carbowax 20M plus 2.5%
KOH (prepd in MeOH) on 80-100 mesh Chromosorb VV(HP).
Condition new column 2 days at 210°. Typical operating con-
ditions — temps (°): column 180, injection port 185, detector
185; flow rates (mL/min): N carrier gas 60, O 15, air 125, H
200; electrometer sensitivity 1 x 10" 9 amp full scale deflection
with 1 mv recorder. Approx. retention time of S-butylated ETU
under these conditions is 4 min; 12 ng gives ca 50% full scale
deflection. Change glass wool plug in injection port daily be-
fore use, and clean out inside of column at injection port weekly.
AOAC Official Methods of Analysis (1990)
Ethylenethiourea
301
(d) High-speed blender . — Waring Blendor, orequiv. {Cau-
tion: See safety notes on blenders.)
(e) Pipets. — Disposable glass capillary pipets, 145 x 6 (id)
mm (Arthur H. Thomas Co., or equiv.).
(f) Rotary evaporator. — Use with 150 mm ¥ 24/40 Vi-
greux column and place vac. release valve in line.
(g) Silanized glass wool. — Applied Science Laboratories,
Inc., or equiv.
B. Reagents
(a) Aluminum oxide. — Fisher No. A-540, or equiv., 80-
200 mesh, for chromatgc adsorption. (Available from Fisher
Scientific Co. as "Alumina, Adsorption, Fisher.") Use as re-
ceived.
(b) l-Bromobutane. — Fisher Scientific Co., or equiv. Re-
distil between 101 and 101.5°.
(c) Diatomaceous earth. — Celite 545. Do not acid-wash.
(d) Eluant.— 4% alcohol in CHC1 3 . Dil. 40 mL alcohol to
1 L with CHC1 3 , and mix well.
(e) Solvents. — CHCI3, MeOH, and toluene, distd in glass
(see statement regarding solvs, 970. 52A).
(f) Ethylenethiourea std solns. — (7) Stock soln. — 10 (xg
ETU/mL. Transfer 100 mg ETU ref. std (available from Pes-
ticides and Industrial Chemical Repository (MD-8), Environ-
mental Protection Agency, Research Triangle Park, NC 27709)
to 100 mL vol. flask, and dil. to vol. with H 2 0. Pipet 1 mL
this soln into another 100 mL vol. flask, and dil. to vol. with
H 2 0. Prep, fresh monthly. (2) Working soln.— 2 juug ETU/
mL. Pipet 10 mL stock soln into 50 mL vol. flask, and dil.
to vol. with H 2 0. Prep, fresh weekly.
C. Extraction
(Samples must be started and completed on same day.)
(a) For crops, canned goods, and milk. — Blend 100 g
chopped crop (vegetables and fruits) or 100 g milk, 150 mL
H 2 0, 15 g NaCl, 10 g diat. earth, (c), and 200 mL MeOH in
high-speed blender 2 min. Filter with vac. thru 1.3 cm bed of
diat. earth spread dry and evenly on 9 cm double sharkskin
filter paper in 91 mm (id) buchner. Transfer 87 mL (20 g)
aliquot to previously weighed 500 mL ¥24/40 r-b flask. Add
50-70 mL MeOH, insert Vigreux column into flask, and cone.
on rotary vac. evaporator immersed in 60-65° H 2 bath. If
substantial initial frothing occurs, add 4-5 drops octanol. If
much frothing occurs during last stages of concn, add addnl
4-5 drops octanol, 25 mL alcohol, or both. Cone, to ca 10 g.
Disconnect flask, weigh, and add enough H 2 to bring wt to
13 g. Proceed immediately as in 978. 16D.
(b) Optional extraction for crops. — (Applicable when pres-
ence of parent ethylenebisdithiocarbamate (EBDC) fungicide
is suspected in sample. Provides measure of potential ETU
residues which may be converted from EBDCs in home cook-
ing.) Place 100 g chopped crop, 150 mL H 2 0, and 1 mL NH 4 OH
in 1 L beaker, and record total wt. Cover with large watch
glass, place on 600-720 watt hot plate turned to high, and heat
15 min, reducing heat to low after initial boiling. Cool, re-
move watch glass, and reweigh. Add H 2 to beaker to restore
to original wt. Transfer quant, to high-speed blender, using
200 mL MeOH. Add 10 g diat. earth, (c), and blend 20 sec.
Proceed as in (a), beginning "Filter with vac. thru 1 .3 cm bed
. . .", except cone, sample to ca 8 g and add H 2 to bring wt
to 10 g. Proceed immediately as in 978. 16D.
(c) Optional extraction for milk. — (Applicable when pres-
ence of parent EBDC fungicide is suspected in sample.) Place
100 g milk, 25 mL H 2 0, 5 g NaCl ? and 1 mL NH 4 OH in 1 L
beaker, and record total wt. Proceed as in (b), beginning "Cover
with large watch glass, ..." except use 275 mL MeOH, stir
thoroly 1 min, and, after filtering, transfer 80 mL (20 g) ali-
quot to r-b flask. Proceed immediately as in 978. 16D.
D. Cleanup
Add 10 g Gas-Chrom S to sample ext, stopper, and shake
vigorously until lump-free (30-60 sec), tapping on cork ring,
if necessary. Add 50 mL eluant, (d), stopper, and shake 1-2
min. Pour mixt., including as much of Gas-Chrom S as pos-
sible, into chromatgc tube, 978.16A(a), contg 4-5 g Al 2 3 ,
(a), held in place with 1.3 cm glass wool plug. Rinse flask
with 3 addnl 50 mL portions eluant, adding each rinse to tube.
Collect eluate in 500 mL J 24/40 r-b flask contg 10 mL H 2
and 20 mL alcohol. Cone, eluate to ca 20 mL using rotary
vac. evaporator with Vigreux column. Add 5 mL H 2 and 20
mL alcohol, and cone, to 10 mL to eliminate CHC1 3 . Rinse
column and flask with 5 mL H 2 followed by 20 mL alcohol .
Proceed immediately as in 978. 16E.
E. Derivatization
(a) Sample. — To sample flask add 1.5 g KOH, 2 mL 1-
bromobutane, (b), and 5-6 boiling chips. Reflux on pre-heated
hot plate 10 min, using cold H 2 condenser and clamp to sup-
port flask. Cool, and transfer quant, to 250 mL separator with
10 mL H 2 followed by 50-60 mL CHC1 3 . Shake 1-2 min,
and let layers sep. completely (ca 5 min). Collect lower CHC1 3
layer in clean 250 mL I 24/40 r-b flask. Add 2 drops HC1
and evap. to near dryness on rotary vac. evaporator, using Vi-
greux column and 60-65° H 2 bath. Rinse neck of flask with
2-3 mL MeOH, and evap. again on rotary evaporator. Re-
move flask and evap. to dryness, using air jet for 2-3 min.
All MeOH must be removed by this final evapn or it will in-
terfere with GC detn. Remove flask from air jet, pipet in I
mL toluene and add 1.0-1.5 mL 10% KOH, stopper, and shake
1-2 min. Remove 0.5-0.7 mL of toluene layer with dispos-
able pipet, (e), without collecting any of lower aq. layer. Place
toluene sample ext in clean, dry calibrated test tube, and re-
cord vol. At this point, 1 mL ext is equiv. to 20 g sample.
However, if final ext is dild for GC, vol. recorded with respect
to original 1 mL toluene must be used for sample wt. calcns:
Wt sample in final diln
= [(vol. obtained from 1 mL)/(l mL toluene)] X 20 g
Do not cone, sample ext at this point due to volatility of
butyl derivative of ETU.
(b) Standard. — Pipet 1 mL ETU working std soln into 500
mLl 24/40 r-b flask. Add 15 mL H 2 0, 20 mL alcohol, 1.5
g KOH, 2 mL 1-bromobutane, and 5-6 boiling chips, and pro-
ceed as in (a), beginning "Reflux on preheated hot plate . . . ".
F. Determination
Initially, inject 4-6 \xL std ext, 978.16E(b), into gas chro-
matograph, and then inject 5 \xL sample ext, 978.16E(a). Ad-
just injection vol. of sample ext until peak hts of std and sam-
ple are approx. equal, and continue with alternate injections
of sample and std. (S detector is non-linear; therefore, do not
prep, std curve.)
ETU, ppm - (PH/PH') x (W /W)
where PH = peak ht of sample, PH' - peak ht of std, W —
ng ETU in std aliquot, and W = mg sample represented by
sample aliquot.
G. Regeneration of Gas-Chrom S
Shake all used Gas-Chrom Strom cleanup column, 978. 16D,
into large beaker or flask. Discard A1 2 3 and glass wool plug.
Wash thoroly with H 2 0, and decant after each wash. Wash
thoroly with MeOH, decant, and vac. dry in large buchner.
302
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
Air dry in hood and transfer to 80° oven overnight. Gas-Chrom
S may now be re- used.
Refs.: JAOAC60, 1105, 1111(1977).
CAS-96-45-7 (ethylenethiourea)
963.23 Glyodin Pesticide Residues
Spectrophotometry Method
First Action 1963
Final Action 1964
(Applicable to apples and pears. Not applicable to fruits with
extensive softening or decomposition. All glassware must be
free of soap or detergent.)
A. Reagents
(a) Bromophenol blue soln. — Prep, just before use. Trans-
fer 50 mg bromophenol blue powder into 500 mL vol. flask
with small amt of H 2 0. Add 2 mL HO Ac and swirl until dye
is completeJy dissolved. Oil. to vol. with H 2 0, and mix.
(b) Glyodin std solns. — Prep, from 2-heptadecyl glyoxali-
dine, purified grade. (7) Stock soln. — 1 mg/mL. Dissolve 100.0
mg 2-heptadecyl glyoxalidine in CHC1 3 in 100 mL vol. flask,
dil. to vol. with CHC1 3 , and mix. (2) Working soln. — 0.05
mg free base/mL. Transfer 5.0 mL stock soln to 100 mL vol.
flask, dil. to vol. with CHC1 3 , and mix.
B. Preparation of Standard Curve
Add 0, 2, 4, 6, 8, and 10 mL working std soln to six 50
mL vol. flasks. Add exactly 1 mL HO Ac to each flask and
dil. to vol. with CHC1 3 . Place 25 mL of each std, measured
in graduate or fast-flow pipet, in 125 mL separator. Add 25
mL bromophenol blue soln, (a), from graduate or fast-flow
pipet to each separator, and shake vigorously 1 min. Let sep.
>20 min. Filter CHC1 3 layer thru pledget of glass wool in stem
of separator into small g-s erlenmeyer. Det. A at 415 nm in
spectrophtr, using 1 cm cells and std as ref. Plot A against
mg 2-heptadecyl glyoxalidine.
C. Preparation of Sample
Fill tared wide-mouth gal. (3.8 L) glass jar with whole fruit
so that little or no slack is present (to prevent battering of fruit).
Weigh, and add 250 mL isopropanol. Screw cap on tightly
with double thickness of cellophane placed over mouth of jar
before cap is screwed on to help prevent leakage. Tumble or
shake 10 min. Filter into 500 mL vol. flask thru small layer
of glass wool in funnel. Drain off as much liq. as possible.
Repeat stripping with second 250 mL portion of isopropanol,
and filter into vol. flask. Wash glass wool and funnel with
small portions of isopropanol and dil. to vol.
D. Determination
Transfer 25 mL aliquot of strip soln to 50 mL beaker and
evap. to dryness on steam bath under air jet. To residue add
exactly 1 mL HOAc, allowing acid to drip slowly down sides
of beaker so that all residue is wetted. Cover beaker with watch
glass and heat gently on steam bath with swirling, until residue
at bottom loosens and disintegrates. Thoroly rinse down sides
with few mL CHC1 3 and transfer to 50 mL vol. flask. Rinse
beaker with 4 addnl small portions CHC1 3 , and transfer to voL
flask. Dil. to vol. with CHC1 3 (disregard turbidity and slight
color in soln).
Transfer 25 mL CHC1 3 soln, measured in graduate or fast-
flow pipet, to 125 mL separator. Proceed as in 963. 23B, be-
ginning, "Add 25 mL bromophenol blue soln, (a), . . ."
Perform detns along with prepn of std curve, using std as
ref. when detg sample A. Det. amt of 2-heptadecyl glyoxali-
dine in aliquot from std curve.
Glyodin (2-heptadecyl glyoxalidine acetate)
= 2-heptadecyl glyoxalidine x 1.195
Ref.: JAOAC 46, 238(1963).
CAS-556-22-9 (glyodin)
977.19 Hexachlorobenzene and Mirex
Pesticide Residues in Fatty Products
Gas Chromatographic Method
First Action 1977
Final Action 1978
A. Reagents
(a) Solvents. — Hexane, CH 2 CL, CH 3 CN, and pet ether. See
970.52B
(b) Florisil. — 60-100 mesh PR grade. (7) [Inactivated, for
partition chromatography . — Use as received from manufac-
turer. (2) Activated.— See 970.52B(i).
(c) Eluant mixture . — For Florisil column cleanup. Dil. 200
mL CH 2 C1 2 with hexane. Let reach room temp, and adjust to
1 L with hexane.
B. Apparatus
(a) Gas chromato graph. — With electron capture detector.
See 970.52H(a) and (c).
(b) Column. — 1.85 m X 4 (id) mm glass column with 80-
100 mesh Chromosorb W (HP) support, N flow 120 mL/min,
and injection temp. 220°. For HCB analysis, use 15% OV-210
liq. phase; for Mirex, 3% OV-101 . For HCB, use column temp.
180°, detector, 200°; for Mirex, use column temp. 210°, de-
tector 220°. Operate 3 H electron capture detector at dc voltage
which produces half scale deflection for 0.5 ng HCB or Mirex
when electrometer sensitivity is 1 x 10~ 9 amp. Or, operate
63 Ni detector to produce stable, reproducible, linear response
and adjust amt of injected sample, 970. 52R, to accommodate
difference in instrument sensitivity.
(c) Chromatographic tubes. — (1) Plain. — 250 x 22 (id) mm.
(2) With stopcocks.— See 970.52E(b).
(d) Kude ma-Danish concentrators. — See 970.52E(e).
(e) Micro-Vigreux Column. — See 970.52E(h). Use for concn
to vols < 5 mL.
C. Extraction of Fat
Ext >3 g fat as in 970. 52L. For products reported on fat
basis, use 3 g fat. For products reported on as-is basis, record
wt, W, of fat extd. Corresponding wt sample analyzed = (wt
fat taken for cleanup/ W) x wt original sample.
D. Cleanup
(Caution: See safety notes on acetonitrile and petroleum ether.)
Weigh 3 g fat into 250 mL beaker, add 20 g unactivated
Florisil, and stir with spatula or glass rod until free-flowing
powder is obtained. Place glass wool plug in bottom of plain
chromatgc tube and add 3 g unactivated Florisil. Completely
transfer fat-Florisil mixt. to tube. Settle column by repeatedly
tapping tube. Place glass wool plug on top of Florisil. Place
1 L separator under column as receiver. Elute with 150 mL
CH 3 CN by gravity.
When elution is complete, add exactly 100 mL pet ether to
separator, and shake vigorously 1—2 min. Add 10 mL satd
NaCl soln and 500-600 mL H 2 0, and shake vigorously 1 min.
Let sep. and discard aq. (lower) layer. Wash pet ether with
AOAC Official Methods of Analysis (1990)
Hexachlorobenzene
303
two 100 niL portions H 2 0. Discard washings, transfer pet ether
to 100 mL g-s graduate, and record vol., P. Calc.
Wt sample in eluate
= (wt sample taken for cleanup x P)/100
where 100 = mL pet ether added.
Complete cleanup on column of activated Florisil, 970.520,
using amt Florisil detd from 1 auric acid test, 970.52B(i). Sam-
ple ext must be dry and free from polar solvs when placed on
column. Elute at ca 5 mL/min with 200 mL eluant mixt., (c),
and cone. Det HCB and Mirex as in 970. 52R, using column,
977.196(b)
Refs.: JAOAC 58, 557(1975); 60, 229(1977); 63, 277(1980).
CAS-1 18-74-1 (hexachlorobenzene)
CAS-2385-85-5 (mirex)
980.22 Hexachlorobenzene and Mirex
Pesticide Residues in Adipose Tissue
Gas Chromatographic Method
First Action 1980
Final Action 1982
(Caution: See safety notes on potassium hydroxide, pesticides,
hexane, and pyridine.)
A. Reagents
(a) Hexane, 2-propanol. — See sol v. purity test, 970.52B.
(b) Sodium sulfate. — Anhyd. granular, Soxhlet-extd with
hexane.
(c) Sodium sulfate. — 2% aq. soln.
(d) Florisil column. — Packed with 100 mm Florisil and 12
mm Na 2 S0 4 on top. Hold in 130 ± 2° oven >16 hr prior to
use. (Remove stopcocks before placing columns in oven.) Pre-
wash with 50 mL hexane immediately before use. Predeter-
mine HCB and mirex elution on each lot of Florisil.
(e) "Keeper" soln. — 1% paraffin oil in hexane.
(f) Pyridine. — Burdick and Jackson, or equiv., suitable for
liq. and gas chromatgy.
(g) Potassium hydroxide soln. — 10% KOH in 2-propanol.
B. Apparatus
(a) Gas chromato graph. — With 3 H or 63 Ni electron capture
detector and 1.8 m x 4 mm id borosilicate glass columns packed
with 1.5% OV-17/1.95% OV-210 or 5% OV-210 on 80-100
mesh Gas-Chrom Q. Typical operating conditions . — Carrier
gas flow rate 60 mL/min, column temp. 200° (OV-17/OV-
210); 180° (5% OV-210).
(b) Glassware. — 300 X 25 mm od Chromoflex column for
Florisil (Kontes, K-420530), Kuderna-Danish (K-D) concen-
trator assembly (K-570000) fitted with 25 mL graduated tube
(K-570050, size 2525, 19/22, 25 mL), modified micro Snyder
column (K-569250), disposable pipets.
C. Determination
Accurately weigh ca 0.5 g rendered or extd fat in tube. Dis-
solve fat in ca 0.5 mL hexane and transfer quant, to Florisil
column pre-washed with hexane. Rinse tube with two 0.5 mL
portions hexane. Let column elute until sol v. level is just at
top of Na 2 S0 4 . Rinse column sides with 2-3 mL hexane. Elute
with 200 mL hexane at 5 mL/min.
Collect eluate in K-D flask equipped with 25 mL concen-
trator tube. Evap. on steam bath to >:10 mL. Use warm (50°)
H 2 bath and gentle N stream for further vol. reduction.
Inject 3-8 (xL stds and samples into OV-17/OV-210 col-
umn. Alternatively, OV-210 column at 180° may be used for
mirex quantitation. Adjust sample vol. as required to produce
major peak responses. Peak hts of stds and samples should not
vary >25%. Do not quantitate <20% full scale deflection. Amts
injected must fall within linear range of detector. Work at same
attenuation .
D. Confirmation of HCB
Prep, sample for derivatization by evapg hexane ext to 0. 1-
0.2 mL with warm H 2 bath and gentle N stream. Derivative
^3 stds along with samples. (HCB stds must bracket HCB
concn in samples as detd by initial GC analysis. Response of
HCB stds must be linear.) Add 2-3 drops 1% paraffin oil in
hexane to stds as "keeper" before evapg to 0.1-0.2 mL.
Add 0.5 mL 10% KOH in 2-propanol and 0.2 mL pyridine
to each sample and std. Gently tap tube to mix. Place modified
micro Snyder columns on concentrator tubes and place tubes
in boiling H 2 bath 45 min. Remove tubes and cool under cold
tap H 2 0. Add ca 10 mL 2% Na 2 S0 4 soln to each tube and
exactly 2 mL hexane. Mix vigorously 1 min. Let sol v. phases
sep. completely. Inject 3™ 8 u,L hexane phase into gas chro-
matograph for quantitation. Adjust hexane vol. as required.
Calc. concn HCB. Mirex will not be recovered thru derivative
procedure.
Refs.: JAOAC 63, 1128(1980); 64, 531(1981).
CAS-1 18-74-1 (hexachlorobenzene)
CAS-2385-85-5 (mirex)
957.15* Malathion Pesticide Residues
Colorimetric Method
First Action
Surplus 1974
See 29.116-29.120, 12th ed.
963.24 Maleic Hydrazide
Pesticide Residues
Spectrophotometric Method
First Action 1963
Final Action 1965
(Applicable to whole, dehydrated mashed, and frozen french
fried potatoes, and potato chips; whole cranberries, onions,
and peaches; and tobacco dust)
A. Principle
Sample is boiled in alk. soln to drive off volatile basic in-
terferences. Distn with Zn with N sweep expels hydrazine lib-
erated from maleic hydrazide. Hydrazine is reacted in acid soln
with p-dimethylaminobenzaldehyde to form yellow compd.
B. Apparatus
(a) Distillation apparatus. — See Fig. 963.24. Flask is 300
mL capacity, flat- bottom, double thickness, with thermometer
well. Thermometer is 90-220° (Tinius Olsen No. 718636
"Yellow Bak," or equiv. in temp, range and length; available
on special order from Accuracy Scientific Instrument Co., 335
E Chew Ave, Philadelphia, PA 19120). Use 5" wire gauze
with 4" diam. asbestos center. Centrf. tube receiver (50 mL)
is graduated in 1 mL divisions.
(b) Spectrophotometer. — Beckman Model DU, or equiv.
C. Reagents
(a) p-Dimethylaminobenzaldehyde soln. — Dissolve 2 g in
100 mL 17V H 2 S0 4 . Soln is stable.
304 Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
WATER RESERVOIR
50 ML
CENTRIFUGE
TUBE
FIG. 963.24— Distillation apparatus for maleic hydrazide
determination
(b) Zn granules. — "10 mesh."
(c) Maleic hydrazide std soln. — 10 fxg/mL. Dissolve 0.0100
g maleic hydrazide in 100 mL 0. IN NaOH and dil. to 1 L.
Soln is stable.
D. Preparation of Sample
Grind sample to soup-like consistency in high-speed blender,
adding measured wt of H 2 if necessary. Prepd samples may
be frozen for storage.
E. Determination
Transfer wt ground sample specified in Table 963.24 to 300
mL distn flask. Dry socket neck joint, and add 50 g NaOH
pellets, antifoam agent indicated in Table 963.24, and 40 mL
H 2 0. Add 1 mL high bp oil to thermometer well and insert
thermometer. Heat flask on high-temp, hot plate and swirl ca
Table 963.24 Sample Weights of Commodities and Use of An-
tifoam Agents
Commodity
Wt Sample,
9
Antifoam Agent
Cranberries
(whole)
French fries
(frozen)
Mashed potatoes
(dehydrated)
Onions (whole)
Peaches (whole)
Potatoes (whole)
Potato chips
Tobacco (dust)
2.0
1.0
1.5
4.2
5.0
2.5
1.1
1.0
1 g paraffin wax + 1 mL Antifoam A
none
1 g paraffin wax + 1 mL Antifoam A
1 mL Antifoam A
1 g paraffin wax + 1 mL Antifoam A
0.5 mL vegetable oil
none
1 g paraffin wax + 1 mL Antifoam A
every 20 sec until pellets dissolve and gentle boiling begins.
When temp, reads 160°, (180° with cranberries), remove flask
and let cool 5 min. Wipe socket joint clean and dry; add 0.5
g ferrous chloride and 5 mL (equiv. to ca 5 g) Zn.
Quickly grease socket joint with light film of high-vac. sil-
icone grease and attach flask to app. (Fig. 963.24). Center
flask firmly on asbestos pad. Place 4 mL /?-dimethylamino-
benzaldehyde soln in 50 mL centrf. tube (ice-cooled) and im-
merse condenser tip. Adjust N flow {dry N) to 3 bubbles/sec
in receiver. With rapid flow of condenser H 2 0, heat flask with
Bunsen burner, centering tip of outer cone of flame on asbes-
tos pad. When boiling begins, adjust distance of burner so
foaming contents fill ca 2 / 3 of flask. Distil until temp, reads
173°, slowly add H 2 from reservoir until temp, drops to 168°,
turn off H 2 0, and distil to 173°. Continue H 2 6 addn and distn
at these temps until receiver contains ca 40 mL. Remove re-
ceiver. (If during distn receiver soln becomes turbid or ppt
appears, add 2 drops H 2 S0 4 and shake.)
Record vol. of distillate and det. A at 430, 460, and 490
nm, using 1 cm cells and 4 mL p-dimethylaminobenzaldehyde
soln dild to 40 mL as ref.
After distn, remove hot distg flask from app. with heat-re-
sistant gloves, remove thermometer, and seal well with small
cork. Rinse N-H 2 inlet tube free of caustic with HC1 from
plastic squeeze bottle followed by H 2 0. Then (with gloves and
safety glasses) pour molten contents of distg flask into Fe can
in sink to trap Zn granules. Rinse flask 3 times with H 2 and
2 times with HO to remove encrusted caustic and Zn granules.
Fill flask with HC1 (1 + 9) to remain until next use. Rinse 3
times with H 2 before reuse. (Careful removal of all Zn gran-
ules with HO is essential because residual Zn would cause
premature destruction of maleic hydrazide in precook of next
sample. Because of corrosion by the caustic soln, flasks may
last for only ca 30 detns.)
F. Preparation of Standard Curve
To clean 300 mL distn flasks, add 50 g NaOH pellets, 40
mL H 2 0, and std soln equiv. to 0, 5, 10, 20, 30, 50, 100,
150, and 200 |xg maleic hydrazide. Precook, distil, and mea-
sure A as in 963. 24E. Det. A A for each std as follows:
AA - [A 4
((A 430 + v4 490 )/2)] X mL color soln/40
Plot A A of each std against \xg maleic hydrazide to obtain std
curve. If desired, derive simple factor from curve slope, K 7
converting A A to (xg maleic hydrazide; thus, \xg maleic hy-
drazide = AA x K.
G. Calculations
Multiply A A of sample by K to derive u,g maleic hydrazide;
ppm = |ULg/sample wt (g).
Refs.: JAOAC 46, 261(1963); 48, 744(1965); 49, 87(1966);
64, 394(1981).
CAS-6915-15-7 (maleic hydrazide)
954.09*
Methoxychlor
Pesticide Residues
Colorimetric Method
Final Action 1965
Surplus 1970
See 24.148-24.152, 10th ed.
AOAC Official Methods of Analysis (1 990)
MONOFLUOROACETIC ACID
305
949.09 Monofluoroacetic Acid
Pesticide Residues
Qualitative Test
Final Action 1975
(Sodium salt, "1080")
(Monochloroacetic acid also responds to this test. Confirm
presence of org. F by 949.10.)
A. Reagents
(a) Decolorizing carbon. — To 10 g high-grade C (Nuchar
C-190-N, Suchar, or Darco G60) in 600 mL beaker, add ca
200 mL H 2 and 30 mL IN HC1, and keep on steam bath 20
min, agitating continuously with air passed thru cotton. Filter
on buchner and suck as dry as possible, tamping with flat-end
rod. Transfer cake to beaker, add ca 200 mL H 2 0, mix tho-
rolv, and refilter. Repeat washing and filtering twice, and dry
at 100°.
(b) Thiosalicylic acid soln. — Dissolve 300 mg thiosalicylic
acid (Eastman P2805 is suitable) in mixt. of 2 mL \N NaOH
and 18 mL H 2 0.
(c) Potassium ferricyanide soln. — Dissolve 1 g K 3 Fe(CN) 6
in H 2 and dil. to 50 mL with H 2 0.
5. Test
Prep, sample and ext as in 949.10D-E. If convenient, ext
large enough sample to obtain 2-10 mg 1080. With very low
levels of 1080, e.g., 1-5 ppm, ext large enough sample to
obtain >0.5 mg 1080.
Sep. ether ext from any aq. sludge which may have been
carried over in extn, add ca 5 g anhyd. Na 2 S0 4 and 0.5 g
decolorizing C/100 mL ether, and shake vigorously. Let stand
ca 15 min at room temp, with occasional shaking, and decant
thru fluted paper into separator. Add ca 25 mL H 2 and enough
NaOH soln (ca IN) to make aq. layer alk. after vigorous shak-
ing (outside test paper). Drain aq. layer into 125 mL erlen-
meyer and aerate to remove dissolved ether. Using pH test
paper and ca \N solns of H 2 S0 4 and NaOH, adjust to pH 4~
6. Add 0.5 g C and place on steam bath for 15 min.
Cool under tap and filter thru fluted paper into ca 25 X 150
mm test tube. Add I mL thiosalicylic acid soln and 2 drops
NaOH (1 + 1), and mix. Cone, soln to small vol. by placing
on steam bath under gentle air current. Completely dry residue
in oven at 130° or, if time is not factor, in 100° oven. (When
convenient, overnight drying is satisfactory, with or without
prior concn of soln.)
Dissolve thoroly dry residue in 2-3 mL H 2 0, add 1 mL
K 3 Fe(CN) 6 soln, and mix. Red ppt, which forms at once when
>1 mg 1080 is present, or upon standing when only fraction
of mg is present, is pos. test for 1080.
Employ chromatgc instead of C purification in following
cases:
(/) With pineapple juice when <2 mg 1080 can be extd.
(2) With grape juice even when >2 mg of 1080 can be con-
veniently extd.
(J) With any food or material when 1080 is strongly sus-
pected and neg. test is obtained using C purification technic.
For chromatgc purification, follow 949.1 OF for sepg 1080
from other acids. Discard forerun, which may contain HO Ac
and other extraneous materials. Collect fraction large enough
to contain all the 1080 as detd by preliminary detn. Ext fluo-
roacetic acid from eluate with 25 mL H 2 and enough alkali
to cause aq. layer to retain alky after vigorous shaking (outside
test paper). Drain org. layer and discard. Drain aq. layer into
125 mL erlenmeyer and aerate to remove CHC1 3 . Pour soln
into test tube and continue as above, beginning "Add 1 mL
thiosalicylic acid soln ..."
Refs.: JAOAC 32, 788(1949); 33, 608(1950); 34, 827(1951);
37, 581(1954).
CAS- 144-49-0 (monofluroacetic acid)
CAS-79-11-8 (monochloroacetic acid)
949.10 Monofluoroacetic Acid
Pesticide Residues
Quantitative Method
Final Action
(Sodium salt, "1080")
A. Principle
After suitable sample prepn, acid is extd with ether and sepd
from inorg. fluorides (partially ether-sol.) by partition chro-
matgy on silicic acid, using 0.5/V H 2 S0 4 as immobile sol v.
and CHC1 3 contg 10% rerf-amyl alcohol or n-BuOH as mobile
sol v. Monofluoroacetic acid in eluate is converted to its Na
salt, and quantity is detd by micro F detn, 944.08E(a), 944.08F,
and 944.08G.
B. Apparatus
(a) Chromatographic tubes. — 18 mm od X 250 mm long,
prepd from Pyrex tubing.
(b) Pressure source. — Compressed air or cylinder of N or
C0 2 , and means of keeping pressure const, such as Hg column
or diaphragm-type pressure regulator.
(c) Mixer.- — High-speed blender.
C. Reagents
(a) Silicic acid. — Mallinckrodt analytical reagent grade pptd
powder, or equiv.
(b) Mobile solvent. — Add 100 mL tert-amy\ alcohol or n-
BuOH to 900 mL CHC1 3 , and mix.
(c) Phosphotungstic acid soln. — Dissolve 20 g in H 2 and
dil. to 100 mL.
D. Preparation of Sample
This will vary with type of material. Dissolve sugars in H 2 0,
acidify with H 2 S0 4 , and ext directly. Following methods for
different type materials will be suggestive. Simple H 2 wash
may be adequate to prove contamination of certain foods.
(a) Sugar, — Dissolve 100 g sample in enough H 2 to give
ca 350 mL.
(b) Flour. — Place 100 g sample in mixer, add 400 mL H 2
and 5 g pancreatin, and comminute ca 2 min. Adjust to pH
7-8, using satd Na 3 P0 4 .12H 2 soln and suitable indicator pa-
per. Transfer comminuted material to tared I L erlenmeyer,
washing mixer with three 25 mL portions H 2 0. Incubate mixt.
>3 hr at 35-40°. Add 5 mL H 2 S0 4 (1 + 1) and swirl. Add
20 mL phosphotungstic acid soln and swirl again. Dil. to 750
g with H 2 0, stopper, and shake vigorously ca 2 min. Filter
thru fluted paper or with suction thru buchner (16 cm size is
convenient). Or, more quickly, centrf. and decant supernate.
Use >375 g aliquot of filtrate. (Since sp gr of filtrate is very
close to 1, measuring out aliquot in graduate is satisfactory.)
(c) Wheat. — Finely grind sample in suitable mill, such as
Wiley mill. Proceed as in (b).
(d) Corn meal. — Proceed as in (b), except omit pancreatic
digestion.
(e) Corn. — Grind sample and proceed as in (d).
(f) Peanuts. — Grind sample finely (like peanut butter) and
306
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
proceed as in (d), except use 100 mL phosphotungstic acid
soln. If necessary, refilter thru folded paper to remove oil.
(g) Cheese, — Proceed as in (d), except use 40 mL phos-
photungstic acid soln.
(h) Other foods such as chili peppers, cacao beans, etc. —
Treat in manner similar to one of preceding foods.
(i) Biological tissue. — If material is tough or fibrous, grind
it twice thru food chopper. (Soft tissues, e.g., brain and liver,
need not be ground.) Place 100 g ground tissue in 800 mL
beaker, add ca 300 mL H 2 0, cover with watch glass, and boil
gently ca 30 min. Transfer material to mixer, rinsing beaker
with two 25 mL portions H 2 0, and comminute thoroly (ca 2
min). Transfer comminuted material to tared 1 L erlenmeyer,
rinsing mixer with two 25 mL portions H 2 0. Add 5 mL H 2 S0 4
(1 + 1) and mix. Add enough phosphotungstic acid soln (50-
75 mL) to ppt all proteins, then H 2 to make 600 g. Shake
vigorously ca 2 min, and filter thru fluted paper or with suction
thru buchner. If material does not filter rapidly, return mixt.
to flask, add ca 10 mL addnl phosphotungstic acid soln, shake
vigorously, and refilter.
Alternative method. — Place 100 g ground tissue in mixer,
add 300 mL H 2 and 15 g pancreatin, and comminute thoroly
(ca 2 min). Adjust to ca pH 8 with satd Na 3 P0 4 .12H 2 soln,
using suitable indicator paper. Transfer comminuted material
from mixer to tared erlenmeyer, washing mixer with two 25
mL portions H 2 and incubate ca 3 hr at 35-40°. Ppt proteins
and make to wt as directed previously.
E. Extraction
Transfer soln (of sugar) or wt-aliquot of protein-free filtrate
(of protein-contg materials) to 200 mL continuous extractor,
Fig. 937.05. (Tube is 115-120 cm long and 33-34 mm od,
side arm, attached ca 63 cm from bottom, is 15-16 mm od;
and inner tube is 12-13 mm od flared at top to ca 25 mm
diam.; L5 L extractors of this type have been used success-
fully. Extra coarse fritted filter tip on bottom end of inner tube
aids in getting smaller droplets of extg sol v.) For each 50 g
soln, add 1 mL H 2 S0 4 (1 + 1). Ext with ether until all fluo-
roacetic acid has been extd (detd by preliminary experiment;
usually 3-4 hr with 400 mL extractor). Transfer ether ext to
separator of appropriate size.
To extn flask add ca 20 mL H 2 0, 2 drops phthln, and enough
1.0N NaOH from buret to give strong alk. color of indicator
after swirling. Pour rinse soln into separator and add addnl
alkali until alk. color of indicator persists in aq, phase after
vigorous shaking. Record vol. alkali required. Drain aq. layer
into 100 mL beaker and wash ether with two 10 mL portions
H 2 0, rinsing extn flask each time with the H 2 before pouring
it into separator. Add washings to beaker. Carefully adjust alky
of ext just to alk. color of phthln with 0. IN H 2 S0 4 and NaOH
so Ins. Evap. neutzd ext to dryness on steam bath (current of
air hastens evapn). If during evapn alk. color of indicator should
disappear, add just enough 0. IN NaOH to give alk. color again.
Do not continue heating after residue is apparently dry. Slightly
moist residue is permissible.
F. Chromatography
To 5 g silicic acid, (a), in mortar, add max. amt of 0.5N
H 2 S0 4 that it will hold without becoming sticky (50-80% of
its wt). Mix well with pestle; then add ca 35 mL of the mobile
sol v. and work up into smooth slurry. (If Si0 2 agglomerates
in sol v., too much H 2 S0 4 was used.) Place small cotton plug
in bottom of chromatgc tube and pour in slurry, tilting tube
slightly to avoid air bubbles. Let silicic acid pack down under
2-10 lb (14-69 kPa) pressure applied thru gas pressure reg-
ulator. When excess solv. has drained thru (column firm and
viscous enough to resist pouring when tipped), column is ready
for use. In prepg column take care to avoid cracking or drying
out of the gel caused by leaving pressure on after column packs
down and all solv. sinks into gel.
To dry or slightly moist residue in 100 mL beaker add enough
H 2 S0 4 (1 + 1) (ca 18A0, usually 0.5-1.0 mL, to give excess
of ca 0.25 mL over vol. necessary to convert all salts to free
acid, as calcd from amt of IN NaOH required to neutze acid
extd by the ether. Wet salts thoroly with the acid, using small,
narrow blade spatula (steel or Monel metal) to loosen salts
from glass, and using flat-end glass rod to break up solid par-
ticles and mix resulting slurry. Add 5-10 g anhyd. granular
Na 2 S0 4 to take up excess liq. Stir well with tamping rod,
breaking up any lumps. Add 10 mL mobile solv., (b), stir
thoroly, and decant solv. carefully onto column.
Catch eluate in graduate. Apply pressure until all solv. sinks
into gel; then release pressure. Add 5 mL mobile solv. to beaker
and again stir thoroly. Carefully decant solv. onto column and,
with aid of narrow-blade spatula, transfer bulk of material in
beaker, mostly Na 2 S0 4 , to column. Renew pressure. When
solv. passes ca halfway thru Na 2 S0 4 , release pressure. Rinse
out beaker with addnl 5 mL solv. and transfer to column. After
this washing sinks ca halfway into Na 2 S0 4 , fill tube with mo-
bile solv. and complete collection, under pressure, of enough
eluate to obtain all monofluoroacetic acid, as detd by test run
on silicic acid used (ca 50 mL). Collect dropwise; 3-4 mL/
min is convenient rate.
Transfer eluate to 1 25 mL separator; add ca 20 mL H 2 and
enough 1 .ON NaOH to give alk. color of phthln (phthln is pres-
ent in eluate and no further addn is required) in aq. phase,
after vigorous shaking. Drain aq. layer into 125 mL erlen-
meyer and return solv. layer to separator. Wash solv. with two
10 mL portions H 2 and add washings to erlenmeyer. Aerate
soln with current of air to remove traces of CHC1 3 . (If excess
CHC1 3 is not removed, excessive CI may complicate F distn
in next step.)
G. Determination
Transfer aq. ext to Pt dish with little H 2 and mix with ca
20 mL lime suspension, 944.08D(a), evap. to dryness, and
ash 15-20 min at 600°. (Little C in ash will not interfere in
detn.) Proceed as in 944.08E(a), beginning "When clean ash
is obtained, ..." and 944.08F-G (100 mL Nessler tubes are
preferable). Convert F results to fluoroacetic acid (x4.11) or
to Na monofluoroacetate (1080) (x5.26) as desired, and cor-
rect for aliquot taken, if any, in extn. Ignore vol. occupied by
insol. solids.
Refs.: JAOAC 32, 788(1949); 33, 608(1950); 34, 827(1951);
37, 581(1954).
CAS-144-49-0 (monofluoroacetic acid)
970.54 Naphthyleneacetic Acid
Pesticide Residues
Spectrophotometry Method
First Action 1970
Final Action 1976
(AppJicabJe to apples and potatoes)
A, Apparatus
(a) Spectrophotometer. — Cary 15 (current Model Cary 219,
Varian Instrument Group), or equiv., with 5 cm cells.
AOAC Official Methods of Analysis (1990)
Nicotine
307
(b) Chromatographic tube. — Glass, 22 mm id x 200 mm.
(c) Food chopper. — Hobart No. 84141 (Hobart Manufac-
turing Co., 711 Pennsylvania Ave, Troy, OH 45374), or equiv.
(d) Blender cups. — Stainless steel, 1 L capacity, with air-
tight screw cover (Scientific Products, Inc. No. S8390) for
high-speed blender.
B. Reagents
(a) Sodium phosphate soln.—0.5M. 134 g Na 2 HP0 4 .7H 2
or 70.5 g anhyd. salt/L.
(b) Permanganate soln. — 0.027V/. 31.6 g KMn0 4 /L.
(c) Florisil. — 60-100 mesh PR grade activated at 675°
(1250° F) (Floridin Co.); use as received.
(d) Naphthyleneacetic acid (NAA) soln. — 0.1 mg ot-NAA/
mL CH.C1 3 .
C. Extraction
Chop sample in food chopper and transfer 200 g to blender
cup. Add 20 mL 17V H 2 S0 4 and 400 mL CHC1 3 , screw top on
blender, and blend 2 min at low speed. Pour mixt. into 500
mL centrf. bottle and centrf. 10 min at 1600 rpm. Take 200
mL aliquot from CHC1 3 layer.
D. Cleanup
(a) Apples. — Place glass wool plug into chromatgc tube,
add 10 cm Florisil, and top Florisil with glass wool plug.
Transfer 200 mL CHC1 3 ext to column with min. amt CHC1 3 .
Rinse inside of tube twice with ca 5 mL CHC1 3 . Elute column,
in order, with 100 mL portions of CH 3 CN, ether, NH 3 -satd
CHCI3, and CHCI3 and discard eluates. Using 500 mL sepa-
rator as receiver, elute NAA with 100 mL 1% HO Ac in CHC1 3
followed by 100 mL CHC1 3 . Discard column, add 50 mL 17V
H 2 S0 4 to separator, and shake vigorously. Transfer CHC1 3 layer
to 250 mL separator contg 50 mL H 2 and shake vigorously.
Transfer CHC1 3 layer to 250 mL separator contg exactly 50
mL 0.5M Na 2 HP0 4 , shake vigorously, and discard CHC1 3 layer.
(b) Potatoes.— Proceed as in (a). Add 2 mL 85% H 3 P0 4
and 2 mL 0.0'^M KMn0 4 to separator contg Na 2 HP0 4 phase,
mix, and let stand exactly 5 min. Ext NAA with two 25 mL
portions CHC1 3 , transfer CHC1 3 exts to 125 mL separator contg
exactly 50 mL 0.5M Na 2 HP0 4 , shake vigorously, and discard
CHC1 3 layer.
E. Determination
(a) Apples. — Transfer 1 mL NAA std soln to 125 mL sep-
arator, add exactly 50 mL 0.5M Na 2 HP0 4 and 50 mL CHC1 3 ,
and shake vigorously. Let layers sep. and discard CHC1 3 layer.
Obtain UV spectra (230-330 nm) of cleaned up apple ext and
NAA std ext, using 5 cm cells, against 0.5M Na 2 HP0 4 . Use
peak at 283 nm to compare apple ext and NAA std ext, cor-
recting for baseline A, and calc. ppm NAA present.
(b) Potatoes. — Transfer 1 mL NAA std soln to 125 mL
separator, add 50 mL 0.5M Na 2 HP0 4 and 50 mL CHC1 3 , and
shake vigorously. Let layers sep. and discard CHC1 3 layer. Add
2 mL 85% H 3 P0 4 and 2 mL 0.02M KMn0 4 to separator, mix,
and let stand exactly 5 min. Ext NAA with two 25 mL portions
CHCI3, transfer CHC1 3 exts to 125 mL separator contg exactly
50 mL 0.5M Na 2 HP0 4 , shake vigorously, let layers sep., and
discard CHC1 3 layer. Obtain UV spectrum and calc. ppm NAA
as in (a). (If there is excessive interference in sample spectra,
repeat 5 min oxidn for both sample and std, beginning with
"Add 2 mL 85% H 3 P0 4 . . .".)
Ref.: JAOAC 53, 149(1970).
CAS-86-87-3 (naphthyleneacetic acid)
964.20 Nicotine Residues
Spectrophotometric Method
First Action 1964
Final Action 1965
(Applicable to apples, cabbage, and spinach)
A. Reagents
(a) Dilute hydrochloric acid. — Approx. 0.057V. Dil. 4.1 mL
HC1 to 1 L.
(b) Nicotine std solns. — (7) Stock soln.— I mg/mL. Dil.
100 mg nicotine (Eastman Kodak Co. No. 1242, or equiv.) to
100 mL in vol. flask with ca 0.05N HC1. (Caution: nicotine
is very toxic.) (2) Working soln. — 0.01 mg/mL. Pipet 1 mL
stock soln into 100 mL vol. flask and dil . to vol . with ca 0.057V
HC1.
(c) Stripping soln. — Dil. 20 mL NH 4 OH to 2 L in vol. flask.
Prep, at time of use.
Leafy Crops
B. Preparation of Sample
(Caution: See safety notes on toxic solvents, benzene, and
chloroform.)
Weigh 500 g chopped sample (spinach, cabbage) into clean,
dry jar (3-5 gal.; 11-20 L). Add 800 mL benzene, 200 mL
CHCI3, and 10 mL NH 4 OH. Close, tumble or roll ca 10 min,
and drain soln as completely as possible into 1 L beaker. Filter
thru folded 38.5 cm paper into flask and proceed immediately
with detn.
C. Determination
Place 400 mL filtered soln in 500 mL separator. Add 25 mL
ca 0.057V HO and 2 mL HO, and shake vigorously. Let phases
sep. (ca 5 min) and drain lower layer into 250 mL separator.
Swirl large separator, let stand ca 2 min, and drain any addnl
ext into 250 mL separator. Repeat several times. Then ext soln
with 25, 25, 15, and 10 mL portions ca 0.057V HO, repeating
swirling as above. Drain all acid exts into 250 mL separator.
Make exts just alk. to litmus with 10% NaOH soln. Ext with
two 50 mL and four 25 mL portions CHC1 3 , combining exts
in 250 mL separator.
Add 2 mL HO to exts and make sure soln is acid to litmus.
Ext with 25, 25, 20, 10, and 5 mL portions ca 0.057V HO,
combining all exts in short-stem 125 mL separator. Wash exts
with 15 mL pet ether. Drain aq. layer into second 125 mL
separator and wash pet ether with 5 mL ca 0.057V HO, adding
wash to combined acid soln. Ext soln with another 15 mL pet
ether, drain aq. layer into 100 mL vol. flask, and wash pet
ether with 5 mL ca 0.05N HO. Drain acid into vol. flask and
dil. to vol. with ca 0.057V HO. Mix, pour portion into 50 mL
beaker, and let stand 10-15 min. Det A at 236, 259, and 282
nm with ca 0.057V HC1 as ref. Confirm presence of nicotine
by reading at 2 nm intervals and plot absorption curve, or use
recording spectrophtr. Det. A of std nicotine soln against ca
0.05N HO as ref.
Waxy Crops
D. Preparation of Sample
Weigh 2-2.5 kg apples into clean, dry, jar (3-5 gal.; 11-
20 L). Add 1 L stripping soln, tumble or roll ca 10 min, and
drain carefully into 1 L beaker. Filter thru folded 38.5 cm pa-
per into flask and proceed immediately with detn.
308
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
E. Determination
Place 400 mL filtered soln in 500 mL separator. Add 50 mL
CHC1 3 , invert separator back and forth gently ca 2 min, and
let phases sep. Drain clear portion of ext into 250 mL sepa-
rator. (With fruits, emulsions may form which are very hard
to break. Break emulsions by drawing CHC1 3 layer into dry
125 mL separator and shaking vigorously. Separator must be
dry.) Let phases sep. and drain clear portion into the 250 mL
separator. Add 35 mL CHC1 3 to the 125 mL separator, shake
gently, and drain into the 500 mL separator. Ext as above and
combine clear ext in the 250 mL separator. Ext with 35, 35,
and 10 mL CHCl r , combining exts in the 250 mL separator.
Add >1 mL HO to exts until definitely acid to litmus. Then
ext with three 15 mL portions ca 0.057V HC1, combining acid
exts in a 125 mL separator. Wash the 250 mL separator with
10 mL ca 0.05/V HC1 after each extn and add to 125 mL sep-
arator used to break emulsions. Shake, but do not attempt to
break any emulsions in this separator. Combine all acid exts
in 125 mL separator and shake with 15 mL pet ether. Let stand
ca 5 min and drain aq. layer into another 125 mL separator.
Wash pet ether with 5 mL ca 0.057V HO (do not shake vig-
orously) and add washings to separator. Repeat washing with
15 mL pet ether and drain aq. ext into 100 mL vol. flask.
Wash pet ether as before, add washings to flask, and let stand
10-15 min. Dil. to vol. with ca 0.05N HC1 and det. A at 236,
259, and 282 nm, against ca 0.05/V HCl as ref. Confirm pres-
ence of nicotine as in 964. 20C.
Take A of std soln as:
^std = A' 259 ~ 0.5(A' 236 + A'282)
and A of sample soln as:
Asamp. = >*259 ~ 0.5(i4 23 6 + ^282?
Then:
mg Nicotine = (A,, dmp jA std ) x 2.5
Refs.: JAOAC 44, 177(1961); 47, 303(1964).
CAS-54-11-5 (nicotine)
960.42* Parathlon Pesticide Residues
Colorimetric Method
First Action 1960
Final Action 1965
Surplus 1970
See 29.139-29.144, 11th ed.
985.24 Pentachlorophenol in Gelatin
Gas Chromatographic Method
First Action 1985
A. Principle
Sample is acid-hydrolyzed and PCP is extd with hexane,
partitioned into KOH soln, acidified, and extd into hexane.
Compd is sepd and detd by gas chrornatgy with electron cap-
ture detection.
B. Apparatus
(a) Test tubes. — 25 X 150 mm with Teflon-lined screw caps
(Thomas Scientific, no. 9212-K75).
(b) Disposable pipets. — Pasteur type, 23 cm long (Thomas
Scientific).
(c) Centrifuge. — IEC Model UV (replacement model PR-
7000) with head for spinning 25 X 150 mm test tubes at 1000
x g (International Equipment Co., Needham Heights, MA
02194).
(d) Shaker.— (Optional). Shaker-in-the-Round Model S-500
(Glas-Col Apparatus Co., 711 Hulman St, PO Box 2128, Terre
Haute, IN 47802).
(e) Gas chromato graph.— Equipped with 63 Ni electron cap-
ture detector. Conditions: column oven 180°; injector 250°; de-
tector 350°; 5% CH 4 in Ar carrier gas flow 60 mL/min. Adjust
electrometer setting to obtain 0.5 FSD from 0.1 ng PCP (re-
tention time ca 10 min).
(f) Chromatographic column. — 1.8 m x 4 mm id glass
column contg 1% SP-1240DA on 100-120 mesh Supelcoport
(Supelco Inc.). Place small plug (2-3 mm) acid-washed glass
wool in detector end of column (silanized glass wool may be
substituted; however, peaks will be slightly broader). Install
packed column for on-column injection. Condition by purging
with carrier gas at ambient temp, for 10-15 min. Program from
70 to 190° at r/min, holding at 190° for 6-8 h. Lower temp,
to 180° for PCP detn. Note: Use only recently prepd and tho-
roly conditioned column.
C. Reagents
Store all reagents in ground glass-stopper or Teflon-lined
screw-cap containers.
(a) PCP std solns.— Dissolve 2.5 mg PCP ref. std (Stan-
dard No. 5260, Pesticides and Industrial Chemical Repository
(MD-8), Environmental Protection Agency, Research Triangle
Park, NC 27709) in 100 mL benzene (pesticide quality). Make
appropriate dilns with hexane to give std solns ranging from
0.004 to 0.4 fjig PCP/mL.
(b) Extraction solvent mixture. — Hexane-isopropanol (pes-
ticide quality) (4 + 1).
(c) Acid-washed glass wool. — Phosphoric acid-treated
(Supelco).
D. Extraction and Cleanup
Weigh 2.0 g gelatin into 25 X 150 mm screw -cap test tube
and add 10 mL 12N H 2 S0 4 . Tightly cap and heat 1 h in 100°
H 2 bath in fume hood. Remove tube periodically during hy-
drolysis, wrap in cloth towel, and mix sample by carefully
shaking.
Prep, reagent blank with each set of samples.
After 1 h, remove tube, let cool, add 10 mL hexane-iso-
propanol (4 + 1), and shake by hand or by shaker 2 min.
Centrf. 2 min at 1000 x g and transfer upper hexane layer to
second test tube with Pasteur pipet. Repeat extn and centrf gn
2 addnl times, combining all hexane exts in second test tube.
To combined exts, add 5 mL 1.0N KOH, cap, shake 2 min,
centrf. as before, remove upper layer with Pasteur pipet, and
discard. Add 10 mL hexane, cap, shake, and centrf. as before;
remove hexane wash with Pasteur pipet and discard. Add 5
mL I2N H 2 S0 4 , cap, and mix by carefully swirling tube. Ext
3 times by shaking 2 min each with 5,2, and 2 mL hexane.
After each extn, centrf. as before and transfer exts with Pasteur
pipet to 10 mL vol. flask. Adjust to vol. for GC detn.
£ GC Determination
Before each injection, rinse syringe by pumping 3-5 times
with soln to be injected. Inject 5 jxL sample soln (equiv. to
1.0 mg sample) into gas chromatograph. Measure area or ht
of PCP peak and det. amt of residue by comparison to peak
area or ht obtained from injection of known amt of PCP ref.
std. To ensure valid measurement of PCP residue, size of PCP
peak from sample and std should be within ± 10%. Make dilns
as needed. Following each injection, rinse syringe by pumping
5-10 times with hexane. After each injection of sample or std
soln, inject 5 (xL hexane. Appearance of ghost PCP peaks may
be noted following injection of high PCP soln. Repeat injec-
AOAC Official Methods of Analysis (1990)
PlPERONYL BUTOXIDE 309
tion of sotv. until ghost PCP peak becomes negligible. Repeat
sample and std injections until consistent responses are ob-
tained.
Correct sample results by subtracting reagent blank. Max.
acceptable reagent blank for satisfactory performance of method
is 0.01 ppm.
Ref.: JAOAC 68, 419(1985).
CAS-87-86-5 (pentachlorophenol)
960.43 PiperonyB Butoxide Residues
Spectrophotometry Method
First Action 1960
Final Action 1963
(Applicable to Alaska peas, barley, hulled rice, oats, pinto
beans, and wheat)
A. Principle
Strong H 2 S0 4 liberates HCHO, which is detd colorimetri-
cally with chromotropic acid.
B. Reagents
(a) Chromotropic acid reagent. — Dissolve 100 mg Na 1 ,8-
dihydroxynaphthalene-3,6-disulfonate/mL of H 2 0, filter, and
keep in dark. Prep, daily. (I mL required for each detn.)
(b) Dilute sulfuric acid. — Carefully mix 5 vols H 2 S0 4 with
3 vols H 2 0. Cool to room temp, and store in tight g-s con-
tainer.
(c) Methanolic potassium hydroxide. —Dissolve 1.4 g KOH
in 5 mL H 2 and add 95 mL MeOH (HCHO-free).
(d) Methanol. — If necessary, purify as follows: Reflux 1 L
MeOH 1 hr with ca 10 g powd Al and ca 10 g NaOH and
distil ca 800-900 mL.
(e) Hexane. — Redistd.
(f) Chloroform. — Reagent or redistd (for wheat extn).
(g) Piperonyl butoxide std solns. — (1) Stock soln. — 1 mg/
mL. Dissolve 0.1000 g in 100 mL benzene. (2) Intermediate
soln. — 100 |xg/mL. Dil. 10 mL stock soln to 100 mL with
benzene. (3) Working soln.— 20 |xg/mL. Dil. 20 mL inter-
mediate soln to 100 mL with benzene.
C. Preparation of Standard Curve
Add 0, 20, 40, 60, 80, and 100 juug piperonyl butoxide, resp.,
to each of 6 g-s test tubes (15 X 150 mm) (25-50 mL g-s
centrf. tubes are also satisfactory) and evap. on steam bath
with small air jet. Evap. last 1-2 mL benzene without heat.
Into each tube pipet both 1 mL chromotropic acid reagent
and 5 mL dil. H 2 S0 4 , (b). Stopper loosely and place tubes in
beaker of boiling H 2 45 min, remove, and cool in beaker of
cold H 2 0. When cool, pipet 5 mL H 2 into each test tube,
mix well, and read A in spectrophtr at 575 nm against reagent
blank prepd similarly. Plot |mg piperonyl butoxide against A.
D. Determination
Ext 25 g sample with CHC1 3 in soxhlet app. or shake larger
samples with suitable amts of CHC1 3 in centrf. bottle, centrf.
and decant solv. Repeat extn once or twice according to sam-
ple size. Measure total vol. of exts. With current of air, evap.
25 mL (or suitable size aliquot) ext in small beaker just to
dryness. Add 5 mL methanolic KOH. Warm gently just enough
to melt wax (do not boil). Let stand 30 min, swirling vigor-
ously at ca 10 min intervals. Transfer to small separator, rinse
beaker with two 5 mL portions H 2 0, and add to separator. Add
15 mL hexane to separator, shake vigorously 1 min, and let
sep. Drain aq. layer and discard. Quant, transfer hexane layer
to g-s test tube or centrf. tube and evap. to dryness with air
jet. Small amt of heat may be used, but evap. last 1-2 mL
with air alone. (Warmth of hand at this point is enough.)
Into dried residue pipet both 1 mL chromotropic acid re-
agent and 5 mL of the dil. H 2 S0 4 . Swirl vigorously to ensure
that reagent contacts all of sample and place test tube in boil-
ing H 2 bath. Stopper tube, lightly at first and then tighten.
After 45 min in H 2 bath, remove, and cool to room temp.
in beaker of cold H 2 0. Pipet in 5 mL H 2 0, mix well, and
measure A in spectrophtr at 575 nm against reagent blank prepd
similarly. From std curve, calc. piperonyl butoxide in aliquot.
Refs.: JAOAC 43, 707(1960); 46, 244(1963).
CAS-5 1-03-6 (piperonyl butoxide)
972.29 Thiram Pesticide Residues
Spectrophotometric Method
First Action 1965
Final Action 1972
(Applicable to corn, apples, tomatoes, strawberries, celery, and
similar fruits and vegetables)
A. Principie
Thiram is extd from sample with CHCl 3 . Treatment with
solid Cul results in formation of brown, CHCJ 3 -sol. Cu di-
methyldithiocarbamate, and its A is measured at 440 nm. Other
commonly used pesticides do not interfere, with exception of
metal dithiocarbamates sol. in CHC1 3 , such as ferbam or ziram.
Moderate amts of color, waxes, and other extd plant matter
do not interfere,
B. Reagents
(a) Chloroform.— Either reagent or tech. grade may be used.
(b) Thiram std solns. — (/) Stock soln. — 500 fxg/mL. Dis-
solve 50.0 mg thiram (available from E. I. du Pont de Ne-
mours & Co., Biochemicals Dept., 1007 Market St, Wil-
mington, DE 19898) in CHC1 3 and dil. to 100 mL with CHC1 3 .
(2) Working soln. — 25 jig/mL. Dil. 5 mL stock soln to 100
mL with CHC1 3 .
(c) Cuprous iodide. — If not available, prep, as follows: To
soln of 10 g CuS0 4 .5H 2 in ca 100 mL H 2 0, slowly add ex-
cess of KI soln. Remove liberated I 2 by adding Na 2 S 2 3 soln
in slight excess. Filter, and wash ppt thoroly with H 2 and
with alcohol. Dry at room temp, and crush to fine powder.
(d) Attapulgus clay. — Available from MC/B Manufactur-
ing Chemists, No. AX1799.
C. Apparatus
(a) Spectrophotometer . — Suitable for measuring A in UV
and at 440 nm.
(b) Glassware. — Avoid contamination by rinsing with CHC1 3
and drying before use. Rinse app. that may have contained
Cul from previous detns with dil. acid, H 2 0, alcohol, and
CHCI3.
D. Preparation of Standard Curve
(To minimize errors due to evapn of solv., keep flasks closed
as much as possible, and cover funnels with watch glasses dur-
ing filtrations.)
Using buret, add 2.0, 5.0, 10.0, and 15.0 mL working std
soln to 25 mL vol. flasks. Dil. to vol. with CHCl 3 , and mix.
Solns contain 2, 5, 10, and 15 p,g thiram /mL, resp.
Transfer ca 10 mL portions of std solns to 125 mL g-s er-
lenmeyers, add 10 mg Cul to each, stopper, and let stand 1
hr with occasional mixing. Filter, using 9 cm quant, paper,
310
Pesticide and Industrial Chemical Residues
AOAC Official Methods of Analysis (1990)
and read A at 440 nm against CHC1 3 as ref. Plot A against
thiram concn in |xg/mL.
E. Isolation
(Avoid contact of solv. with rubber.)
(a) Com. —Ext 200 g by shaking with 100 mL CHC1 3 5
min in 500 mL g-s erlenmeyer. Decant ext thru small funnel
(to retain corn kernels) into flask.
(b) Apples, pears, and similar firm fruits . — Weigh 2-3 kg
into clean, dry jar (ca 3 gal.; 11 L). Add 500 mL CHC1 3 and
stopper with tight-fitting cork, wooden bung, or plastic screw
cap faced with gasket of sheet cork or other suitable solv.-
re si sting material. Ext 5 min by tumbling or other agitation.
Decant ext into flask.
(c) Tomatoes, berries, and similar soft fruits and vegeta-
bles. — Weigh 1-3 kg into suitable container. Add 500 mL
CHCI3 and stopper with solv. -resisting closure. Ext 5 min by
gentle shaking and decant into g-s erlenmeyer thru loose plug
of glass wool.
(d) Celery. — Cut 2-3 kg into 3~8 cm pieces. Mix thoroly
and ext 500 g sample with 500 mL CHC1 3 as above.
Add anhyd. Na 2 S0 4 , ca 5 g/100 mL, to decanted ext. Stop-
per flask, shake 5 min, and filter thru folded Whatman No.
12, or equiv., paper.
F. Determination
(Thiram in CHC1 3 soln, particularly in presence of plant ex-
tractives, may decompose. Make detns as soon as possible.)
Transfer ca 10 mL filtered ext to g-s erlenmeyer and develop
color as in 972. 29D, beginning "... add 10 mg Cul . . ."As
ref., use another portion of filtered ext., untreated with CuL
From std curve, obtain thiram concn in (xg/mL. If developed
color is too intense, dil. with CHCJ 3 , making similar diln of
ref. ext, and multiply thiram value found by appropriate diln
factor.
ppm Thiram - (u>g thiram /mL) X mL CHC1 3 used for
extn/g sample.
G. Qualitative Test
Adjust concn of ext, 972. 29E, if necessary, to 10-15 \xg
thiram/mL by evapn on steam bath or by diln with CHC1 3 .
Add 0.25-1 .0 g Attapulgus clay, depending on color of ext,
to 50 mL of adjusted ext in beaker. Mix well and filter thru
Whatman No. 12 folded paper, or equiv. Transfer 25 mL fil-
trate to g-s erlenmeyer, add 0.2 mL ca 0.1 N AgN0 3 to ppt
thiram and other CHCl 3 -sol. dithiocarbamates, stopper, and
shake vigorously 30 sec. Add ca 1 g anhyd. Na 2 S0 4 and shake
30 sec. Let settle, decant carefully into 1 cm quartz cell, and
use as ref. soln, adjusting to A at 350 nm. Det. UV ab-
sorption curve over range 250-350 nm on clarified and filtered
ext untreated with AgN0 3 . Thiram gives curve with plateau at
270-283 nm, dropping sharply after peaking at ca 283. Fer-
bam and ziram give characteristic curves distinguishable from
thiram.
Refs.: JAOAC 42, 545(1959); 45, 410(1962).
CAS- 137-26-8 (thiram)
Common Names and Chemical Names of Pesticides in this Chapter
Common Name
Chemical Name
Common Name
Chemical Name
Ace p hate
Aldicarb
Aldicarb sulfone
Aldrin
Amitrole
Aroclor
Azinphos-methyl
Benfluralin
BHC
Bufencarb
Captan
Carbanolate
Carbaryl
Carbofuran
Carbophenothion
Chloramben
Chlordane
Chlorpyrifos
DDE
DDT
Acetylphosphoramidothioic acid O, S-dimethyl es-
ter
2-Methyl-2-(methylthio) propanal 0[methyl
amino)carbonyl]oxime
2-Methyl-2-(methylsulfonyl) propanal 0[methyl
amino)carbonyl]oxime
1 ,2,3,4, 1 0, 1 0-Hexachloro-1 ,4,4a,5,8,8a-hexahydro-
exo-1 ,4-endo-5,8-dimethanonaphthalene, not
less than 95%
3- Amino- 1 ,2,4-triazole
Polychlorinated btphenyl compound
0,0-Dimethyl 5-[(4-oxo-1,2,3-benzotriazin-3(4H)-
yl)methyl]phosphorodithioate
/V-Butyl-A/-ethyl-a,a,a-trifluoro-2,6-dinitro-p-toluidine
1,2, 3,4,5, 6-Hexachlorocyclohexane (mixture of iso-
mers)
3-(1-Ethylpropyl)phenyl methylcarbamate mixture
with 3-(i-methylbutyl) phenyl methylcarbamate
(1:3)
A/-[{Trichloromethyl)thio]-4-cyclohexene-1,2-dicar-
boximide
6-Chloro-3,4-xylyl-methylcarbamate
1-Naphthyl A/-methylcarbamate
2,3-Dihydro-2,2-dimethyl-7-benzofuranol methylcar-
bamate
S-[(p-Chlorophenylthio)methyl] 0,0-diethyl phos-
phorodithioate
3-Amino-2,5-dichlorobenzoic acid
1,2,4,5,6,7,8,8-Octachlor-2,3,3a,4,7,7a-hexahydro-
4,7-methanoindane
0,0-Diethyl 0-(3,5,6-trichloro-2-pyridyl) phosphoro-
thioate
Dichlorodiphenyldichloroethylene
1,1 '-(2,2,2-Trichloroethylidene)bis[4-chlorobenzene]
Diazinon 0,0-Diethyl 0-(2-isopropyl-4-methyl-6-pyrimidinyl)
phosphorothiote
Dichlone 2.3-Dichloro-1 ,4-naphthoquinone
Dieldrin 3,4,5,6,9,9-Hexachloro-l a,2,2a,3,6,6a,7,7a-octahy-
/ dro-2,7:3,6-dimethanonaphth(2,3-b)oxirene
Dodine n-Dodecylguanidine acetate
Endosulfan 6,7,8,9, 1 0, 1 0-Hexachloro- 1 ,5,5a,6,9,9a-hexahydro-
6,9-methano-2,4,3-benzodioxathiepin-3-oxide
Endosulfan sulfate 1 ,4,5,6,7,7-Hexachloro-5-norbornene-2,3-dime-
thanol cyclic sulfate
Endrin Hexachloroepoxyoctahydro-endo, endo-dimethan-
onaphthalene
EPN O-Ethyl 0-(4-nitrophenyl) phenylphosphonothioate
Ethion 0,0,0,0-Tetraethyl S,S- methylene bisphosphoro-
dithioate
Ethylan 1,1-Dichloro-2.2-bis(4-ethylphenyl) ethane
Glyodin 2-Heptadecyl-2-imidazoline acetate
HCB Hexachlorobenzene
Heptachlor 1 ,4,5,6,7,8,8-Heptachloro-3a,4,7,7a-tetrahydro-4,7-
methanoindene
Heptachlor epoxide Oxidation product of heptachlor
3-Hydroxycarbofuran 2,3-Dihydro-2,2-dimethyl-3-hydroxy-7-benzofuranyl
methylcarbamate
Lindane Gamma isomer of 1 ,2,3,4,5,6-hexachlorocyclohex-
ane (BHC)
Malathion 0,0-Dimethyl S-(1,2-dicarbethoxyethyl) phospho-
rodithioate
Maleic hydrazide 6-Hydroxy-3(2H)pyridazinone
Methamidophos Phosphoramidothioic acid 0,S-dimethyl ester
Methiocarb 3,5-Dimethyl-4-(methythio)phenyl methylcarbamate
Methomyl A/-[[(Methylamino)carbonyl]oxy]ethanimidothioic
acid methyl ester
Methoxychlor 2,2-Bis(p-methoxyphenyl)-1 ,1 ,1 -trichloroethane
AOAC Official Methods of Analysis (1990)
Common Names
311
Common Names and Chemical Names of Pesticides in this Chapter (Continued)
Common Name
Chemical Name
Common Name
Chemical Name
Methyl parathion
Mi rex
Monocrotophos
NAA
Nicotine
Omethoate
Oxamyl
Oxychlor epoxide
(also oxychlor-
dane)
OODimethyl O-p-nitrophenyl phosphorothioate
Dodecachlorooctahydro-1,3,4-metheno-2H-cyclo-
buta(cd)pentalene
Phosphoric acid dimethyl [1-methyl-3-(methylam-
ino)-3-oxo-1-propenyl] ester
Naphthalene acetic acid
3-(1 -Methyl-2-pyrrolidyl) pyridine
0,0-Dimethyl S-[2-(methylamino)-2-oxoethylj phos-
phorothioate
2-(Dimethylamino)-/V-[[methylamino)carbony|joxy]-
2-oxoethanimidothioic acid methyl ester
1-exo,2-endo-4,5,6,7,8,8-Octachloro-2,3-exo-epoxy-
2,3,3a,4,7,7a-hexahydro-4,7-methanoindene
Paraoxon
Parathion (same as
ethyl parathion)
PCBs
Piperonyl butoxide
Propoxur
Ronnel
TDE
Tetrad if on
Tetrasul
Thiram
0,0-Diethyl O-p-nitrophenyl phosphate
0,0-Diethyl O-p-nitrophenyl phosphorothioate
Some mixture of chlorinated biphenyl compounds
having various percentages of chlorine
a-[2-(2-Butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-
propyltoluene
2-(1-Methylethoxy) phenol methylcarbamate
0,0-Dimethyl 0-(2,4,5-trichloro-
phenyl)phosphorothioate
1 ,r-(2,2-Dichloroethylidene)bis[4-chlorobenzene]
4-Chlorophenyl 2,4,5-trichlorophenyl sulfone
4-Chlorophenyl 2,4,5-trichlorophenyl sulfide
Zinc dimethyldithiocarbamate
Sources: The Merck Index (1983) 10th ed., Merck & Co., Inc., Rahway, NJ; The Agrochemicals Handbook (1987) 2nd ed., The Royal Society of Chemis-
try, Nottingham, UK; Farm Chemicals Handbook (1988) 74th ed., Meister Publishing Co., Willoughby, OH.
SPECIAL REFERENCES
U.S. Dept. of HHS, Food and Drug Admin., "Pesticide An-
alytical Manual," Vols I and II. Available from FDA, Pub-
lic Records and Documents Center HFI-35, 5600 Fishers
Lane, Rockville, MD 20857.
Burke, "Development of the Food and Drug Administration's
Method of Analysis for Multiple Residues of Organo-
chlorine Pesticides in Foods and Feeds." Residue Reviews
34, 59-90(1971).
11. Waters; and Salt
Andrew Saufer Associate Chapter Editor
Henderson, Nevada
Kenneth Stoub, Associate Chapter Editor
Waste Management, Inc.
WATER
955.37 Specific Gravity of Water
Pycnometer Method
Final Action
Det. sp gr at 20/20°, using pycnometer, as in 945. 06C.
973.40
Specific Conductance
of Water
First Action 1973
A. Principle
Conductivity of sample is compared with that of std KC1
soln. Method is applicable to drinking, surface, and saline
waters, and domestic and industrial wastes.
Synthetic H 2 samples contg increments of inorg. salts ana-
lyzed by 41 analysts in 17 laboratories showed following re-
sults:
Increment
Std deviation
Bias
as sp
conductance,
(jimhos/cm
%
|i,mhos/cm
%
|x mhos /cm
100
7.6
7.55
-2.0
- 2.0
106
77
8.14
-0.8
~~ 0.8
808
7.5
66.1
-3.6
-29.3
848
9.4
79.6
-4.5
-38.5
1640
6.5
106
-5.4
-87.9
1710
7.0
119
-5.1
-86.9
B. Apparatus and Reagent
(a) Conductivity meter. — Self-contained, Wheatstone bridge-
type, capable of being read to ±1%.
(b) Specific conductance cell. — Choose cell according to
expected sp conductance so that measured cell resistance is
500-10,000 ohms. Cell const should be ca 0.1 for solns of
low conductivity (<100 |xmhos), 1 for moderate, and 10 for
highly conducting, such as brines. Check complete assembly
with KCI solns of known conductance shown in Table 973.40.
Clean new cells with chromic acid cleaning soln and platinize
new electrodes before use. Reclean and platinize electrodes
whenever readings become erratic or if inspection shows any
Pt black has flaked off. To platinize, connect both electrodes
together to neg. terminal of 1 .5 v dry cell and immerse in soln
of 1 g chloroplatinic acid and 12 mg Pb(OAc) 2 in 100 mL
H 2 0. Connect pos. terminal to piece of Pt wire and dip into
soln. Control current so that only small amt gas is evolved.
Discontinue electrolysis when both electrodes are coated. Soln
may be saved for subsequent use. Rinse electrodes thoroly and
keep immersed in H 2 when not in use.
(c) Potassium chloride std soln. — 0.0 IM. Dissolve 745.6
mg KCI in freshly boiled double-distd H 2 and dil. to 1 L at
25°. Soln has sp conductance of 1413 fxmhos at 25°. It is sat-
isfactory for most waters when using cell with const of 1-2.
With other cells, use soln in Table 973.40 and corresponding
sp conductance in calcn. Store in g-s Pyrex bottle.
C. Determination
Temp, must be const thruout detn since sp conductance var-
ies ca 2% /degree. Use 25° if possible; otherwise use near room
temp, but between 20-30°.
Place 4 tubes std KCI soln and 2 tubes of each sample in
H 2 bath and let stand 30 min. Rinse cell in 3 tubes of KCI
soln and measure resistance of soln 4, R K c\- Rinse cell thoroly
with tube 1 of sample and measure resistance of tube 2, R s .
Do not repeat measurement of KCI soln unless temp, drift of
more than few tenths degree occurs. If samples differ in con-
ductivity by factor of >5, minimize carry-over by rinsing in
2 tubes of sample and measuring third.
D. Calculation
Calc. cell const, C, in mhos/cm = R KC] x 0.001413 at 25°.
Specific conductance of sample at 25° = C/R s in mhos /cm.
Multiply by 10 6 to obtain jxmhos/cm.
If temp, is not exactly 25°, measure R KCl and /? s at same
temp, and calc. sp conductance = 1413 X R KCl /R s in fimhos/
cm.
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study 1; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). JAOAC 56, 295(1973).
973.41
pH of Water
First Action 1973
A. Principle
pH, which is accepted measure of acidity or aJky, is detd
by change in potential of glass-satd calomel electrodes, as
Table 973.40 Conductances of KC! Solns at 25°
Concn,
Conductance,
(xmhos/cm
M
Equiv.
Specific
149.85
0.0001
149.43
14.94
0.0005
147.81
73.90
0.001
146.95
147.0
0.005
143.55
717.8
0.01
141.27
1,413
0.02
138.34
2,767
0.05
133.37
6,668
0.1
128.96
12,900
0.2
124.08
24,820
0.5
117.27
58,640
1.0
111.87
111,900
312
AOAC Official Methods of Analysis (1990)
Acidity 313
measured by com. app. stdzd against std buffer solns whose
pH values are assigned by NIST. pH of most natural H 2 falls
within 4-9. Majority of waters are slightly basic from pres-
ence of CO3-HCO3 system.
Method is applicable to drinking, surface, and saline waters,
and domestic and industrial wastes. Oils and greases, by coat-
ing electrodes, may cause sluggish response.
Buffered synthetic H 2 samples analyzed by 44 analysts in
20 laboratories showed following results:
Std deviation,
Bias,
PH
pH units
pH units
3.5
0.10
-0.01
3:5
0.11
0.00
7.1
0.20
+0.07
7.2
0.18
-0.002
8.0
0.13
-0.01
8.0
0.12
+0.01
B. Apparatus and Reagent
(a) pH meter. — Com. instrument with flow-type electrodes
(preferred for relatively unbuffered samples such as conden-
sates) or immersion electrodes. Operate in accordance with
manufacturer's instructions.
(b) Std buffer solns.See 964.24 and Table 964.24.
C. Determination
Thoroly wet electrodes and prep, in accordance with man-
ufacturer's instructions. Stdze instrument with std buffer with
pH near that of sample and then with 2 others to check lin-
earity of electrode response.
Analyze sample as soon as possible, preferably within few
hr. Do not open sample bottle before analysis. With immersion
electrodes, wash 6-8 times with portions of sample, particu-
larly when unbuffered soln follows buffered so In. Equilibrium,
as shown by absence of drift, must be established before read-
ings are accepted.
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study 1 ; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). JAOAC 56, 295(1973).
973.42
Acidity of Water
Titrimetric Method
First Action 1973
A. Principie
Sample is titrd to pH 8.3, using phthln as indicator, and
results are reported as mg CaC0 3 /L. Method is applicable to
drinking and surface waters, domestic and industrial wastes,
and saline waters. Synthetic H 2 contg increments of HC0 3
analyzed by 40 analysts in 17 laboratories showed following
results:
Added,
Std deviation
Bias
mg
CaC0 3 /L
%
mg
CaC0 3 /L
%
mg
CaC0 3 /L
20
21
9.0
8.2
1.79
1.73
+2.8
+0.5
+0.55
+0.11
B. Apparatus
(a) Illumination. — Daytime fluorescent lamps provide uni-
form lighting conditions.
(b) Potentiometric equipment. — Automatic titrators and pH
meters, suitably calibrated, may be substituted for visual titrn
and end point.
C. Reagents
(a) Carbon dioxide-free water. — If pH is <6.0, prep, as in
936.16B(a). Deionized H 2 may be substituted if conductance
is <2 fjimhos/cm and pH >6.0.
(b) Sodium hydroxide std soln. — 0.02vV. Dil. 20.0 mL \N
NaOH with C0 2 -free H 2 to 1 L. Store in tightly stoppered
Pyrex bottle protected by soda-lime tube. Prep, weekly. Stdze
against 0.02CXW KH phthalate soln (4.085 g/L) or against stdzd
0.02N HC1 or H 2 S0 4 . Use vol. soln to give acidity approx.
that of samples titrd, dild to vol. of sample, with same vol.
indicator, and same time intervals as in detn. 1 mL 0.0200W
NaOH = 1.00 mg CaCO 3 /1.00 mL.
D. Preparation of Samples
Collect and store samples in Pyrex or polyethylene bottles.
Refrigerate at 4° and perform detn as soon as possible, pref-
erably within 24 hr.
E. Determination
Use sample vol. requiring <25 mL titrant. If indicator is
used, remove free CI with 1 drop 0.1W Na 2 S 2 3 , 942. 27A.
To 50 or 100 mL sample in white porcelain casserole or in
erlenmeyer over white surface, add 0. 15 mL phthln. Titr. with
stdzd 0.02N NaOH to faint pink (pH 8.3).
mg CaC0 3 /L - mL NaOH
X normality NaOH X 50,000/mL sample
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study 1 ; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). JAOAC 56, 295(1973).
973.43
Alkalinity of Water
Titrimetric Method
First Action 1973
A. Principie
Unaltered (undild, unconcd, unfiltered) sample is titrd po-
tentiometrically to pH 4.5. Applicable to drinking and surface
waters, domestic and industrial wastes, and saline waters.
Suitable for all concn ranges.
Synthetic H 2 contg increments of HCO3 analyzed by 40
analysts in 17 laboratories showed following results:
Std deviation
Bias
Added,
mg
mg
mg
CaC0 3 /L
%
CaC0 3 /L
%
CaC0 3 /L
8
16
1.3
+ 10.6
+0.85
9
12
1.1
+ 22.3
+2.0
113
4.7
5.3
- 8.2
-9.3
119
4.5
5.4
- 7.4
-8.8
0. Apparatus
See 973.428(b).
314
Waters; and Salt
AOAC Official Methods of Analysis (1990)
C. Reagents
(a) Carbon dioxide-free water. — See 973.42C(a).
(b) Acid std soln. — Q.02N. Prep, ca 0. IN stock soln by dilg
8.3 mL HC1 or 2.8 mL H 2 S0 4 to 1 L. Dil. 200 mL stock soln
to 1 L with C0 2 -free H 2 0. Stdze against 0.027V Na 2 C0 3 (1 .060
g Na 2 C0 3 /L, 936.15F(c)) or stdzd 0.02N NaOH, 973.42C(b).
Use vol. soln to give alky approx. that of samples titrd, dild
to vol. of sample, with same vol. indicator and same time in-
tervals as in detn. I mL 0.02^ acid = 1.00 mg CaC0 3 /L.
D. Preparation of Samples
See 973.42D.
E. Determination
Use sample vol. requiring <25 mL titrant. Titr. potentiom-
etrically to pH 4.5.
mg CaC0 3 /L = mL acid x normality acid
x 50,000/ mL sample
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study 1; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). JAOAC 56, 295 (1973).
973.44 Biochemical Oxygen Demand
(BOD) of Water
Incubation Method
First Action 1973
4. Principle
Sample is incubated 5 days at 20° in presence of acclimated
biological system. Comparison of O content of sample at be-
ginning and end of incubation is measure of BOD.
Method is applicable to raw or treated domestic wastes, in-
dustrial water, and industrial waste water. Following classes
of materials exert O demand: (7) org. material usable as food
by aerobic organisms (source of BOD of many waste waters);
(2) oxidizable N from nitrites, NH 3 , and org. N compds which
serve as food for specific bacteria (e.g. , Nitrosomonas and Ni~
trobacter) (a source of some of O demand of biologically treated
effluents); (3) chemically oxidizable materials (e.g., Fe + \ S™ 2 ,
S0 3 ~ 2 ) (when present, test must be based upon calcd initial
dissolved O content).
Many synthetic org. components of industrial wastes are not
degraded by common organisms. Without special seeding ma-
terial, effect is manifested as retardation of aerobic metabolism
because of toxic effect or deficiency or absence of appropriate
microorganism. Toxic compds in distd H 2 0, frequently Cu,
may result in low BOD.
Distd H 2 contg known increments of oxidizable org. ma-
terial analyzed by 74 analysts in 50 laboratories showed fol-
lowing results:
Increment org.
Std deviation
Bias
material,
mg/L
% mg/L
% mg/L
2.2
194
33 0.7
15 26
- 4 - 0.08
-10 -19
B. Apparatus
(a) Incubation bottles. — 250 or 300 mL with glass stop-
pers.
(b) Incubator. — Air or H 2 bath maintained at 20 ± 1° and
which excludes light. {Caution: Check H 2 bath to assure that
it is electrically grounded.)
C. Reagents
(a) Water. — Contg <0.01 mg Cu/L, obtained by double
demineralization of distd H 2 or distn from all-glass or Sn-
lined system.
(b) Calcium, chloride soln. — 27.5 g anhyd. CaCl 2 /L.
(c) Ferric chloride soln.— 0.25 g FeC] 3 .6H 2 0/L.
(d) Magnesium sulfate soln. — 22.5 g MgS0 4 .7H 2 0/L.
(e) Phosphate buffer soln. — pH 7.2. Dissolve 8.50 g
KH 2 P0 4 , 21 .75 g K 2 HP0 4 , 33.40 g Na 2 HP0 4 .7H 2 0, and 1.70
g NH4CI in ca 500 mL H 2 and dil. to 1 L.
(f ) Seeding material. — Satisfactory seed may sometimes be
obtained or developed from supernate of domestic sewage stored
24-36 hr at 20°, from receiving H 2 downstream from point
of discharge, or, in case of industrial wastes contg org. compds
not amenable to oxidn by domestic sewage seed, from accli-
mated seed developed in laboratory.
(g) Sodium hydroxide soln. — 50 g NaOH/L.
(h) Sodium sulfite soln. — L575 g Na 2 S0 3 /L. Prep, fresh
as needed.
D. Preparation of Dilution Water
Store H 2 0, 973.44C(a), in cotton-plugged bottles long enough
to sat. with atm. O at 20°, or aerate with air filtered to remove
any oil from compressor (<1 hr may be required for 19 L (5
gal.)). Add desired vol. of O-satd H 2 to suitable bottle and
add 1 mL each of phosphate buffer, MgS0 4 , CaCl 2 , and FeCl 3
solns/L. Seed this diln H 2 with seeding material and with
vol. found by experience to be niost satisfactory for particular
waste being examined. Use seeded diln H 2 within 24 hr of
prepn.
Periodically check quality of diln H 2 0, effectiveness of seed,
and technic with particular org. compd if known to be present
in waste or, for general work, with mixt. of glucose and glu-
tamic acid (150 mg each/ L) which should show BOD ca 220
± 30 mg/L in 95% of detns. Appreciable divergence requires
examination of quality of H 2 0, viability of seeding material,
or technic.
E. Preparation of Samples
Keep time between collection of sample and start of analysis
to absolute min. Protect samples from atm. O. If necessary,
pretreat samples as follows:
(a) Acidity or caustic alkalinity .— Neutze to ca pH 7 with
dil. H 2 S0 4 or 5% NaOH, using pH meter or bromothymol blue
as external indicator. pH of seeded diln H 2 should not be
changed by diln of sample.
(b) Residual chlorine. — Let stand 1-2 hr to dissipate CI. If
not effective, use Na 2 S0 3 treatment. Det. vol. to be used by
adding 10 mL HOAc (1 + 1) or H 2 S0 4 (1 + 49) and 10 mL
10% Kl to 1 L sample. Titr. to starch -1 end point with Na 2 S0 3
soln. Add indicated vol. to sample, and test small portion with
starch-1 soln to check that treatment is complete.
(c) Toxic substances. — Remove or neutze. Test for toxicity
as follows: Add same amt seed to duplicate set of BOD bot-
tles. Add diln H 2 to each bottle, leaving room for amt of
sample to give final concns of 0.06, 0.12, 0.25, 0.50, 1.0,
2.5, 5, 1.0, 20, and 40%. Neutze sample, add required vol.
sample to duplicate bottles, and fill with diln H 2 0. Det. dis-
solved O in 1 series ca 15 min after prepn of diln. Det. dis-
solved O in second series after 3 days. Plot consumption of
dissolved O against concn. Magnitude of O concn change will
depend on amt of food available and toxicity of sample. If
toxicity is factor, O consumption will decrease at higher concns.
AOAC Official Methods of Analysis (1990)
Biochemical Oxygen Demand 315
(d) Super saturation with oxygen. — Samples contg >9.2 mg
O/L at 20° may be encountered during winter or where algae
are actively growing. To prevent loss of O during incubation,
reduce O content to satn by transferring sample at ca 20° to
partially fill bottle and shake vigorously.
F. Determination
Sample must be dild with seeded diln H 2 so that at least
1 diln will achieve dissolved O depletion of 1 mg/ L (ppm)
during 5 day test period but will not reduce residual dissolved
O to <1 mg/L. (Preliminary chemical O demand (COD) detn,
973.46, may serve as guide to est. range of BOD.)
Carefully siphon seeded diln H 2 into 1 or 2 L graduate,
filling it V2 full. Add vol. of carefully mixed sample to desired
diln and fill to mark with diln H 2 0. Mix well with plunger-
type mixing rod, avoiding entrainment of air. If possible BOD
range is large, prep, geometric series of dilns to cover possible
range. Siphon, with continued mixing, dild sample to com-
pletely fill 3 BOD bottles — 1 for incubation, 1 for detn of
dissolved O content, and 1 for detn of immediate dissolved O
demand (IDOD). Insert stoppers without entrainment of any
bubbles. Det. dissolved O by method indicated in 973. 45 A.
Alternatively, prep, dild samples directly by pipetting sam-
ple with wide-tip pipet into BOD bottles of known capacity
and filling bottles with seeded diln H 2 0. If diln > 1:100 is re-
quired, prep, in graduate before adding to BOD bottles.
Prep, blank of seeded diln H 2 contg vol. used for diln of
samples for detn of initial dissolved O content. Prep, control
of 2 BOD bottles with unseeded diln H 2 0. Stopper and H 2 0-
seal 1 bottle for incubation. (If special H 2 0-sealed bottles are
not used, H 2 0-seal by immersion in tray of H 2 0.) Det. dis-
solved O in other bottle before incubation. Quality of unseeded
diln H 2 is satisfactory if depletion obtained is <0.2 mg/L,
preferably <0.1. Do not use this value as blank correction.
If diln H 2 is seeded, det. O depletion of seed used in such
diln that will result in 40-70% depletion in 5 days. Use this
depletion, not seeded blank, to calc. correction due to small
amt of seed in diln H 2 0.
Incubate prepd mixts, H 2 0-sealed, 5 days at 20 ± 1° and
det. final dissolved O content.
G. Calculation
Calc. in mg/L (ppm) as follows:
Immediate dissolved O demand {IDOD) = (D c - D x )f P
When seeding is not required, BOD = (D y - D 2 )/P
When using seeded diln H.O, BOD = [(D, - D 2 ) - (B { -
B 2 )f\/P
Including IDOD, if small or not detd, BOD = (D c -
D 2 )/P
Where
D = dissolved O (DO) of original diln H 2 0,
£>, = DO in dild sample 15 min after prepn,
D 2 — DO of dild sample after incubation,
S = DO of original undild sample,
D c = DO available in diln at zero time = (D p) + SP,
p — decimal fraction of diln H 2 used,
P = decimal fraction of sample used,
B x — DO of the diln of seed control before incubation,
B 2 — DO of the diln of seed control after incubation,
/ = ratio of seed in sample to seed in control == (% seed
in £>,)/(% seed in B { ).
H. Interpretation
Arbitrary std 5 day incubation period is satisfactory mea-
surement of the O load on receiving water for raw or treated
domestic sewage. It may be misleading for wastes contg org.
compds not easily amenable to biological oxidn. Studies with
3 incubation periods on series of dilns of the waste will pro-
vide information on lag periods, suitability of inocula, rate of
biochem. oxidn, ultimate O demand, and amenability to
biochem. self-purification. Particularly important is ratio of 5
day BOD to ultimate O demand.
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
Method Research Study 3; Demand Analyses, 1971
(available from National Technical Information Ser-
vice, 5285 Port Royal Rd, Springfield, VA 22161 , NTIS
PB230275/BE). JAOAC 56, 295(1973).
973.45 Oxygen (Dissolved) in Water
Titrimetric Methods
First Action 1973
(Caution: See safety notes on pi pets.)
A. Applications
Azide (Alsterberg) method is ordinarly used; it is not af-
fected by most common interference, nitrite, but most other
oxidizing or reducing agents should be absent. Effect of Fe +3
is eliminated with F™. Permanganate (Rideal-Stewart) method
is used in presence of Fe t2 but not org. matter. Pomeroy-
Kirshman-Alsterberg method is used for waters supersatd with
O or contg high org. matter content.
Method I, Azide Method
B. Reagents
(a) Alkaline iodide-sodium azide soln. — Dissolve 500 g
NaOH (or 700 g KOH) and 135 g Nal (or 150 g KI) in H 2 0,
dil. to 950 mL, and cool. Slowly, with stirring, add soln of
10 g NaN 3 in 40 mL H 2 0. Dild and acidified soln must not
give color with starch indicator. Store in dark bottle with rub-
ber stopper.
(b) Manganese sulfate soln. — Dissolve 364 g MnS0 4 .H 2
in H 2 0, filter, and dil. to 1 L. No more than trace of I should
be liberated when soln is added to acidified KI soln.
(c) Potassium biiodate std soln. — 0.025/V. Dissolve 0.8125
g KH(I0 3 ) 2 in H 2 in 1 L vol. flask and dil. to vol.
(d) Potassium fluoride soln. — 40 g KF.2H 2 O/100 mL.
{Caution: KF is toxic and corrosive. See safety notes on toxic
dusts.)
(e) Sodium thiosulfate std solns. — (/) 0.1 N. — Dissolve 25
g Na,S 2 3 .5H 2 in H 2 0, add 1 g NaOH or 5 mL CHCL, and
dil. to 1 L. Stdze against KH(10 3 ) 2 or K 2 Cr 2 7 , 942.27B. (2)
0.025N.—DW. 250 mL Q.W to I L. 1 mL = 0.2 mg O.
(f) Starch indicator soln.— Disperse 5-6 g potato or arrow-
root starch in mortar with few mL H 2 0. Pour into 1 L boiling
H 2 0, boil few min, and let settle overnight. Decant clear soln
and preserve with 1.3 g salicylic acid or few drops toluene.
C. Determination
(Add all reagents, except H 2 S0 4 , well below surface of sample
from 10 mL pipets graduated in 0.1 mL, with tips elongated
ca 50 mm.)
Add 2.0 mL MnS0 4 soln and 2.0 mL alk. I-NaN 3 soln to
sample in 250 or 300 mL BOD bottle, replace stopper, ex-
cluding air bubbles, and invert several times to mix. Let floe
settle and repeat mixing. (Water with high chloride concn re-
quires 10 min contact with ppt.) After floe has settled, leaving
316 Waters; and Salt
AOAC Official Methods of Analysis (1990)
>:100 mL clear supernate, remove stopper and add 2.0 mL
H 2 S0 4 down neck of bottle. (Jf >100 ppm Fe +3 is present,
add 1.0 mL KF soln before acidifying.) Restopper and mix by
inversion until I 2 is uniformly distributed. Immediately titr, 203
mL (3 mL is allowance for added reagents) with 0.0257V Na 2 S 2 3
to pale straw yellow. Add 1-2 mL starch indicator and titr.
to disappearance of blue. Disregard reappearance of blue.
ppm dissolved O
(mL 0.0257V Na 2 S 2 3 X 0.2/200) X 1000
Method II, Permanganate Method
D. Reagents
(a) Alkaline iodide soln. — Prep, as in 973.456(a), except
omit NaN 3 .
(b) Potassium oxalate soln. — 2%. Dissolve 2 g K 2 C 2 04.H 2
in 100 mL H 2 0. 1 mL equiv. to 1.1 mL KMn0 4 soln, (c).
(c) Potassium permanganate soln. — 6.3 g/L.
£ Determination
Add to sample 0.70 mL H 2 S0 4 and then 1.0 mL KMn0 4
soln. If sample is high in Fe, also add 1.0 mL KF soln,
973.45B(d). If necessary, add addnl KMn0 4 soln to maintain
violet tinge 5 min. (If >5 mL is required, prep, stronger KMn0 4
soln to avoid diln of sample.) After 5 min, decolorize with
just enough oxalate soln (usually 0.5-1.0 mL) within 2-10
min. Add 2.0 mL MnS0 4 soln, 973.45B(b), and 3.0 mL alk.
h soln, 973.450(a). Stopper bottle and mix. Let ppt settle,
remix 20 sec, and let settle until 2:100 mL clear supernate is
present. Acidify with 2.0 mL H 2 S0 4 . Titr., using vol. of 205
mL, and calc. as in 973. 45C.
Method III, Pomeroy-Kirshman-Alsterberg Method
F. Reagent
Alkaline iodide-sodium azide soln. — Dissolve 400 g NaOH
in 500 mL freshly boiled and cooled H 2 0. Cool slightly and
then add 900 g Nal; mix. Dissolve 10 g NaN 3 in 40 mL H 2 0.
Add slowly, with stirring, to alk. I 2 soln, bringing total vol.
to >1 L.
G. Determination
To sample add 2.0 mL MnS0 4 soln, 973.45B(b), and 2.0
mL alk. I 2 -NaN 3 soln, 973. 45F. Stopper and mix by inversion.
After ppt has settled, add 2.0 mL H 2 S0 4 and mix. Titr., using
vol. of 203 mL, and calc. as in 973.45C.
Ref.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
JAOAC 56, 295(1973).
973.46 Chemical Oxygen Demand
(COD) of Water
Titrimetric Methods
First Action 1973
A. Principle
Org. substances are oxidized by K 2 Cr 2 7 in H 2 S0 4 (1 + 1)
at reflux temp, with Ag 2 S0 4 as catalyst and HgS0 4 to remove
CI interference. Excess dichromate is titrd with Fe +2 , using
orthophenanthroline as indicator. Method is independent detn
of org. matter in sample and has no definable relationship to
biological oxygen demand (BOD).
Method is applicable to surface and saline waters and in-
dustrial wastes. Apply Method /, using 0.257V reagents, to
samples contg >50 mg COD/L; apply low level modification,
Method 7/, using 0.0257V reagents, to samples in range 5-50
mg/L; apply special modification, Method III, to saline waters
contg >1000 mg Cl/L and >250 mg COD/L.
Org. matter from glassware, atm., and distd H 2 must be
excluded. Condition glassware by using it for blank detn to
eliminate org. matter.
Distd H 2 contg known increments of oxidizable org. ma-
terial analyzed by 89 analysts in 58 laboratories showed fol-
lowing results:
Increment org.
material, mg/L
Std deviation
Bias
% mg/L
% mg/L
12.3
270
34 4
7 18
-2 -5
B. Preparation of Sample
Collect samples in glass bottles if possible; plastic may be
used if it contributes no org. material to sample. Test biolog-
ically active samples as soon as possible. Mix or homogenize
samples contg settleable materials. Samples may be preserved
with H 2 S0 4 , 2 mL/L.
C. Apparatus and Reagents
(a) Reflux apparatus. — 500 mL erlenmeyer or 300 mL r-b
flask with ¥ joint connected to 30 cm (12") Allihn condenser.
(b) Distilled water. — Low in org. matter. Ordinary distd
H 2 is satisfactory; do not use deionized H 2 0.
(c) Potassium dichromate std solns. — (7) 0.25N. — Dis-
solve 12.259 g K 2 Cr 2 O v , primary std grade, previously dried
2 hr at 103°, in distd H 2 and diJ. to 1 L. (2) 0.025N.— Dil.
100 mL 0.257V to 1 L with H 2 0.
(d) Sulfuric acid reagent.— -Dissolve 23,5 g Ag 2 S0 4 in 9 lb
(4.1 kg) bottle H 2 S0 4 . (1-2 days may be required for dissoln.)
(e) Ferrous ammonium sulfate std soln. — (7) 0.25N. — Dis-
solve 98 g Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 in H 2 0, add 20 mL H 2 S0 4 ,
cool, and dil. to 1 L. Stdze daily against 0.25/V K 2 Cr 2 7 , (c)(7).
(2) 0.025N. —D\\. 100 mL 0.25N to 1 L with H 2 6. Stdze daily
against 0. 0257V K 2 Cr 2 7 , (c)(2).
(f) Phenanthroline ferrous sulfate (ferroin) indicator soln. —
Dissolve 1.48 g l,10-(ortho)-phenanthroline.H 2 O and 0.70 g
FeS0 4 .7H 2 in 100 mL H 2 0.
D. Standardization of Ferrous Solutions
(a) Concentrated soln. — Dil. 25.0 mL 0.257V K 2 Cr 2 7 ,
(c)(7), to ca 250 mL with H 2 0. Add 75 mL H 2 S0 4 and cool.
Titr. with 0.257V Fe(NH 4 ) 2 (S0 4 ) 2 , using 10 drops ferroin in-
dicator. Normality = (mL K^Cr ? 7 x normality)/mL
Fe(NH 4 ) 2 (S0 4 ) 2 .
(b) Dilute soln.— To 15 mL H 2 add 10.0 mL 0.025N
K 2 Cr 2 7 , (c)(2). Add 20 mL H 2 S0 4 and cool. Titr. with 0.0257V
Fe(NH 4 ) 2 (S0 4 ) 2 , using 1 drop ferroin indicator. Blue-green to
reddish brown color change is sharp. Calc. normality as in (a).
£. Method I— High Level
(Caution: See safety notes on mercury and mercury salts.)
Place several boiling chips and 1 g HgS0 4 in reflux flask.
Add 5.0 mL H 2 S0 4 and swirl until HgS0 4 dissolves. Place in
ice bath and slowly add, with swirling, 25.0 mL 0.257V K 2 Cr 2 7 .
Slowly, and with swirling, add 70.0 mL H 2 S0 4 -Ag 2 S0 4 re-
agent. While still in bath, pipet in 50 mL sample (or aliquot
dild to 50 mL) with continuous mixing. Attach condenser and
reflux 2 hr. (Shorter period may be used on waste H 2 of const
or known composition where time of max. oxidn has been detd
previously.)
AOAC Official Methods of Analysis (1990)
Carbon
317
Cool, and wash down condenser with ca 25 mL H 2 0. If
r-b flask has been used, quant, transfer soln to 500 mL erlen-
meyer. Dil. to ca 300 mL with H 2 0, and let cool to ca room
temp. Add 8-10 drops ferroin indicator, and titr. excess K 2 Cr 2 7
with 0.25N Fe(NH 4 ) 2 (S0 4 ) 2 to sharp, reddish end point (S mL).
Perform blank detn with all reagents, including refluxing, on
distd H 2 in place of sample and det. mL 0.25N Fe(NH 4 ) 2 (S0 4 ) 2
required (B mL).
mg COD/L - (B ~ S) x N x 8000/V
where N = normality Fe(NH 4 ) 2 (S0 4 ) 2 soln and V = vol. sam-
ple used.
F. Method II — Low Level
Proceed as in high level detn, 973. 46E, except use 0.025N
K 2 Cr 2 7 and Fe(NH 4 ) 2 (S0 4 ) 2 .
G. Method III— Saline Waters
Pipet 50 mL sample of 250-800 mg COD/L and Cl~ >1000
mg/L (or aliquot dild to 50 mL with distd H 2 having Cl~
concn equal to that of sample) into 500 mL erlenmeyer and
add 25.0 mL 0.25vV K 2 Cr 2 7 and 5.0 mL H 2 S0 4 . Add 10 mg
HgS0 4 /mg CI in sample and swirl until dissolved. Carefully
add 70.0 mL H 2 S0 4 -Ag 2 S0 4 reagent with swirling. Add sev-
eral boiling chips, attach condenser, and reflux 2 hr. (If vol-
atile org. compds are present in sample, attach condenser prior
to addn of H 2 S0 4 -Ag 2 S0 4 reagent and add reagent thru con-
denser while cooling flask in ice bath.)
Cool, and proceed as in low level detn, 973. 46F, including
blank. Disregard reappearance of blue-green after end point is
reached.
For saline waters, prep, std curve of COD against mg C\~ /
L, using NaCl solns with intervals of <4000 up to 20,000 mg
Cr/L, carried thru entire detn.
COD, mg/L = \(B - S) x N x 8000 - 50O] x 1.20/V
where D — Cl~ correction from std curve, and 1.20 is com-
pensation factor to account for extent of CI" oxidn which is
dissimilar in org. and inorg. systems. Other symbols are de-
fined in 973.46E.
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
Method Research Study 3; Demand Analyses, 1971
(available from National Technical Information Ser-
vice, 5285 Port Royal Rd, Springfield, VA 22161, NTIS
PB230275/BE). JAOAC 56, 295(1973).
920.193
Solids in Water
Final Action
A. Total Solids
Thoroly shake sample, and pipet 100 mL unfiltered sample
into weighed Pt dish. If sample contains much suspended mat-
ter, shake, pour rapidly into 100 mL graduate, and immedi-
ately transfer to weighed Pt dish. Evap. to dryness and heat
to const wt at 100°.
B. Solids in Solution
Let sample stand until all sediment settles and filter if nec-
essary to secure perfectly clear liq. (Occasionally, clear filtrate
can be obtained only by use of alumina cream; avoid if pos-
sible.) Evap. 100-250 mL to dryness in weighed Pt dish. Heat
to const wt at 100°.
Alumina cream. — Prep, cold satd soln of alum in H 2 0. Add
NH 4 OH with constant stirring until soln is alk. to litmus, let
ppt settle, and wash by decantation with H 2 until wash H 2
gives only slight test for sulfates with BaCI 2 soln. Pour off
excess H 2 and store residual cream in g-s bottle. (Alumina
cream is suitable for clarifying light-colored sugar products or
as adjunct to other agents when sugars are detd by polariscopic
or reducing sugar methods.)
C. Ignited Residue
Ignite residue from 920.193A at 525-550° in furnace or over
burner until dish shows dull red glow and ash is white or nearly
so. Note any odor or change in color produced during ignition.
Det. wt ignited residue and calc. loss on ignition.
973.47 Organic Carbon in Water
Infrared Analyzer Method
First Action 1973
A. Principle
Carbonaceous material of water sample is oxidized to C0 2
in stream of O or air in catalytic combustion tube at 950°.
Calibrated IR analyzer measures C0 2 .
Method is applicable to 1-150 mg org. C in surface and
saline waters and domestic and industrial wastes. Preliminary
treatment of sample defines type of C measured: (/) sol., non-
volatile org. C (e.g., natural sugars); (2) sol., volatile org. C
(e.g., mercaptans); (3) insol., partially volatile C (e.g., oils);
(4) insol., particulate carbonaceous materials (e.g., cellulose
fibers); (5) sol. or insol. carbonaceous materials adsorbed or
entrapped on insol. inorg. suspended matter (e.g., oily matter
adsorbed on silt particles).
Since usefulness of method is in assessing potential O-de-
manding load of org. material, C0 3 and HCO3 carbon must
be removed before analysis or subtracted from final result.
Distd H 2 analyzed by 28 analysts in 21 laboratories showed
following results on exact increments of oxidizable org. compds:
Added total
Std deviation
Bias
org. C,
mg/L
%
mg/L
% mg/L
4.9
107
80
8
3.9
8.3
+ 15 0.75
+ 1 1.1
B. Preparation of Sample
Glass bottles are preferable storage containers but polyeth-
ylene and Cubitainers (Hedwin Corp., 1209 E Lincoln Way,
La Port, IN 46390) may be used if tests show no contribution
of C to samples. Keep interval between collection and analysis
at min., store at 4°, and protect from light and O. If samples
cannot be analyzed within 2 hr, acidify to pH <2 with HCl
or H 2 S0 4 .
C. Apparatus
(a) Organic carbon analyzer. — Dow-Beckman Carbona-
ceous Analyzer (single channel) or Model No. 915B (dual
channel) (Beckman Industrial Corp., 600 S Harbor, LaHabra,
CA 90631), or equiv., with air pump, purification train, flow
controls, nondispersive-type IR stream analyzer specific for C0 2 ,
and recorder.
(b) Syringes. — (1) 0-50 fxL, needle opening ca 150 |xm,
Hamilton No. 705N, or equiv.; (2) 0-500 uX, needle opening
ca 400 |mm, for samples with large particulates, Hamilton No.
750N, or equiv.; or (3) push button syringes which ensure uni-
formity of injection rate, 20 or 200 |jlL size, Hamilton No.
CR700-20 or CR700-200, or equiv.
318
Waters; and Salt
AOAC Official Methods of Analysis (1990)
D. Reagents
(Caution: See safety notes on asbestos.)
(a) Water. — For diln of samples and prepn of stds, blanks,
and reagents. Use C0 2 -free, double distd H 2 0; do not use H 2
purified by ion exchange.
(b) Organic carbon std solas. — (/) Stock phthalate soln. —
1000 mg C/L. Dissolve 0.2128 g KH phthalate, 936.16B(c),
in H 2 0, and dil. to 100 mL. (2) Working solns. — Prep, solns
contg 10, 20, 30, 40, 50, 60, 80, and 100 mg C/L by dilg
1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, and 10.0 mL stock soln to
100 mL with H 2 0.
(c) Carbonate carbon std solns. — (1) Stock soln. — 1000
mg C/L. Dissolve 0.3500 g NaHC0 3 and 0.4418 g Na 2 C0 3
in H 2 and dil. to 100 mL. (2) Working solns. — Prep, iden-
tical series with concns as in (b)(2).
(d) Packing for total carbon tube. — Dissolve 20 g
Co(N0 3 ) 2 .6H 2 in 50 mL H 2 and add to 15 g long fiber
asbestos in porcelain evapg dish. Mix, and evap. to dryness
on steam bath. Place in cold furnace, heat to 950°, and hold
at this temp. 1-2 hr. Cool, break up any lumps, and mix ad-
equately but not excessively. With combustion tube held ver-
tically, taper joint up, add ca I cm untreated asbestos and then
ca 1 g catalyst transferred in small amts with forceps. As it is
added, tap or push material with 6 mm glass rod, using only
wt of rod to compress material. Do not force packing. When
completed, length of packing should be ca 5-6 cm. Test packed
tube by measuring flow rate of gas thru it at room temp, and
at 950°. Rate should drop <20%.
(e) Packing for carbonate tube (dual channel instru-
ment). — Place small wad of quartz wool or asbestos near exit
of carbonate evolution tube. From entrance end add 6-12 mesh
quartz chips to length of 10 cm. Add H 3 P0 4 while holding
tube vertically and let excess drain.
E. Adjustment of Instrument
Turn on 1R analyzer, recorder, and furnaces, setting total C
furnace at 950° and carbonate furnace at 175°. Let warm up
>2 hr; leave on continuously for daily operation. Adjust O
flow to 80-100 mL/min thru total C tube. With recorder set
at appropriate mv range, adjust amplifier gain so that 20 jjlL
sample of 100 mg C/L std gives peak ht ca half scale. Noise
level should be <0.5% full scale; if higher, analyzer or re-
corder may need servicing.
If single channel unit is equipped with large diam. com-
bustion tube and dual channel unit with Hastalloy tube for total
C channel, use 100 uJL sample in range 1-30 mg C/L.
F. Calibration
(a) Dual channel instrument. — Rinse syringe several times
with std soln, fill, and adjust to 20 |xL. Wipe off excess with
soft paper tissue, taking care that no lint adheres to needle.
Remove plug from syringe holder, insert syringe, and inject
soln into tube with single, rapid movement of index finger.
Leave syringe in holder until flow rate returns to normal; then
replace it with plug. Run duplicate detns on each std soln and
on blank. Read ht of each peak. Let recorder return to baseline
between injections. Subtract blank from each peak and prep,
std curve of corrected peak ht against mg C/L.
Turn 4-way valve to direct flow thru low temp, tube and
analyzer. Adjust flow to 80-100 mL/min and let baseline sta-
bilize. Inject in duplicate 20 \xL each of 20, 40, 60, 80, and
100 mg inorg. C/L std solns and blank. Prep, std curve of
corrected peak ht against mg inorg. C/L.
(b) Single channel instrument. — Prep, std curve as in (a),
par. I.
G. Determination
(a) Dual channel instrument. — Mix sample thoroly and dil.
to bring C content within range of std curve. Inject 20 uX
sample in duplicate as in 973.47F(a) and det. peak hts cor-
responding to total and inorg. C. Convert to concn and subtract
inorg. C from total C to obtain total org. C. Results may be
verified by operating unit as single channel system, injecting
acidified, N -purged sample into high temp, furnace, and com-
paring results.
Filter 100 mL aliquot sample thru prerinsed 0.45 |mm fritted
glass filter and repeat detn to obtain dissolved C values. Sub-
tract dissolved or inorg. C to obtain dissolved org. C. Results
may be verified by operating unit as single channel system,
injecting acidified, N -purged, filtered sample into high temp,
furnace, and comparing results.
(b) Single channel instrument. —Transfer 10-15 mL sam-
ple to 30 mL beaker. If sample is not acid-preserved, add 2
drops HC1 to reduce pH to <2 and purge with C0 2 -free N ca
5-10 min. (Do not use plastic tubing.) Place beaker on mag.
stirrer and withdraw 20 |ulL aliquot while stirring. Inject as in
973.47F(a). Prep, and inject filtered samples. Calc. total, in-
org., and dissolved C as in (a).
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
Method Research Study 3; Demand Analyses, 1971
(available from National Technical Information Ser-
vice, 5285 Port Royal Rd, Springfield, VA 22161, NTIS
PB230275/BE). JAOAC 56, 295(1973).
973.48 Nitrogen (Total) in Water
Kjeldahl Method
First Action 1973
A. Principle
Sample is digested with H 2 S0 4 to convert org. N to NH 3 ,
which is distd after alkalinization and detd by nesslerization
or titrimetry. Preserve samples by addn of 40 mg HgCI 2 /L and
store at 4°. Analyze as soon as possible, as conversion of org.
N to NH 3 may occur even with preservation.
Method is applicable to surface and saline waters and do-
mestic and industrial wastes. Some industrial wastes contg ma-
terials such as amines, nitro compds, hydrazones, oximes,
semicarbazones, and some refractory tertiary amines may not
be converted to NH 3 .
Natural H 2 analyzed by 31 analysts in 20 laboratories showed
the following results on exact increments of org. N:
Added,
mg N/L
Std deviation
B
as
Method
%
mg N/L
%
mg N/L
Colorm.
Colorm.
Titr.
Titr.
0.20
0.31
4.10
4.61
100
81
26
26
0.20
0.25
1.06
1.19
+ 15.5
+ 5.5
+ 1.0
- 1.7
0.03
0.02
0.04
-0.08
B. Apparatus
(a) Digestion apparatus. — See 920.02B(a).
(b) Distillation apparatus. — See 920.026(b); or use all-glass
app. with 800 or 1000 mL distg flask and 500 mL g-s erlen-
meyers, marked at 350 and 500 mL, as receivers. Prep, for
use by distg mixt. of NaOH-Na 2 S 2 03 soln and H 2 (1 + 1)
until distillate is NH 3 -free by Nessler reagent, (j). Repeat each
time app. is out of service ^4 hr.
AOAC Official Methods of Analysis (1990)
Nitrogen
319
(c) Nessler tubes. — Matched, ca 300 mm long, 17 mm id,
and marked at 225 ± 1.5 mm inside measurement from bot-
tom.
(d) Spectrophotometer or filter photometer. — For use at 425
nm.
C. Reagents
(Caution: See safety notes on mercury, mercury salts, and toxic
dusts.)
(a) Water. — Distd, NH 3 -free. Pass thru ion exchange col-
umn of mixed strongly acidic cation and strongly basic anion
exchange resins. Regenerate resins according to manufactur-
er's instructions.
(b) Mercuric sulfate so In. — Dissolve 8 g red HgO in 50 mL
H 2 S0 4 (1+5) and dil. to 100 mL with H 2 0.
(c) Digestion soln. — Dissolve 267 g K 2 S0 4 in 1300 mL H 2
and add 400 mL H 2 S0 4 . Add 50 mL HgS0 4 soln, (b), and
dil. to 2 L.
(d) Sodium hydroxide-sodium thiosulfate soln. — Dissolve
500 g NaOH and 25 g Na 2 S 2 3 .5H 2 in H 2 and dil. to 1 L.
(e) Phenolphthalein indicator soln. — Dissolve 5 g phthln in
500 mL alcohol or isopropanol and add 500 mL H 2 0. Add
0.02JV NaOH until faint pink.
(f) Sulfuric acid std soln. — 0.02A/. Prep, and stdze as in
890.01. 1.00 mL - 0.28 mg N.
(g) Ammonia std solns. — (/) Stock soln. — 1 .00 mg N/mL.
Dissolve 3.819 g NH 4 C1 in H 2 and dil. to 1 L. (2) Working
soln. — 0.01 mg N/mL. Dil. 10 mL stock soln to 1 L.
(h) Boric acid soln. — Dissolve 20 g H3BO3 in H 2 and dil.
to 1 L.
(i) Mixed indicator. — Mix 2 vols 0.2% ale. Me red with 1
vol. 0.2% ale. methylene blue. Prep, fresh every 30 days. SDA
3-A or 30 denatured alcohol may be used.
(j) Nessler reagent. — Dissolve 100 g Hgl 2 and 70 g KI in
small amt H 2 0. Add slowly, with stirring, to cooled soln of
160 g NaOH in 500 mL H 2 0, and dil. to 1 L. Reagent is stable
1 year if stored in Pyrex container out of direct sunlight. Re-
agent should give characteristic color, but no ppt, with 0.04
mg NH 3 -N in 50 mL H 2 within 10 min.
D. Digestion and Distillation
Det. sample size as follows:
mg N/L
mL sample
0- 5
500
5~ 10
250
10- 20
100
20- 50
50.0
50-100
25.0
Place sample, or residue from NH 3 detn (for org. Kjeldahl N
only), into 800 mL Kjeldahl flask. Dil., if necessary, to 500
mL and add 100 mL digestion soln, (c). Boil until S0 3 fumes
are evolved and soln becomes colorless or pale yellow. Cool,
and dil. with 300 mL H 2 0. Add NaOH-Na 2 S 2 3 soln slowly
down neck of tilted flask to underlay acid soln in amt sufficient
to make final soln strongly alk. as shown by phthln (60 mL
NaOH-Na 2 S 2 3 soln will neutze 20 mL H 2 S0 4 ). Connect flask
to condenser, with tip of condenser dipping into 50 mL 2%
H3BO3 soln in 500 mL g-s erlenmeyer. If soln is to be titrd,
100 or 200 mL H 3 B0 3 may be used. Mix solns and distil 300
mL at 6-10 mL/min. If NH 3 concn is >\ mg/L, det. titri-
metrically, 973.48E; if less, det. colorimetrically, 973. 48F.
E. Titrimetric Determination
Add 3 drops mixed indicator, (i), to distillate and titr. with
0.02N H 2 S0 4 , (f ), matching end point against blank contg same
vol. NH 3 -free H 2 0, H3BO3 soln, and indicator.
mg Total N/L = [(mL std H 2 S0 4 for sample
- mL std H 2 S0 4 for reagent blank) x normality std H 2 S0 4
x 14.01 X 1000]/mL sample digested
F. Colorimetric Determination
Prep, series of stds contg 0.0, 0.2, 0.5, 1.0, 1.5,2.0,3.0,
and 4.0 mL NH 3 working std soln, (g)(2), dild to 50 mL with
NH 3 -free H 2 (contains 0.0, 0.04, 0.10, 0.20, 0.30, 0.40, 0.60,
and 0.80 mg NH 3 N/L). Add 1 mL Nessler reagent, (j), and
mix. After 20 min, read A at 425 nm against 0.0 (blank) std,
and plot A against concn to obtain std curve. Distil 1 or more
high and low std solns daily to ensure adequate recoveries.
As estd by preliminary detn, det. NH 3 in 50 mL aliquot, or
aliquot dild to 50 mL, as above, and read NH 3 concn from std
curve.
mg Total N/L
= [(mg NH 3 -N from curve X 1000)/ mL sample
taken for distn] X (mL final distillate,
including H 3 B0 3 soln/ mL distillate taken for nesslerization)
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
Method Study No. 2; Nutrient Analyses, Manual Meth-
ods, 1970 (available from National Technical Infor-
mation Service, 5285 Port Royal Rd, Springfield, VA
22161, NTIS PB230828/BE). JAOAC 56, 295(1973).
CAS-7727-37-9 (nitrogen)
973.49 Nitrogen (Ammonia) in Water
Colorimetric Method
First Action 1973
A. Principle
Sample buffered at pH 9.5 is distd into H3BO3 soln. De-
pending upon concn, NH 3 is detd colorimetrically (0.05-1.0
mg N/L) by nesslerization or titrimetry (1.0-25 mg N/L).
Hg, if present as preservative, and residual CI must be re-
moved by addn of Na 2 S 2 3 before distn.
Method is applicable to surface and saline waters and do-
mestic and industrial wastes. A number of volatile amines will
cause turbidity with Nessler reagent. Some volatile compds,
such as certain ketones, aldehydes, and alcohols, may cause
off color on nesslerization. Some of these, such as HCHO,
may be eliminated by boiling at pH 2-3 before distn. Volatile
compds, such as hydrazine, influence titrimetric results.
Natural and distd H 2 analyzed by 24 analysts in 16 labo-
ratories showed the following results on exact increments of
ammonium salt:
Added,
Std
deviation
Bias
mg
mg
mg
Method
Type
N/L
%
N/L
%
N/L
Colorm.
Distd.
0.21
58
0.122
- 5.54
-0.01
Colorm.
Nat.
0.26
27
0.070
-18.12
-0.05
Titr.
Distd.
1.71
14
0.244
+ 0.46
+0.01
Titr.
Nat.
1.92
15
0.279
- 2.01
-0.04
B. Apparatus
See 973.488(b), (c), and (d).
C. Reagents
See 973.48C(a), (f)-(j), and following:
(a) Borate buffer.— pH 9.5. Add 88 mL 0. IN NaOH to 500
mL 0.025M Na 2 B 4 7 (5.0 g anhyd. salt/L), and dil. to 1 L.
320 Waters; and Salt
AOAC Official Methods of Analysis (1990)
(b) Sodium hydroxide soln. — IN. Dissolve 40 g NaOH in
NH 3 -free H 2 and dil. to 1 L.
(c) Dechlorinating reagent. — Dissolve 3.5 g Na 2 S 2 3 in NH 3 -
free H 2 and dil. to 1 L. 1 mL will remove 0.5 mg residual
CI in 500 mL sample.
D. Distillation
Add 500 mL NH 3 -free H 2 and few boiling chips previously
treated with NaOH soln to Kjeldahl distg flask.
Adjust 400 mL sample to pH 9.5 with IN NaOH, using pH
meter or short range test paper. If sample contains residual CI,
remove by adding equiv. amt dechlorinating reagent, (c).
Transfer to distg flask and add 25 mL buffer, (a). Distil 300
mL at 6-10 mL/min into 50 mL H 3 B0 3 soln, 973.48C(h).
Dil. distillate to 500 mL in receiving flask. Det. NH 3 in 50
mL aliquot as in colorimetric detn. If NH 3 concn is >1 mg/
L, det. titri metric ally, 973. 49E; if less, det. colorimetrically,
973.49F.
E. Titrimetric Determination
Proceed as in 973. 48E, using remaining 450 mL distillate.
mg NH 3 -N/L = (mL 0.02/V H 2 S0 4
x 1000) /equiv. mL sample in aliquot titrd
F. Colorimetric Determination
Proceed as in 973.48F.
mg NH 3 -N/L
= (NH 3 concn from std curve X 1000)/
(0.8 X mL distillate taken for detn)
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
Method Study No. 2; Nutrient Analyses, Manual Meth-
ods, 1970 (available from National Technical Infor-
mation Service, 5285 Port Royal Rd, Springfield, VA
22161, NTIS PB230828/BE). JAOAC 56, 295(1973).
973.50 Nitrogen (Nitrate) in Water
Brucine Colorimetric Method
First Action 1973
A. Principle
Nitrate ion reacts with brucine in H 2 S0 4 at 100° to form
colored compd whose A is measured at 410 nm. Temp, control
of reaction is critical. Applicable to 0.1-2 mg NO3-N/L in
surface and saline waters and domestic and industrial wastes.
Org. matter developing color with H 2 S0 4 and natural color
are compensated for by blank; effect of salinity is compensated
for by addn of NaCl. Strong oxidizing and reducing agents
interfere. Det. presence of free CI with o-tolidine reagent.
Eliminate residual CI by addn of NaAs0 3 soln. Effect of Fe +2 ,
Fe +3 , and Mn +4 is negligible at <1 mg/L.
Natural H 2 analyzed by 27 analysts in 15 laboratories showed
the following results on exact increments of inorg. nitrate:
Added,
Std deviation
Bias
mg N/L
%
mg N/L
%
mg N/L
0.16
58
0.092
-6.8
-0.01
0.19
44
0.083
+ 8.3
+0.02
1.08
23
0.245
+4.1
+0.04
1.24
17
0.214
+ 2.8
+ 0.04
(b) Tubes. — Matched tubes for conducting reaction and
measuring A .
(c) Racks. — Neoprene, wire coated, evenly spaced, to per-
mit uniform flow of bath H 2 between tubes.
(d) Water baths.— (1) 100°.— Boiling H 2 bath of suffi-
cient size so that when tubes are inserted, temp, drop is <1—
2°. Should have tight-fit cover, preferably of gable construc-
tion, with circulator or stirrer to maintain uniform temp. Uni-
form temp, control of this bath is critical. (Caution: Check
H 2 bath to assure it is electrically grounded.) (2) 10-15°. —
For cooling tubes.
C. Reagents
(a) Water. — Use distd or deionized H 2 for prepn of all
reagents and stds.
(b) Salt soln.— Dissolve 300 g NaCl in H 2 and dil. to
1 L.
(c) Sulfuric acid. — UN. Carefully add 500 mL H 2 S0 4 to
125 mL H 2 0. Cool, and keep tightly stoppered.
(d) Brucine -sulfanilic acid reagent. — Dissolve 1 g brucine
sulfate. 7H 2 and 0.1 g sulfanilic acid.H.O in 70 mL H 2 0.
Stored in dark bottle at 5°, soln is stable several months. Slowly
developing pink does not affect usefulness.
(e) Nitrate std solns. — (1) Stock soln. — 100 mg N/L. Dis-
solve 0.7218 g KNO3 in H 2 and dil. to 1 L. (2) Working
soln. — 1 mg/L. Dil. 10 mL stock soln to 1 L. Prep, fresh
weekly.
D. Determination
(Caution: See safety notes on pipets and sulfuric acid.)
Preserve samples with 40 mg HgCl 2 /L and store at 4°. Ad-
just pH to ca 7 with HO Ac (1 +3) and, if necessary, filter
thru 0.45 |xm filter.
Prep, set of matched tubes for blanks, stds, and samples. If
necessary to correct for color or for org. matter which will
cause color on heating, add extra set of tubes to which all
reagents except brucine will be added.
Pipet 10 mL sample, or aliquot dild to 10 mL, into sample
tubes. For saline sample, add 2.0 mL 30% NaCl soln to sam-
ples, stds, and blank tubes. Swirl tubes and place in 0-10°
bath. Pipet 10 mL 137V H 2 S0 4 into each tube and swirl. Let
all tubes come to thermal equilibrium. Pipet 0.5 mL brucine
reagent to all tubes except color control tubes and swirl. Then
place entire rack contg all tubes in boiling H 2 bath for exactly
25 min. Remove rack and transfer to cold H 2 bath and let
cool to 20-25°. Dry tubes and read A against reagent blank at
410 nm.
Prep, set of stds contg 0.1-2 mg N/L and conduct stds along
with samples. Color may not follow Beer's law. If necessary,
subtract A of color controls from A of samples.
Refs,: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
Method Study No. 2; Nutrient Analyses, Manual Meth-
ods, 1970 (available from National Technical Infor-
mation Service, 5285 Port Royal Rd, Springfield, VA
22161, NTIS PB230828/BE). JAOAC 56, 295(1973).
973.51
0. Apparatus
(a) Spectrophotometer or filter photometer. — Capable of
accommodating 25 mm diam. tubes and measuring A at 410
Chloride in Water
Mercuric Nitrate Method
First Action 1973
A. Principle
Chloride titrd with mercuric ions forms sol., slightly dis-
sociated HgCl 2 . In pH range 2.3-2.8, diphenylcarbazone in-
AOAC Official Methods of Analysis (1 990)
Elements
321
dicates end point by forming purple complex with excess Hg +2 .
Xylene cyanol FF serves as pH indicator and background color
to facilitate end point detection. NaHC0 3 added to both blank
and sample followed by const amt of HN0 3 added with in-
dicators provides pH of 2.5 ± 0.1. Increasing strength of ti-
trant and modifying indicator mixt. permits detn of high CI
concns common in waste water.
Br and I titr. as chloride. Chromate, Fe +3 , and S0 3 ~ 2 in-
terfere when present at >10 mg/L. Sulfites may be removed
with 0.5-1 mL H 2 O 2 /50 mL sample. Methods are applicable
to drinking, surface, and saline waters, and domestic and in-
dustrial wastes at all CI concns. However, to avoid large titrn
vols, use sample contg <20 mg Cl/50 mL.
Synthetic H 2 samples analyzed by 42 analysts in 18 lab-
oratories showed the following results on exact increments of
CI:
Std deviation
Bias
Added,
mg Cl/L
%
mg Cl/L
%
mg Cl/L
17
9.1
1.54
+ 2.2
+0.4
18
7.3
1.32
+3.5
+0.6
91
3.2
2.92
+ 0.1
+0.1
97
3.3
3.16
-0.5
-0.5
382
3.1
11.7
-0.6
-2.3
398
3.0
11.8
-1.2
-4.7
B. Reagents
(a) Sodium chloride std soln. — 0.01417V. Dissolve 824.1
mg NaCl, dried at 140°, in Cl-free H 2 0, and dil. to 1 L. 1
mL = 0.500 mg CI.
(b) Chlorine-free water. — Red i std or deionized.
For Low Chloride Concentration
(c) Indicator-acidifier reagent. — (Neutzes 150mgCaCO 3 /
L in 100 mL sample.) Dissolve, in order given, 250 mg s-
diphenylcarbazone, 4.0 mL HN0 3 , and 30 mg xylene cyanol
FF in 100 mL alcohol or isopropanol. Store in dark bottle in
refrigerator. For routine analysis of samples with very high or
low alky, HN0 3 concn may be adjusted so that final pH when
added to samples is 2.5 ± 0.1.
(d) Mercuric nitrate std soln. — 0.01417V. Dissolve 2.3 g
Hg(N0 3 ) 2 or 2.5 g Hg(N0 3 ) 2 .H 2 in 100 mL H 2 contg 0.25
mL HN0 3 . Dil. to just under 1 L. Stdze as in 973.51C(a),
using 5.00 mL aliquot NaCl std soln, (a), and 10 mg NaHC0 3 ,
dild to 100 mL. Adjust soln to exactly 0.014 W and perform
final stdzn. Store in dark bottle away from light. 1 mL = 0.500
mgCl.
For High Chloride Concentration
(e) Mixed indicator.— Dissolve 5 g s-diphenylcarbazone and
0.5 g bromophenol blue in 750 mL alcohol or isopropanol and
dil. to 1 L with same sol v.
(f) Mercuric nitrate std soln. — 0.141/V. Dissolve 25 g
Hg(N0 3 ) 2 .H 2 in 900 mL H 2 contg 5.0 mL HNO3. Dil. to
just under 1 L. Stdze as in 973.51C(b), using 25.00 mL ali-
quots NaCl std soln, (a), and 25 mL H 2 0. Adjust soln to ex-
actly 0.1 417V and perform final stdzn. Store in dark bottle away
from light. 1 mL = 5.00 mg CI.
C. Determination
(a) For low chloride (drinking water). — To <100 mL sam-
ple contg ^10 mg CI, add 1.0 mL indicator-acidifier, (c). Color
should be green-blue. If not, adjust pH of sample to 8 before
addn of reagent. Titr. with 0.014 17V Hg(N0 3 ) 2 to definite pur-
ple end point. (Soln becomes blue few drops before end point.)
Det. blank by titrn of equal vol. H 2 contg 10 mg NaHC0 3 .
(b) For high chloride. — To 50.0 mL sample (5.00 mL if
^5 mL titrant needed) in 150 mL beaker, add 0.5 mL mixed
indicator, (e), and mix well. Color should be purple. Add 0. 17V
HNO3 dropwise until just yellow. Titr. with 0.1417V Hg(N0 3 ) 2
to first permanent dark purple. Det. blank by titrn of equal
vol. H 2 0.
(c) Calculation. —
mg Cl/L = [(mL sample titrn - mL blank titrn) x
normality Hg(N0 3 ) 2 X 35,340]/mL sample
mg NaCl/L = (mg Cl/L) X 1.65
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study 1; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). JAOAC 56, 295(1973).
939.11
Fluoride in Water
Colorimetric Method
Final Action
A. Reagents
(a) Fluoride std soln. — 0.01 mg F/mL. Dissolve 2.21 g
NaF (min. purity 98%) in 1 L H 2 0. Dil. 10 mL of this soln
to 1 L.
(b) Thorium nitrate soln. — Dissolve 0.25 g Th(N0 3 ) 4 .12H 2
or 0.2 g Th(N0 3 ) 4 .4H 2 in 1 L H 2 0.
(c) Alizarin red indicator. — 0.01% aq. soln Na alizarin sul-
fonate (alizarin red S).
(d) Hydrochloric acid. — Exactly 0.Q5N.
(e) Sodium hydroxide soln. — Exactly 0.05N.
(f) Hydroxy lamine hydrochloride soln. — \.0 g/100 mL.
B. Apparatus
(a) Claisen flask. — 250 mL.
(b) Nessler tubes. — 6 long-form 50 mL tubes with double
optically plane disks fused to tubes. Match tubes for length
and test for optical similarity as follows: Add ca 40 mL H 2 0,
1 mL indicator, 2 mL 0.05^ HCl, and H 2 to mark on tube.
To 1 tube add amt of Th(N0 3 ) 4 soln such that, after dilg to
mark and mixing, color is barely changed to faint pink. Note
amt of Th(N0 3 ) 4 soln used. Add same amt of Th(N0 3 ) 4 soln
to each of remaining 5 tubes. Reject tubes showing detectable
differences in shade or intensity.
See also 944.08C
C. Preparation of Sample
If sample has odor of H 2 S, oxidize with 0.1 mL 30% H 2 2
soln before evapn.
Place 100 mL sample in porcelain or Pt dish, make alk. to
phthln with 10% NaOH soln (avoid excess), and evap. to 20
mL over burner at temp, just below bp. During evapn keep
sample alk. by adding small amts of 0.05N NaOH from time
to time. Transfer the 20 mL evapd sample to Claisen flask
contg glass beads or boiling tube previously rinsed with boil-
ing 10% NaOH soln to eliminate all traces of gelatinous Si0 2
accumulating in flask.
Place flask contg sample on insulating board (15 x 15 x
0.6 cm with 2.5 cm center hole) over burner adjusted for me-
dium flame. Close straight neck of flask with 2-hole rubber
stopper thru which pass thermometer and stem of small sep-
arator with outlet constricted to 2 mm diam. (Adjust thermom-
eter and outlet tube of separator to extend almost to bottom of
322
Waters; and Salt
AOAC Official Methods of Analysis (1990)
flask.) Close other neck of flask with solid rubber stopper.
(Alternatively, all -glass distn assembly may be used.)
Connect flask with H 2 condenser; add 20 mL 60% HC10 4
(Caution: See safety notes on perchloric acid.) to flask, rinsing
evapg dish and separator; then add amt of satd AgCl0 4 soln
that will ppt chlorides (detd previously by titrn with std AgN0 3
soln), and distil at 132 ± 3°, adding H 2 dropwise thru sep-
arator to maintain temp, during distn. Collect nearly 200 mL
distillate. Dil. to vol. (200 mL) and mix well. To det. acidity,
use 40 mL distillate, add 1 mL indicator, mix thoroly, and
note mL 0.05jY NaOH required for neutzn.
Repeat prepn and distn, using 100 mL H 2 in place of sam-
ple, to det. blank.
D. Determination
Prep. 1 std, 1 color comparison tube, and 1 or more sample
tubes as follows:
(a) Color comparison tube. — To 40 mL H 2 add 2 mL 0,057V
HO, 1 mL alizarin red indicator, 1 mL NH 2 OH.HCl soln, and
enough Th(N0 3 ) 4 soln to give faint but definite pink end point.
Compare all end point colors with this color.
(b) Sample tube. — To sample tube contg 40 mL distillate
add 1 mL indicator, I mL H 2 NOH.HCl soln, and vol. 0.05iV
HC1 such that total vol. acid in tube (acidity previously detd
plus vol. 0.057V HC1 added) equals 2 mL 0.057V HCl. Dil. to
vol. and mix. If in preliminary acidity detn it is found that the
40 mL distillate requires >2 mL 0.05/V NaOH soln for neutzn,
do not add the HCl soln to sample tube, but add to std tube
same amt of acid as was found present in sample tube. If 40
mL distillate requires >5 mL 0.057V NaOH, repeat distn under
conditions favorable to low acidity. From 10 mL buret, grad-
uated to 0.05 mL, add Th(N0 3 ) 4 soln with frequent mixing
until faint pink appears, comparable to comparison tube, (a).
Note vol. Th(N0 3 ) 4 soln used.
(c) Std tube. — To std tube contg 40 mL H 2 add 1 mL
indicator, 1 mL H 2 NOH.HCl soln, and >2 mL 0.057V HCl,
as was required in sample tube in (b). If aliquot chosen for
detn already contains 2-5 mL 0.057V acid, add exactly same
amt to std tube. Add exactly same amt of Th(N0 3 ) 4 soln as
was added to sample tube. To std tube (now more highly col-
ored than sample tube), add std F soln from 10 mL buret with
mixing until color matches that of sample tube. Dil. contents
of both std and sample tubes to same vol. Mix soln in each
tube and let all air bubbles escape before making color com-
parisons. Check end point by adding 1-2 drops std F soln to
std tube. Distinct color change should develop.
£. Calculation
Subtract mL F soln required by blank from mL F soln re-
quired by sample.
mL F soln x mL total distillate X 10
= F (ppm)
mL aliquot titrd x wt sample taken
Example: 100 mL sample, evapd and distd to 200 mL, of
which 40 mL aliquot corresponds to 5 mL F soln, gives:
(5 X 200 X 10) /(40 X 100) - 2.5 F (ppm)
Ref.: J AOAC 22, 482(1939).
926.15* Hydrogen Sulfide in Water
lodometric Method
Final Action
Surplus 1965
920,194 Carbonate and Bicarbonate
in Water
Titrimetric Method
Final Action
To 100 mL sample add few drops phthln, and if pink is
produced, titr. with 0.05/V HCl or H 2 S0 4 , adding drop every
2-3 sec until color disappears. Multiply buret reading by fac-
tor 3 to obtain mg C0 3 ion in 100 mL. To colorless soln from
this titrn, or to original soln if no color is produced with phthln,
add 1-2 drops Me orange, continue titrn without refilling bu-
ret, and note total reading. If C0 3 is absent, multiply total
buret reading by factor 3.05 to obtain value of HC0 3 ion in
mg/100 mL. If C0 3 is present, multiply reading with phthln
by 2 and subtract from total reading of buret. Multiply differ-
ence by 3.05 to obtain HC0 3 ion in mg/100 mL. Express re-
sults as mg/L.
920.195
Silica in Water
Gravimetric Method
Final Action
See 31.016-31.017, 10th ed.
Make preliminary examination, using 100-250 mL sample,
to det. approx. amt of Ca and Mg present, in order to det. amt
of sample to be evapd for final analysis.
Evap. amt of sample equiv. to 0.1-0.6 g CaO or 0.1-1 g
Mg 2 P 2 7 (usually 1-5 L). Acidify sample with HCl and evap.
on steam bath to dryness in Pt dish. Continue drying ca 1 hr.
Thoroly moisten residue with 5-10 mL HCl. Let stand 10-15
min and add enough H 2 to bring sol. salts into soln. Heat on
steam bath until salts dissolve. Filter to remove most of Si0 2
and wash thoroly with hot H 2 0. Evap. filtrate to dryness and
treat residue with 5 mL HCl and enough H 2 to dissolve sol.
salts, as before. Heat, filter, and wash thoroly with hot H 2 0.
Designate filtrate as Soln X.
Transfer the two residues to Pt crucible, ignite, heat over
blast lamp, and weigh. Moisten contents of crucible with few
drops H 2 0, add few drops H 2 S0 4 and few mL HF, and evap.
on steam bath under hood. Repeat treatment if all Si0 2 is not
volatilized. Dry carefully on hot plate, ignite, heat over blast
lamp, and weigh. Difference between the two wts is wt Si0 2 .
Add wt residue (Fe 2 3 + A1 2 3 ) to that of A1 2 3 and Fe 2 3
obtained in 920.196. (If residue weighs >0.5 mg, BaS0 4 may
be present in sample. If so, make necessary correction and add
to wt Fe 2 3 and A1 2 3 in 920.196.)
920.196 Aluminum and Iron in Water
Gravimetric Method
Final Action
Cone. Soln X, 920.195, to 200 mL; while still hot, slowly
add NH 4 OH, stirring constantly, until alk. to Me orange. Boil,
filter, and wash 3 times with hot H 2 0, Dissolve ppt in hot HCl
(1 + 1). Dil. to ca 25 mL, boil, and again ppt with NH 4 OH.
Filter, wash thoroly with hot H 2 0, dry, ignite, and weigh as
A1 2 3 and Fe 2 3 . (In presence of H 3 P0 4 , wt of this residue
must be corrected for P 2 5 equiv. to H 3 P0 4 found in 973. 55E,
allowing for difference in vols of the water used for these detns.)
Designate filtrate as Soln Y.
CAS-7429-90-5 (aluminum)
CAS-7439-89-6 (iron)
AOAC Official Methods of Analysis (1990)
Hardness
323
920.197
Iron in Water
Final Action
A. Colorimetric Method
(Iron <1 mg; not applicable in presence of phosphates)
Fuse, in Pt crucible, ignited ppt of Fe 2 3 and A1 2 3 , 920.196,
with fused KHS0 4 , dissolve in H 2 0, and ppt Fe and Al with
NH 4 OH. Filter, dissolve ppt on filter paper in HC1 and HNO3,
dil. soln, add 3 mL 5% NH 4 SCN soln, dil. to suitable vol.,
and compare color developed with that of calibrated color disks
or stds contg known aints of Fe treated similarly.
B. Titrimetric Method
(Caution: See safety notes on hydrogen sulfide.)
Fuse residue of Fe 2 3 and A1 2 3 , 920.196, in Pt crucible
with ca 1 g fused KHSO4. (Fusion takes only few min, and
must not be continued beyond time actually needed.) When
fusion is complete, set crucible aside to cool. Add H 2 S0 4
(1 + 4) and heat crucible until fused mass dissolves. Evap.
on steam bath as far as possible; then heat gradually until copi-
ous fumes of S0 3 evolve. Dissolve in H 2 and let stand on
steam bath. Cool, transfer to erlenmeyer, and dil. to such vol.
that soln contains <2.5% free H 2 S0 4 .
Pass H 2 S thru soln to reduce Fe and ppt any Pt contami-
nating residue from fusion. (Zn may be used instead of H 2 S
for reducing Fe.) Filter, wash, and again pass H 2 S thru soln
to reduce all Fe. Expel H 2 S by boiling, at same time passing
current of C0 2 thru soln. Test escaping gas with Pb(OAc) 2
paper to confirm complete removal of H 2 S. Discontinue boil-
ing and let flask cool without discontinuing current of C0 2 .
Titr. reduced Fe with std KMn0 4 soln (1 mL = 1 mg Fe) and
calc. as Fe.
CAS-7439-89-6 (iron)
973.52
Hardness of Water
First Action 1973
4. Calculation Method
Calc. hardness as sum of CaC0 3 equivs (mg/L) obtained
by multiplying concn (mg/L) found of following cations by
factor shown:
Cation
Factor
Cation
Factor
Ca
2.497
Al
5.564
Mg
4.116
Zn
1.531
Sr
1.142
Mn
1.822
Fe
1.792
EDTA Titrimetric Method
B. Principle
Ca and Mg at pH 10 in presence of dye eriochrome black
T are wine red. When completely complexed with EDTA, soln
becomes blue. Mg must be present for satisfactory end point
and is added as MgEDTA. End point sharpness increases with
pH, but high pH may cause pptn of Ca(OH) 2 or Mg(OH) 2 and
cause color changes of dye. pH of 10.0 ± 0.1 is satisfactory
compromise. Limit of 5 min for titrn minimizes pptn. Heavy
metal interference is minimized by complexing with cyanide.
Method is applicable to drinking and surface waters and do-
mestic and industrial wastes. To avoid large titrn vols, use
aliquot contg <25 mg CaCG 3 .
Synthetic H 2 samples contg exact increments of Ca and
Mg salts analyzed by 43 analysts in 19 laboratories showed
the following results:
Increment,
total hardness
Std deviation
Bias
as mg CaC0 3 /L
%
mg
CaC0 3 /L
%
mg
CaC0 3 /L
31
9.4
2.9
-0.87
- 0.003
33
7.6
2.5
-0.73
- 0.24
182
2.7
4.9
-0.19
- 0.4
194
1.5
3.0
-1.04
- 2.0
417
2.3
9.7
-3.35
-13.0
444
2.0
8.7
-3.23
-14.3
C. Reagents
(a) Buffer soln.— Dissolve 16.9 g NH 4 C1 in 143 mL NH 4 OH,
add 1.25 g MgEDTA, and dil. to 250 mL with H 2 0. (1.179
g Na 2 EDTA.2H 2 and 0.780 g MgS0 4 .7H 2 or 0.644 g
MgCl 2 .6H 2 dissolved in 50 mL H 2 may be substituted for
1.25 g MgEDTA.) Store in tightly stoppered Pyrex or plastic
bottle. Dispense from bulb-operated pipet. Discard after 1 month
or when 1-2 mL added to sample fails to produce pH 10.0 ±
0.1 at end point of titrn.
(b) Indicator. — Mix 0.5 g eriochrome black T and 100 g
NaCl to prep, dry powd mixt. If end point change is not clear
and sharp, prep, new mixt.
(c) EDTA stdsoln.-~~~0.0lM. Weigh 3.723 g Na 2 EDTA.2H 2
and dil. to 1 L with H 2 0. Stdze against Ca std soln as in
973. 52D. Store in polyethylene bottle and restdze periodically.
(d) Calcium std soln.— 1. 000 mg CaC0 3 /mL. Weigh 1.000
g CaC0 3 (primary std or special reagent low in heavy metals,
alkalis, and Mg) into 500 mL erlenmeyer. Place funnel in neck
and add, little at a time, HC1 (1 + 1) until all CaC0 3 has
dissolved. Add 200 mL H 2 and boil few min to expel C0 2 .
Cool, add few drops Me red indicator, and adjust to inter-
mediate orange with 3N NH 4 OH or HC1 (1 + 1), as required.
Transfer quant, to 1 L vol. flask and dil. to vol.
D. Determination
Dil. 25 mL sample (or such vol. as to require <15 mL ti-
trant) to ca 50 mL with H 2 in porcelain casserole, add 1-2
mL buffer soln, 250 mg NaCN (pH of soln should be 10 ±
0.1), and ca 200 mg indicator powder, and titr. with EDTA
std soln slowly, with continuous stirring, until last reddish tinge
disappears, adding last few drops at 3-5 sec intervals. Color
at end point is blue in daylight and under daylight fluorescent
lamp. Complete titrn within 5 min from time of buffer addn.
For waters of low hardness (<5 mg/L), use 100-1000 mL
sample, proportionately larger amts of reagents, microburet.
and blank of distd H 2 equal to sample vol.
Hardness (EDTA) as mg CaC0 3 /L =
T X B x 1000/mL sample
where T — mL EDTA std soln and B
1.00 mLEDTA std soln.
mg CaC0 3 equiv. to
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study 1; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). J AOAC 56, 295(1973).
324
Waters; and Salt
AOAC Official Methods of Analysis (1990)
920.198
Aluminum in Water
Gravimetric Method
Final Action
920.200
To obtain wt Al 2 3 , in absence of phosphates, subtract from
wt Fe 2 3 and Al 2 3 , 920.196, the Fe, 920.197A or B, calcd
as Fe 2 3 . Calc. as Al.
CAS-7429-90-5 (aluminum)
920.199
Calcium in Water
Gravimetric Method
Final Action
Cone. Soln Y, 920.196, to 150-200 mL, and to this soln,
contg equiv. of ^0.6 g CaO or 1 g Mg 2 P 2 7 , add 1-2 g
H 2 C 2 4 .2H 2 and enough HC1 (1 + 1) to clear soln. Heat to
bp and neutze with NH 4 OH, stirring constantly. Add NH 4 OH
in slight excess and let stand 3 hr in warm place. Filter su-
pernate and wash ppt once or twice by decantation with 1%
(NH4) 2 C 2 04 soln. Dissolve ppt in HCl (1 + 1), dil. to 100-
200 mL, add little more H 2 C 2 4 , and ppt as above. After let-
ting ppt stand 3 hr, filter, wash with 1% (NH 4 ) 2 C 2 4 soln, dry,
ignite, heat over blast lamp at >950°, and weigh as CaO and
SrO. From this wt subtract wt SrO equiv. to the Sr, 911.03.
Difference is wt CaO. Calc. as Ca. Designate combined fil-
trates and washings as Soln Z.
As check on CaO, evap. to dryness filtrate from the Sr(N0 3 ) 2
in 911.03, beginning "Filter, and wash with ether-alcohol mixt.
..." Dissolve the Ca(N0 3 ) 2 in H 2 0, ppt as oxalate, filter,
wash, ignite at 950°, and weigh as CaO. CaO x 0.7147 =
Ca.
CAS-7440-70-2 (calcium)
911.03
Strontium in Water
Spectroscopic Method
Final Action
Dissolve oxides, 920.199, in HN0 3 (1 + 1) and test with
spectroscope for Sr. If Sr is present, transfer HN0 3 soln to
small erlenmeyer. Evap. nearly to dryness over low flame, and
heat 1-2 hr at 150-160° after H 2 is evapd. Break up dried
material with stirring rod and add 10-15 mL mixt. of absolute
alcohol and ether (1 + 1) to dissolve the Ca(N0 3 ) 2 . Cork flask
and let stand with frequent shaking ^2 hr. Decant soln thru
5.5 cm filter, reserving filtrate. Wash residue several times by
decantation with small portions of the ether- alcohol mixt. Dry
residue and paper, and repeatedly wash paper with small por-
tions of hot H 2 0, collecting filtrate in flask contg main portion
of Sr(N0 3 ) 2 residue. Add 1 or 2 drops HN0 3 (1 + 1), evap.,
dry, pulverize, and treat with 10-15 mL ether-alcohol mixt.
Cork flask and let stand ca 12 hr, shaking occasionally.
Filter, and wash with ether-alcohol mixt. until few drops
filtrate evapd on watch glass leave practically no residue. Dry
paper and ppt. Dissolve Sr(N0 3 ) 2 in few mL hot H 2 0. Add
few drops H 2 S0 4 and then add vol. alcohol equal to vol. soln
and Jet stand 12 hr. Filter, ignite, weigh as SrS0 4 , and calc.
to Sr. Test spectroscopically for Ca and Ba. If these elements
are present, det. amt and make necessary correction.
Refs.: Chem. Ztg. 35, 337(1911). JAOAC 1, 97, 458(1915);
2, 113(1916).
CAS-7440-24-6 (strontium)
Magnesium in Water
Gravimetric Method
Final Action
Cone. Soln 2, 920.199, to 200 mL, acidify with HCl (1 +
1), and add 2-3 g (NH 4 ) 2 HP0 4 and enough HCl (1 + 1) to
produce clear soln when all (NH 4 ) 2 HP0 4 is dissolved. When
cold, make slightly alk. with NH 4 OH, stirring constantly. Add
2 mL excess of NH 4 OH and let stand ca 12 hr. Filter supernate
and wash 4 times by decantation with NH 4 OH (1 -I- 10). Dis-
solve ppt in HCl (1 + 1), dil. to ca 150 mL, add little
(NH 4 ) 2 HP0 4 , and ppt with NH 4 OH as before. Let stand 12 hr,
filter, wash Cl-free with NH 4 OH (1 + 10), place in porcelain
crucible, ignite, heat over blast lamp, and weigh as Mg 2 P 2 7 .
Calc. to Mg. Mg 2 P 2 7 X 0.21842 - Mg.
CAS-7439-95-4 (magnesium)
974.27 Cadmium, Chromium,
Copper, Bron, Lead, Magnesium,
Manganese, Silver, Zinc in Water
Atomic Absorption Spectrophotometric Method
First Action 1974
Final Action 1984
A. Principle
Metals in soln are detd directly by A A spectrophotometry;
suspended metals are sepd by membrane filtration, or suspen-
sion is dissolved and analyzed; Pb and Cd in low concn are
chelated, coned, and then extd with org. sol v. prior to A A
detn. Applicable to surface and saline waters, and domestic
and industrial wastes. Three synthetic water samples contg be-
tween 0.05 and 1 .0 mg each metal /L analyzed by 8-23 lab-
oratories showed results given in Table 974. 27 A.
Table
974.27A
Bias
and Standard Deviations of Determina-
tion
of Metals by Atomic Absorption
Added
mg/L
Std deviation
Bias
Metal
%
mg/L
%
mg/L
Cd
0.01
53
0.007
+ 27.5
+ 0.003
0.01 (extn)
61
0.006
0.0
0.0
0.05
8
0.004
+ 2.0
+ 0.001
0.05 (extn)
10
0.005
+ 1.2
+ 0.001
0.10
8
0.008
+ 3.4
+ 0.003
0.10 (extn)
52
0.045
-15.0
-0.015
Cr
0.05
26
0.013
- 2.3
-0.001
0.10
22
0.021
- 2.9
-0.003
0.20
12
0.024
- 3.0
-0.006
Cu
0.05
42
0.023
+ 8.3
+ 0.004
0.25
8
0.020
+ 2.2
+ 0.006
1.00
6
0.060
+ 0.6
+ 0.006
Fe
0.10
34
0.032
- 5.3
-0.005
0.30
18
0.050
- 5.0
-0.015
0.50
6
0.031
+ 1.1
+ 0.006
Pb
0.05
76
0.036
- 5.0
-0.002
0.05 (extn)
53
0.028
+ 3.0
+0.002
0.10
67
0.057
-16.0
-0.016
0.10 (extn)
55
0.053
- 5.0
-0.005
0.20
30
0.052
-14.0
-0.028
0.20 (extn)
48
0.088
- 8.0
-0.017
Mg
0.05
10
0.006
+ 8.5
+ 0.004
0.10
10
0.011
+ 8.2
+ 0.008
0.20
7
0.014
+ 5.0
+ 0.010
Mn
0.05
14
0.007
+ 6.0
+0.003
0.25
12
0.030
+ 4.4
+ 0.011
0.50
8
0.043
+ 1.3
+ 0.007
Ag
0.05
17
0.010
+ 10.6
+ 0.005
0.10
11
0.010
- 7.1
-0.007
0.20
8
0.016
+ 7.3
+ 0.015
Zn
0.05
46
0.021
- 9.3
-0.005
0.50
3
0.016
+ 1.4
+0.007
1.00
5
0.051
- 0.1
-0.001
AOAC Official Methods of Analysis (1990)
Metals 325
B. Apparatus
(Use Pyrex, quartz, or Teflon labware exclusively; clean tho-
roly with detergent and H 2 0; soak in HN0 3 (1 + 1) for 1 week;
rinse with H 2 0, dil. HN0 3 , and H 2 0, in that order. Use deion-
ized, distd H 2 whenever H 2 is specified.)
Atomic absorption spectrophotometer. — Spectrophtr capa-
ble of operating at conditions given in Table 974. 27B. Op-
erator must become familiar with settings and operations of
his app., using table only as guide. Use Boling burner for aq.
solns, and premix burner with solv. (Caution: See safety notes
on A AS.)
C. Reagents
(a) Deionized distilled water. — See 973.48C(a).
(b) Nitric acid. — Dil. 500 mL redistd HN0 3 to 1 L with
H 2 0. {Caution: Perform distn in hood with protective sash in
place.)
(c) Hydrochloric acid.—DW. 500 mL HC1 to 1 L with H 2
and distil in all-Pyrex app.
(d) Metal std solns. — (7) Stock solns. — Accurately weigh
amt of metal specified in Table 974. 27C into beaker and add
dissolving medium. When metal is completely dissolved,
transfer quant, to 1 L vol. flask and dil. to vol. with H 2 0. (2)
Working solns . — -Prep . daily. Dil. aliquots of stock solns with
H 2 to make >4 std solns of each element within range of
detn, Table 974. 27B. Add 1 .5 mL HNO3/L to all working std
solns before dilg to vol. Add 1 mL LaCl 3 /10 mL Mg working
std soln.
(e) Lanthanum stock soln.— 50 g La/L ca 5% HCl. Slowly
add 250 mL HCl to 58.65 g La 2 3 (99.99%, Ventron Corp.,
Alfa Products, 8 Congress St, Beverly, MA 01915, or equiv.),
dissolve, and dil. to 1 L.
(f) Ammonium pyrrolidine dithiocarbamate (APDC) soln. —
Dissolve 1 g APDC in 100 mL H 2 0. Prep, fresh daily.
D. Preparation of Sample
(a) Dissolved metals. — As soon as practicable after collec-
tion, filter known vol. sample thru 0.45 fim membrane. Use
Table 974.27B Operating Parameters
Wavelength,
Optimum
Metal
nm
Flame
range,
mg/L
Cd
328.1
Oxidizing
air-
~C2rl2
0.1
- 2
Cr
357.9
SI. reducing
air-
-CgHg
1
-200
Cu
3247
Oxidizing
air-
-C2H2
0.1
- 10
Fe
248.3
Oxidizing
air-
-C2H2
0.1
- 20
Pb
217.0
SI. oxidizing
air-
-C2H2
1
- 10
Mg a
285.2
Reducing
air-
-CgHj
0.01
- 2
Mn
279.5
Oxidizing
air-
-C2H2
0.1
- 20
Ag
328.1
Oxidizing
air-
"^2^2
0.1
- 20
Zn
213.9
Oxidizing
air-
-G2H2
0.1
- 2
a With 1%
La soln.
first 50-100 mL to rinse flask and discard. Collect filtrate and
preserve soln by adding 3 mL HN0 3 (l-fl)/L.
(b) Suspended metals. — Transfer residue and membrane from
(a) to 250 mL beaker and add 3 mL HN0 3 . Cover with watch
glass and heat gently to dissolve membrane. Increase heat and
evap. to dryness. Cool, and add 3 mL HN0 3 , and heat until
digestion is complete, generally indicated by light colored res-
idue. Add 2 mL HCl (1 + 1), and heat gently to dissolve res-
idue. Wash watch glass and beaker with H 2 and filter. Wash
filter and discard. Dil. filtrate with H 2 to concn within range
of instrument.
(c) Total metal. — Transfer aliquot of well mixed sample to
beaker and add 3 mL HNO3. Heat, and evap. to dryness. (Do
not boil.) Continue as in (b), beginning "Cool, and add 3 mL
HNO3, ..."
E. Determination
(P interference in Mg detn is eliminated by adding La stock
soln to sample and working std solns so that final dilns contain
1% La.)
(a) General method. — Set up instrument as in Table
974. 27B, or previously established optimum settings. Second-
ary or less sensitive lines (Spectrochim Acta 17, 710(1961))
may be used to reduce necessary diln, if desired. Read 4 std
solns within range before and after each group of 6- 1 2 sam-
ples, and re-establish A each time. Prep, calibration curve
from av. of each std before and after sample group. Read sam-
ple concn from plot of A against mg/L.
(b) Special extraction method. — When Pb or Cd concn is
too low for direct detn, transfer sample aliquot to 250 mL beaker
and dil. to 100 mL with H 2 0. Prep, blank and stds in same
manner. Adjust pH of sample and std solns to 2.5 with HCl,
using pH meter. Transfer quant, to 200 mL vol. flask, add 2.5
mL APDC soln, and mix. Add 10 mL methyl isobutyl ketone
and shake vigorously 1 min. Let layers sep.; then add H 2
until ketone layer is in neck of flask. (Centrfg may be nec-
essary.) Aspirate ketone layer and record readings of stds and
samples against blank. (Fuel -to-air ratio should be adjusted to
as blue a flame as possible, since org. solv. adds to fuel sup-
ply.) Prep, calibration curve from av. of each std and read
sample concn from plot (mg/L).
F. Calculations
(a) General method. —
mg Metal /L = (mg metal in aliquot/L) X F
where F - final diln/mL aliquot.
(b) Special extraction method. —
mg Metal /L - mg metal in aliquot/L
Table 974.27C Preparation of Metal Standard Solutions
Dissolving medium
(1 L total)
Metal a
Wt, g Compd
Cd
1.142
CdO
Cr
1.923
Cr0 3
Cu
1.000
Cu, electrolytic
Fe
1.000
Fe wire
Pb
1.599
Pb(N0 3 ) 2
Mg
0.829
MgO
Mn
1.583
Mn0 2
Ag
1.575
AgN0 3
Zn
1.000
Zn
5 mL redistd HN0 3
H 2 + 10 mL redistd HN0 3
5 mL redistd HN0 3
5 mL redistd HN0 3
H 2 + 10 mL redistd HN0 3
10 mL redistd HN0 3 b
10 mL HCl
H 2 + 10 mL redistd HN0 3
10 mL HNO,
a Final concn = 1000 mg/L except for Mg (500 mg/L).
b Add 1 mL La stock soln to 10 mL working std soln.
Refs.: Water Metals No. 4, Study No. 30 (1968), Analytical
Reference Service, Public Health Service (available from
National Technical Information Service, 5285 Port Royal
Rd, Springfield, VA 22161, NTIS PB215673/BE).
JAOAC 67, 421(1984).
CAS-7440-43-9 (cadmium)
CAS-7440-47-3 (chromium)
CAS-7440-50-8 (copper)
CAS-7439-89-6 (iron)
CAS-7439-92-1 (lead)
CAS-7439-95-4 (magnesium)
CAS-7439-96-5 (manganese)
CAS-7440-22-4 (silver)
CAS-7440-66-6 (zinc)
326
Waters; and Salt
AOAC Official Methods of Analysis (1 990)
977.22 Mercury an Water
Flameless Atomic Absorption Spectrophotometric Method
First Action 1977
Finai Action 1978
A, Principle
Org. Hg is oxidized to inorg. Hg by KMn0 4 , K 2 S 2 8 , and
heat. The Hg is reduced to elemental state with stannous ion,
and Hg is aerated from soln thru measuring cell in closed sys-
tem. A is measured in A A spectrophtr.
Method is applicable to detn of 0.2-10 u,g Hg/L of drink-
ing, surface, and saline waters and domestic and industrial
wastes. Interference from CI or <20 mg S/L is eliminated by
oxidn with KMn0 4 ; 10 mg Cu/L does not interfere. Analysis
without reagents will det. if absorbing interfering volatile org.
compds are present.
Natural waters analyzed by 76-82 laboratories showed the
following results on exact increments of org. and inorg. Hg
compds:
Std deviation
Bias
Added,
MS Hg/L
%
M-g Hg/L
%
H-9 Hg/L
0.21
79
0.28
+ 66
+0.14
0.27
67
0.28
+ 53
+0.14
0.51
79
0.54
+ 32
+0.16
0.60
55
0.39
+ 18
+0.11
3.4
44
1.5
+ 0.34
0.0
4.1
29
1.1
_ 7.1
-0.3
8.8
42
3.7
- 0.4
0.0
9.6
39
3.6
- 5.2
-0.5
B. Apparatus
(Rinse all glassware with chromic acid cleaning soln or HN0 3
(1 + 1) and Hg-free H 2 before use.)
(a) Atomic absorption spectrophotometer. — Equipped with
Hg hollow cathode lamp and gas flow-thru cell (Fig. 977.22),
115 x 25 (id) mm with quartz windows cemented in place.
Use at 253.7 nm with operating conditions specified by man-
ufacturer.
(b) Air pump. — Peristaltic pump capable of delivering ca
1 L air/min. Use Tygon tubing for all connections.
(c) Flowmeter. — Capable of measuring air flow of .1 L/min.
(d) Lamp, — Small reading lamp contg 60 watt bulb to pre-
vent condensation of moisture inside cell. Position lamp to warm
cell (See Fig. 977.22.). Alternatively, use Mg(C10 4 ) 2 -filled
drying tube, 977.22C(a).
(e) Digestion flask. — 250 mL flat-bottom boiling flask fit-
ted with 2-hole rubber stopper. Use straight glass frit of coarse
porosity for gas inlet.
C. Reagents
(a) Magnesium perchlorate. — Place 20 g Mg(C10 4 ) 2 in 150
X 18 (id) mm drying tube (Fig. 977.22); replace as needed.
{Caution: See safety notes on magnesium perchlorate.)
(b) Mercury absorbing media. — Add one of following to
250 mL gas-washing bottle fitted with 2-hole rubber stopper
and attach to aeration app. as by-pass (See Fig. 977.22): (1)
Potassium permanganate-sulfuric acid soln. — Equal vols O.LV
KMn0 4 and H 2 S0 4 (1 + 9). (2) Iodine-potassium iodide soln. —
0.25% I 2 -3% Kl soln. Alternatively, vent Hg vapor into ex-
haust hood.
FBG. 977.22 — Apparatus for determination of mercury by flameless atomic absorption closed recirculating system: A, reaction flask;
B, 60 watt light bulb; C, rotameter, 1 L air/min; D, absorption cell with quartz windows; E, air pump, 1 L air/min; F, glass tube
with fritted end; G, hollow cathode Hg lamp; H, atomic absorption detector; J, gas washing bottle contg 0.25% l 2 in 3% Kl soln;
K, recorder, any compatible model.
AOAC Official Methods of Analysis (1 990)
Metals
327
(c) Sodium chloride -hydroxy iamine sulfate soln. — Dissolve
120 g NaCl and 120 g (NH 2 OH) 2 .H 2 S0 4 in H 2 and di.1. to
1 L.
(d) Stannous sulfate soln. — Disperse 100 g SnS0 4 in H 2
contg 14 mL H 2 S0 4 and dil. to 1 L. Stir suspension with mag.
stirrer continuously during use.
(e) Mercury std solns. — (1) Stock soln. — 1000 u,g/mL.
Dissolve 0.1354 g HgCl 2 in 75 mL H 2 0, add 10 mL HN0 3 ,
and dil. to 100 mL with H 2 0. (2) Intermediate soln. — 10 |xg/
mL. Pipet 10 mL stock soln into 500 mL H 2 0, add 2 mL
HN0 3 , and dil. to 1 L with H 2 0. Prep, fresh daily. (3) Work-
ing soln. — 0.1 (xg/mL. Pipet 10 mL intermediate soln into
500 mL H 2 0, add 2 mL HNO3, and dil. to 1 L with H 2 0.
Prep, fresh daily.
D. Preparation of Sample
(a) Dissolved mercury. — Proceed as in 974.270(a).
(b) Suspended mercury. — Transfer residue and membrane
from (a) to digestion flask and proceed as in 977. 22E.
(c) Total mercury. — Preserve soln by adding 2 mL HNO3/
L and proceed as in 977. 22E.
E. Determination
Transfer 100 mL sample or aliquot dild to 100 mL, contg
<1.0 |xg Hg, to digestion flask. Slowly add 5 mL H 2 S0 4 and
2.5 mL HNO3, with mixing. Add 15 mL 5% KMn0 4 soln,
shake, and add addnl KMn0 4 until purple color lasts >15 min.
Add 8 mL 5% K 2 S 2 Q $ soln, heat 2 hr in 95° H 2 bath, and
cool to room temp.
Adjust output of pump to ca 1 L/min and connect app. as
in Fig. 977.22, except for gas inlet. With pump working and
spectrophtr zeroed, add 6 mL NaCl-(NH 2 OH) 2 .H 2 S0 4 soln to
reduce excess KMn0 4 (purple color disappears); let stand 30
sec, and add 5 mL SnS0 4 soln. Immediately connect digestion
flask gas inlet to aeration app. and aerate without manual ag-
itation. A will reach max. within 30 sec. Record A. When pen
levels off (ca 1 min), open by-pass valve and continue aeration
until A returns to min. value. Close bypass valve, remove stop-
per and frit from digestion flask, and continue aeration to flush
system.
Check for interfering volatile org. compds by placing same
vol. sample or dild sample into digestion flask. Connect flask
to aeration app. and aerate without manual agitation and mea-
sure A after 30 sec. Subtract A from reading obtained on sam-
ple with reagents added.
Prep, std curve by dilg 0, 0.5, 1.0, 2.0, 5.0, and 10.0 mL
aliquots Hg working std soln to 100 mL, and adding to series
of digestion flasks. Proceed as in par. 1, beginning "Slowly
add 5 mL H 2 S0 4 ..."
Plot A against |xg Hg. Det. fxg Hg in sample from curve.
fig Hg/L = W x (1000/VO
where W — \xg Hg in sample and V — mL sample.
Refs.: ASTM STP 573, 1975, pp. 566-580. JAOAC 60,
474(1977).
CAS-7439-97-6 (mercury)
Gravimetric Method
A. Reagents
(a) Ammonium dichromate soln. — Dissolve 100 g of the
S0 4 -free salt in H 2 and dil. to 1 L.
(b) Ammonium acetate soln. — Dissolve 300 g of the salt in
H 2 0, neutze with NH 4 OH, and dil. to 1 L.
(c) Dilute ammonium acetate wash soln. — Dil. 20 mL (b)
to 1 L.
(Reaction of acetate solns should be alk. rather than acid.)
B. Determination
Acidify 1-5 L portion of sample with HCl and cone, to ca
200 mL. (If ppt forms, filter off and test for Ba.) Add ca
0.5 g NH 4 C1, and ppt Fe and Al with NH4OH. Boil, filter,
and wash. To filtrate, add excess (10 mL) NH 4 OAc soln, (b),
keeping total vol. ca 200 mL. Heat to bp, and add, with stir-
ring, ca 5 mL (NH 4 ) 2 Cr 2 7 soln. Let settle and cool. Decant
clear liq. thru filter and wash ppt by decantation with dil.
NH 4 OAc soln until filtrate is no longer perceptibly colored (ca
100 mL wash soln).
Place beaker under funnel, dissolve ppt on paper with warm
HNO3 (1 + 1), using as little as possible, and wash paper.
Add little more acid to dissolve ppt in beaker, and then NH4OH
until ppt that forms no longer redissolves. Heat to bp; add,
with stirring, 10 mL NH 4 OAc soln, (b), and 2 mL (NH 4 ) 2 Cr 2 7
soln; let cool slowly, and wash ppt free of chromate with dil.
NH 4 OAc soln by decantation and filtration. Dry ppt, ignite
moderately to const wt, and weigh as BaCr0 4 . Calc. as Ba,
using factor 0.5421.
Ref.: Morse, "Exercises in Quantitative Chemistry/' p. 417.
JAOAC 4, 86(1920).
Titrimetric Method
C. Determination
Proceed as in 920.201B thru "... wash ppt free of chro-
mate with dil. NH 4 OAc soln ..." (after second pptn). Dis-
solve ppt in ca 10 mL HCl (1 + 1) and hot H 2 0. Wash filter,
dil. soln to ca 400 mL, and add ca 50 mL freshly prepd 10%
KI soln. Mix carefully and titr. liberated 1 2 after 3 or 4 min
with 0.17V Na 2 S 2 3 . 1 mL IN Na 2 S 2 3 - 4.578 mg Ba.
973.53 Potassium in Water
Atomic Absorption Spectrophotometric Method
First Action 1973
A. Principle
Method is applicable to detn of 0.01-2 mg K/L of surface
and saline waters and domestic and industrial wastes. Na may
interfere if present at much higher levels than K but effect may
be avoided by approx. matching Na concn of stds with that of
sample.
Synthetic H 2 analyzed by 19 analysts in 10 laboratories
showed the following results on exact increments of K salt:
920.201
Barium in Water
Final Action
(It is not necessary to look for Ba if sulfate is present in ap-
preciable amt unless sample contains large amt of bicarbonate
or chloride, which may hold in soln small amts of both sulfate
and Ba.)
Std deviation
Bias
Added,
mg K/L
%
mg K/L
%
mg K/L
1.5
11
0.17
+ 4.8
0.07
1.4
16
0.22
+ 6.6
0.09
8.0
8
0.64
+7.6
0.60
7.5
9
0.66
+ 8.7
0.64
20.0
6
1.11
+ 7.4
1.5
19.0
8
1.58
+ 7.4
1.4
328
Waters; and Salt
AOAC Official Methods of Analysis (1990)
B. Apparatus
Atomic absorption spectrophotometer. — Equipped with
Boling-type burner, set at 766.5 nm.
C. Reagents
(a) Deionized distilled water. — See 973.48C(a). Use for
prepn of reagents and stds, and as diln H 2 0.
(b) Potassium std solns. — (I) Stock soln. — 100 mg K/L.
Dissolve 0.1907 g KC1, dried at 110°, in H 2 0, and dil. to
1 L. (2) Working solns. — Prep. dil. std solns in range of in-
terest at time of analysis. If HN0 3 is used to preserve samples,
add corresponding amt to working std solns.
D. Determination
(Caution: See safety notes on AAS.)
Follow manufacturer's instructions for app. operation. Op-
timize conditions for max. absorption and stability. Beginning
with blank and working toward highest std, aspirate solns and
record readings. Repeat std solns and samples enough times
to secure reliable av. reading for each soln. If necessary, dil.
sample with H 2 to bring into range for direct reading.
For instruments which read directly in concn , set curve cor-
rector to read out proper concn. Otherwise, plot calibration
curve, using concn range producing absorption of 0-80%. Be-
fore plotting, convert % absorption to absorbance: A = Jog
(100/% T) = 2- log % T, where % T = 100 - % absorption.
Curves are frequently nonlinear. Increase number of stds in
that portion of curve.
Read mg K/L from calibration curve or directly from read-
out system.
mg K/L in sample - (mg K/L) in aliquot x D
D = (mL aliquot + mL H 2 added) /mL aliquot
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study I; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). JAOAC 56, 295(1973).
CAS-7440-09-7 (potassium)
973.54 Sodium in Water
Atomic Absorption Spectrophotometry Method
First Action 1973
A. Principle
Method is applicable to detn of l -200 mg Na/L in surface
and saline waters and domestic and industrial wastes.
Synthetic H 2 analyzed by 22 analysts in 12 laboratories
showed the following results on exact increments of Na salts:
Std deviation
Bias
Added,
mg Na/L
%
mg Na/L
%
mg Na/L
4.1
3.4
0.14
+ 1.9
0.07
3.8
5.0
0.19
+ 2.9
0.11
55.0
3.6
1.99
+0.9
0.5
52.0
3.7
1.93
+ 0.8
0.4
155
2.4
3.75
+ 0.0
0.0
149
2.7
3.97
-0.1
0.0
dizing air-C 2 H 2 flame. For greater sensitivity (0.005-0.2 mg/
L), use 589.0 nm line.
C. Reagents
(a) Deionized distilled water. — See 973.48C(a).
(b) Sodium std solns. — (7) Stock soln. — 1000 mg Na/L.
Dissolve 2.542 g NaCl, dried at 140°, in H 2 0, and dil. to 1
L. (2) Working solns. — Prep. dil. std solns in range of interest
at time of analysis. If HN0 3 is used to preserve samples, add
corresponding amt to working std solns.
D. Determination
Proceed as in 973. 53D, using Na parameters and std solns.
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
FWPCA Method Study 1; Mineral and Physical Anal-
yses, June 1969 (available from National Environmen-
tal Research Center, Environmental Protection Agency,
Cincinnati, OH 45268). JAOAC 56, 295(1973).
CAS-7440-23-5 (sodium)
973.55
Phosphorus in Water
Photometric Method
First Action 1973
B. Apparatus
Atomic absorption spectrophotometer. — See 973. 53B. Use
Na hollow cathode lamp, 330.2 nm, Boling burner, and oxi-
4. Definitions
(a) Phosphorus. — (P). All P present in sample, regardless
of form, measured by persulfate digestion method. (J) Ortho-
phosphate. — (P, ortho). Inorg. P, (P0 4 ~ 3 ), in sample as mea-
sured by direct colorimetric analysis. (2) Hydrolyzable phos-
phorus. — (P, hydro). P as measured by H 2 S0 4 hydrolysis
method minus orthophosphate; includes polyphosphates,
(P 2 7 )~ 4 , (P 3 O 10 )~ 5 , and some org. P. (3) Organic phospho-
rus.— (P, org) = P ~ [(P, ortho) + (P, hydro)].
(b) Dissolved phosphorus. — (P-D). P present in filtrate of
sample filtered thru 0.45 |mm pore filter, measured by persul-
fate digestion method. (7) Dissolved orthophosphate. — (P-D,
ortho). Inorg. P as measured by direct colorimetric method.
(2) Dissolved hydrolyzable phosphorus. — (P-D, hydro). P as
measured by H 2 S0 4 hydrolysis — (P-D, ortho). (3) Dissolved
organic phosphorus. — (P-D, org) - (P-D) — [(P-D, ortho)
+ (P-D, hydro)).
(c) Insoluble phosphorus. — (P-/). When sufficient amt is
present, calc. following: (I) PI = (P) ~ (P-D). (2) Insoluble
orthophosphate. — (P-I, ortho) — \(P, ortho) — (P-D, ortho)].
(3) Insoluble hydrolyzable phosphorus. — (P-I, hydro) — [(P,
hydro) — (P-D, hydro)]. (4) Insoluble organic phosphorus. —
{P-I, org) = [(P, org) - (P-D, org)].
B. Principle
Ammonium molybdate and K antimonyl tartrate react in acid
soln with dil. solns of P0 4 ~ 3 to form Sb phosphomolybdate
complex which is reduced to intensely blue complexes by as-
corbic acid. Method is specific for orthophosphate and for
compds that can be converted to orthophosphate. Various forms
of P are detd, depending on pretreatment, in range 0.01-0.5
mg P/L.
Method is applicable to surface and saline waters and do-
mestic and industrial wastes. Most commonly measured forms
are total P, dissolved P, orthophosphate, and dissolved ortho-
phosphate. Hydrolyzable P is normally found only in sewage-
type samples. Concns of Cu, Fe, silicate, and arsenate many
times greater than those in sea water do not interfere. Inter-
ference of HgCl 2 , used as preservative, is overcome by adding
min. of 50 mg NaCl/L to samples.
AOAC Official Methods of Analysis (1990)
Metals
329
Natural H 2 analyzed by 33 analysts in 19 laboratories showed
following results on exact increments of org. phosphate:
Added,
Std deviation
Bias
mgP/L
%
mgP/L
%
mgP/L
0.110
30
0.033
+ 3.1
+0.003
0.132
39
0.051
+ 12.0
+0.016
0.772
17
0.130
+ 3.0
+0.023
0.882
15
0.128
- 0.9
-0.008
Natural H 2 analyzed by
26 analysts in
16 laboratories showed
following
results
on exact
increments of orthophosphate:
Added,
Std deviation
Bias
mg P/L
%
mg P/L
%
mg P/L
0.029
34
0.010
-5.0
-0.001
0.038
21
0.008
»6.0
-0.002
0.335
5.4
0.018
-2.8
-0.009
0.383
6.0
0.023
-1.8
-0.007
C. Apparatus
(a) Glassware. — Wash all glassware with hot HO (1 + 1)
and rinse with H 2 0. Remove last traces of P by filling with
H 2 contg all color-developing reagents. Use treated glass-
ware only for P detns and after use, rinse with H 2 and keep
covered until used again. Under such conditions, hot HC1 and
reagent treatment need be applied only occasionally. Never use
com. detergents on glassware,
(b) Photometer. — Spectrophtr or filter photometer measur-
ing at 880 nm, using ^2.5 cm light path.
D. Reagents
(a) Dilute sulfuric acid. — 57V. Dil.
70 mL H 2 S0 4 to 500
mL.
(b) Potassium antimony I tartrate soln. — Weigh 1.3715 g
K(SbO)C 4 H 4 O 6 .0.5H 2 O, dissolve in ca 400 mL H 2 0, and dil.
to 500 mL. Store in dark g-s bottle at 4°.
(c) Ammonium molybdate . soln. — Dissolve 20 g
(NH 4 ) 6 Mo 7 0,4.4H,0 in 500 mL H?0. Store in plastic bottle at
4°.
(d) Ascorbic acid soln. — 0. 1M. Dissolve 1 .76 g in 100 mL
H 2 0. Stable 1 week at 4°.
(e) Combined reagent. — Warm reagents (b)-(d) to room
temp., and add with mixing in following order: 50 mL 5N
H 2 S0 4 , 5 mL K antimonyl tartrate soln, 15 mL NH 4 molybdate
soln, and 30 mL ascorbic acid soln. If turbidity forms, shake,
and let stand few min before proceeding. Stable 1 week at 4°.
(f) Hydrolyzing acid soln. — Slowly add 310 mL H 2 S0 4 to
600 mL H 2 O s cool, and dil. to 1 L.
(g) Phosphorus std solns. — (7) Stock soln. — 50 mg P/L.
Dissolve and dil. 0.2197 g KH 2 P0 4 , dried at 105°, to 1 L. (2)
Intermediate soln. — 0.5 mg/L. Dil. 10.0 mL stock soln to I
L. (3) Working solns.— Dil. 0.0, 1.0, 3.0, 5.0, 10.0, 20.0,
30.0, 40.0, and 50.0 mL intermediate soln to 50 mL to prep.
std solns contg 0.0, 0.01, 0.03, 0.05, 0.10, 0.20, 0.30, 0.40,
and 0.50 mg P/L.
E. Determination
Store samples in plastic or Pyrex containers. If analysis can-
not be performed on day of collection, preserve with 40 mg
HgCl 2 /L and refrigerate at 4°. In such case, add 50 mg NaCl/
L before analysis.
(a) Phosphorus. — Add 1 mL hydrolyzing acid soln to 50
mL sample in 125 mL erlenmeyer. Add 0.4 g NH 4 persulfate,
and boil gently on preheated hot plate 30-40 min or until vol
is 10 mL. Do not let sample evap. to dryness. Alternatively,
autoclave 30 min at 121°. Cool, add few drops phthln, adjust
to pink with IN NaOH, and then to colorless with 1 drop
hydrolyzing acid soln. Cool, and dil. to 50.0 mL. If turbid,
filter. Proceed as in (c), beginning "Add 8.0 mL combined
reagent, ..."
(b) Hydrolyzable phosphorus. — Proceed as in (a), except
omit addn of NH 4 persulfate.
(c) Orthophosphate. — Add 1 drop phthln to 50.0 mL sam-
ple; if red develops, add hydrolyzing acid soln drop wise until
color is discharged. Add 8.0 mL combined reagent, and mix
thoroly. After specific time within 10—30 min, measure A at
880 nm against reagent blank as ref.
(d) Std curve and calculation. — Process stds and blank as
in (c) and plot A against mg P/L. Include blank and >2 std
solns with each series of samples. If stds do not agree with
std curve within ± 2%, prep, new std curve. Obtain mg P/L
sample directly from std curve.
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7,
$5). Method Study No. 2; Nutrient Analyses, Manual
Methods, 1970 (available from National Technical In-
formation Service, 5285 Port Royal Rd, Springfield,
VA 22161, NTIS PB230828/BE). JAOAC 56,
295(1973).
CAS-7723-14-0 (phosphorus)
973.56
Phosphorus in Water
Automated Method
First Action 1973
A. Principle
See 973.55. Developed color is measured automatically.
Natural H 2 analyzed by 6 laboratories showed following
results on exact increments of orthophosphate:
Added
Std deviation
Bias
mgP/L
%
rug P/L
%
mg P/L
0.04
0.04
0.29
0.30
47
35
30
22
0.019
0.014
0.087
0.066
+ 16.7
- 8.3
-15.5
-12.8
+0.007
-0.003
-0.05
-0.04
B. Apparatus
(a) Glassware. — See 973.55C(a).
(b) Automatic analyzer. — Auto Analyzer with following
modules (Technicon Instruments Corp.): Sampler I, manifold,
proportioning pump, 50° bath, colorimeter with 50 mm tubular
flowcell and 650 nm filter, and recorder. See Fig. 973.56.
C. Reagents
Prep, reagents 973.55D(a), (d), and (f), and following:
(a) Potassium antimonyl tartrate soln. — Weigh 0.3 g
K(SbO)C 4 H 4 O 6 .0.5H 2 O, dissolve in ca 50 mL H 2 0, and dil.
to 100 mL. Store in dark g-s bottle at 4°.
(b) Ammonium molybdate soln.— Dissolve 4 g
(NH 4 ) 6 Mo 7 24 .4H 2 in 100 mL H 2 0. Store in plastic bottle at
4°.
(c) Combined reagent. — Prep, as in 973.55D(e). 100 mL
is enough for 4 hr operation. Prep, fresh for each series.
(d) Wash water. — Add 40 mL hydrolyzing acid soln,
973.55D(f), to ca 1 L H 2 and dil. to 2 L (not used when
only orthophosphate is detd).
(e) Phosphorus std solns. — (J) Stock soln.— A mg P/L.
Dissolve and dil. 0.4393 g KH 2 P0 4 , dried at 105°, to 1 L. (2)
Intermediate soln 1. — 0.01 mg P/L. Dil. 100 mL stock soln
330
Waters; and Salt
AOAC Official Methods of Analysis (1 990)
LARGE MIXING COIL |LMj
M0000OO
SM
QOOQ
G2
m l/m i
2.90 ^ SAMPLE
0,80 ^ AIR
HEATING BATH
#M
COLORIMETER
50mm TUBULAR f/c
650 fim FILTERS
_L2j)
.DISTILLED WATER
■» * 2 <aU£0 BtA»HT
JJLi
PROPORTIONING PUMP
IX
SAMPLING TIME: 1.0 MIN.
WASH TUBES: TWO
FIG. 973.56— Phosphorus manifold
to 1 L. (3) Intermediate soln 2. — O.OOI rag P/L. Dil. 100
mL intermediate soJn 1 to 1 L. (4) Working solas. — Dil. 0.0,
2.0, 5.0, and 10.0 mL intermediate soln 2 and 2.0, 5.0, 8.0,
and 10.0 mL intermediate soln / to 100 mL to prep, std solns
contg 0.00, 0.02, 0.05, 0. 10, 0.20, 0.50, 0.80, and 1.00 mg
P/L, resp.
D. Determination
Store and prep, samples as in 973. 55E.
(a) Phosphorus. — Proceed as in 973.55E(a), but det. or-
thophosphate as in (c), below.
(b) Hydrolyzable phosphorus. — Proceed as in 973.55E(a),
omitting addn of NH 4 persulfate, and det. orthophosphate as
in (c), below.
(c) Orthophosphate. — Set up manifold as in Fig. 973.56.
Let colorimeter and recorder warm up 30 min. Run baseline
with all reagents but with H 2 thru sample line. Adjust dark
current and operative opening on colorimeter to obtain stable
baseline. Place wash H 2 0, (d), in sampler in pairs, for other
than ortho-P, and H 2 for ortho-P, leaving every third position
vacant. Set sample timing at 1 min. Place std solns in sampler
in vacant positions in order of decreasing concn and complete
filling of sampler tray with unknown samples. Change sample
line from H 2 to sampler and begin analysis.
(d) Std curve and calculation. — Prep, std curve by plotting
peak hts against mg P/L. Obtain sample concn from peak ht.
Reanalyze any sample whose computed value is <5% of its
immediate predecessor.
Ref.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC 20402, Stock No. 055-001-00067-7).
JAOAC 56, 295(1973).
CAS-7723-14-0 (phosphorus)
973.57
Sulfate in Water
Turbidimetric Method
First Action 1973
A. Principie
Sulfate is pptd in dil. HCl with BaCl 2 under controlled con-
ditions to form BaS0 4 crystals of uniform size. A of suspen-
sion is measured and sulfate concn is obtained from std curve.
Method is applicable to drinking and surface waters and do-
mestic and industrial wastes. Dil. samples to concn of <40
mg S0 4 /L. Color and suspended matter interfere. Some sus-
pended matter is removed by filtration. Remaining interference
is corrected by blank which omits BaCL.
Synthetic H 2 samples contg exact increments ofinorg. S0 4
analyzed by 34 analysts in 16 laboratories showed following
results:
Sid deviation
Bias
Increment as
mg SO4/L
%
mg SO4/L
%
mg S0 4 /L
8.6
27
2.3
-3.7
-0.3
9.2
20
1.8
-8.3
-0.8
110
7.1
7.9
-3.0
-3.3
122
6.1
7.5
-3.4
-4.1
188
5.1
9.6
0.0
+ 0.1
199
5.9
11.8
-1.7
-3.4
8. Apparatus
(a) Magnetic-
stirrer.
— Adjustable,
but
once
set
must op-
erate at const speed. Stirring bars must be of identical shape
and size. Exact speed is not critical, but it should be const for
each series of samples and stds and should be at max. at which
no splashing occurs. Timing device to permit operation for
exactly 1 min is desirable.
(b) Photometer. — Nephelometer, spectrophtr set at 420 nm
with 4-5 cm cell, or filter photometer with filter having max.
T near 420 nm with 4-5 cm cell.
C. Reagents
(a) Conditioning reagent.
-Mix 50 mL glycerol with soln
100 mL alcohol or isopropanol,
of 30 mL HCl, 300 mL H 2 0,
and 75 g NaCl.
(b) Barium chloride. — Crystals, 20-30 mesh. Dispense from
0.2-0.3 mL measuring spoon.
(c) Sulfate std soln. — 100 jxg S0 4 /mL. Dil. 10.41 mL
0.020CW H 2 S0 4 to 100 mL, or dissolve 147.9 mg anhyd. Na 2 S0 4
in H 2 and dil. to I L.
D. Determination
Pipet 5 mL conditioning reagent into 100 mL sample or ali-
quot dild to 100 mL in 250 mL erlenmeyer, and mix on mag.
stirrer. While stirring, add spoonful of BaCL crystals and be-
AOAC Official Methods of Analysis (1990)
Metals
331
gin timing. Stir exactly 1 min at const speed. Immediately
transfer some soln into cell and measure turbidity at 30 sec
intervals for 4 min. Record max. reading. Conduct blank detn
without BaCJ 2 and subtract reading.
Prep, std curve by carrying 0-40 mg S0 4 /L, in 5 mg in-
crements, thru entire detn. Introduce std soln with every 3-4
samples.
mg S0 4 /L = mg S0 4 from curve X 1000/mL sample
Refs.: Methods for Chemical Analysis of Water and Wastes,
1971 (available from Superintendent of Documents,
Washington, DC. 20402, Stock No. 055-001-00067-
7). FWPCA Method Study 1; Mineral and Physical
Analyses, June 1969 (available from National Environ-
mental Research Center, Environmental Protection
Agency, Cincinnati, OH 45268). JAOAC 56, 295(1973).
B. Determination
Dissolve insol. residue, 920.202, in excess HN0 3 (1 + 1),
evap. to dryness, treat with H 2 0, and add ca 1 mL HNO3 and
little of the AgN0 3 soln. If ppt of AgCl appears, add addnl
AgN0 3 soln until all C\ is pptd. Add excess of ca 10 mL AgN0 3
soln for each mg Mn present in sample. Filter, add 1 g
(vV// 4 ) 2 5 2 8 to filtrate, and place beaker or flask contg soln on
steam bath until pink color develops (ca 20 min). Compare
color developed with stds similarly prepd by treating solns contg
known amts of std Mn soln with dil. HN0 3 , AgN0 3 soln, and
(NH 4 ) 2 S 2 O s .
C. Bismuthate Method*
—Surplus 1965
See 31.037-31.038, 10th ed.
CAS-7439-96-5 (manganese)
925.54
Sulfate in Water
Gravimetric Method
Final Action
Make preliminary examination, using 100-250 mL sample,
to det. approx. amt of sulfates. (Alkali salts present can be
approximated by calcg amt of Na necessary to combine with
excess of acids — HC1, H 2 S0 4 , and H 2 C0 3 — over Ca and Mg.)
Take enough sample (usually 1-5 L) to yield <1 g BaS0 4
and <0.5 g mixed chlorides. Acidify with HC1 (1 + 1), evap.
to dryness in Pt dish, and remove Si0 2 by 2 evapns as in
920.195, using <2 mL HC1 for final soln. Combine filtrate
and washings from Si0 2 detns and cone, to 150-200 mL. Heat
to bp and ppt with slight excess of 10% BaCl 2 .2H 2 soln,
added very slowly and with const stirring. Cover, and let stand
on steam bath ca 12 hr. Filter, thoroly wash BaS0 4 ppt with
hot H 2 until CI -free, dry, ignite over Bunsen burner and weigh.
If sulfate content of sample is unusually large, proceed as
far as concn of Si0 2 filtrates, as above. Add 50 mL HC1, heat
to bp, and ppt with BaCl 2 soln as before. Evap. to dryness,
take up in H 2 and few drops HO, digest until ppt settles,
wash by decantation, filter, ignite, and weigh. Calc. to S0 4
ion.
920.202 Manganese, Iodine, Bromine,
Arsenic, and Boric Acid in Water
Preparation of Sample
Final Action
Evap. 0.5-2 L sample to dryness after addn of small amts
of solid Na 2 C0 3 . Boil residue thus obtained with H 2 0, transfer
to filter, and wash thoroly with hot H 2 0. Use residue remain-
ing on filter for detn of Mn. Dil. alk. filtrate to definite vol.
and use for detn of I, Br, As, and H3BO3.
920.203
Manganese in Water
Final Action
Persulfate Method
A. Reagents
(a) Silver nitrate soln. — Dissolve 2 g AgN0 3 in H 2 and
dil. to 1 L.
(b) Manganese std soln. — 0.1 mg/mL. Dissolve 0.2877 g
KMn0 4 in ca 100 mL H 2 ; acidify soln with H 2 S0 4 (1 + 1),
and slowly heat to bp. Slowly add enough 10% H 2 C 2 4 .2H 2
soln to discharge color. Cool, and dil. to 1 L.
920.204 Bromide and Iodide
in Water
Cotorimetric Method
First Action
(This method is qua!, and approx. quant. For accurate quant,
methods for iodides, see 925. 56C.)
(Caution: See safety notes on flammable solvents, toxic sol-
vents, chlorine, and carbon disulfide.)
Evap. aliquot of alk. filtrate, 920.202, to dryness; add 2-3
mL H 2 to dissolve residue and enough alcohol to make ca
90% alcohol. (This ppts chlorides.) Heat to bp, filter, and re-
peat soln and pptn once or twice. Add 2 or 3 drops 10% NaOH
soln to combined ale. filtrates and evap. to dryness. Dissolve
last residue in 2-3 mL H 2 and repeat pptn with alcohol, heat-
ing, and filtering. Add drop of 10% NaOH soln to this ale.
filtrate and evap. to dryness.
Dissolve residue in little H 2 0, acidify with H 2 S0 4 (1 + 5),
using 3 or 4 drops excess, and transfer to small flask. Add 4
drops 02% NaN0 2 soln and ca 5 mL CS 2 . Shake until all I
is extd and filter off acid soln from CS 2 . Wash flask, filter,
and contents with cold H 2 and transfer CS 2 contg the I in
soln to Nessler tube, using ca 5 mL CS 2 . In washing filter,
make contents of tube to definite vol., usually 12-15 mL, and
compare color with that of other tubes contg known amts of
I dissolved in CS 2 . Prep, these std tubes by treating measured
amts of soln of known KI content as above, beginning "...
acidify with H 2 S0 4 (1 + 5), . . ."
Sep. transfer acid soln of sample and stds from which I has
been removed to small flasks. To stds add definite measured
amts of bromide soln of known concn, and to each flask contg
sample and stds add 5 mL CS 2 . Add satd and freshly prepd
Cl-H 2 0, 1 mL at time, shaking after each addn until all Br is
set free. Avoid large excess of CI, as a bromo-chloride may
form and change color reaction.
Filter off aq. soln from CS 2 thru moistened filter, wash con-
tents of filter 2 or 3 times with H 2 0, and then transfer to Ness-
ler tube with ca 1 mL CS 2 . Repeat extn of filtrate twice, using
3 mL CS 2 each time. Combined CS 2 exts usually total 11.5-
12 mL. Add enough CS 2 to tubes to make definite vol., usu-
ally 12-15 mL, and compare sample with stds. If, when using
this method near its upper limit, amts of CS 2 recommended
do not ext all Br, make 1 or 2 addnl extns with CS 2 , transfer
exts to another tube, and compare color with some of lower
stds. Add readings thus obtained to others.
Results closely approximating true values for I and Br can
be obtained in shorter time on most samples by omitting extns
332
Waters; and Salt
AOAC Official Methods of Analysis (1990)
with alcohol and comparing color of CS 2 solns directly in extn
flasks.
950.58*
Bromide in Water
First Action
Surplus 1965
(In presence of chloride but not iodide)
See 31.040-31.043, 10th ed.
950.59*
Bromide in Water
Final Action
Surplus 1965
(In presence of chloride and iodide)
See 31.044-31.046, 10th ed.
920. 205
Arsenic in Water
Final Action
A. Reagents and Apparatus
See 963.21A and B.
B. Determination
Take portion of alk. filtrate, 920.202, contg <0.03 mg As 2 3 .
If amt taken is > 10 mL, evap. soln to ca that vol. on steam
bath. Transfer soln into generator of app., 963. 21B, with aid
of ca 10 mL H 2 0, add 20 mL H 2 S0 4 (1 + 2), and proceed as
in 942. 17A or 952. 13 A, beginning with addn of KI reagent.
920.206* Boric Acid in Water
Surplus 1965
A. Quaiitative Test— Procedure
See 31.049, 10th ed.
B. Quantitative Method — First Action
See 31.050—31.051, 10th ed.
965.37 Lead in Water
See 934.07A and B.
922.12 Constituents in Water
Method of Reporting Results
Procedure
Report radicals and anhyd. salts in rng/L or, in case of highly
coned waters, in g/L. For benefit of physicians, in case of
medicinal waters, also report salts in terms of grains/qt, using
factor 0.014600 to convert mg/L to grains/qt. In reporting
salts in terms of grains/qt, convert salts that have H 2 of crystn
to hydrated form as expressed in USP and in NF, and convert
Mg(HC0 3 ) 2 to MgC0 3 and Ca(HC0 3 ) 2 to CaC0 3 . Use follow-
ing factors in these calcns:
Na 2 S0 4 x 2.2683 - Na 2 SO 4 .10H 2 O
MgS0 4 x 2.0477 = MgS0 4 .7H 2
CaS0 4 x 1.2647 = CaS0 4 .2H 2
Mg(HC0 3 ) 2 x 0.5762 = MgC0 3
Ca(HC0 3 ) 2 x 0.6174 - CaCQ 3
When complete analysis is made, report error of analysis and
state how it is distributed. Report only significant figures.
Report Fe and Al together when present in unimportant amts
and in calcns consider them as Fe. When Fe and Al are present
in larger amts, make sepn and report each sep.
In calcg hypothetical combinations of anions and cations,
join N0 2 , N0 3 , B0 3 , and As0 4 to Na; I and Br to K; and P0 4
to Ca. Assign residual cations in following order: NH 4 , Li, K,
Na, Mg, Ca, Sr, Mn, Fe, and Al; to residual anions in fol-
lowing order: CI, S0 4 , C0 3 , and HC0 3 . When not enough
HC0 3 is present to join with all Ca, residual Ca is joined to
Si0 2 to form CaSi0 3 , and Mn, Fe, and A I are calcd to oxides
Mn 3 4 , Fe 2 3 , and A1 2 3 , resp.
Use equiv. combining wts or their reciprocals in uniting rad-
icals and, when necessary for purpose of comparison, in re-
ducing salts to radicals and reuniting radicals in order specified
above. See Table 922.12.
Equiv. combining wt of radical is obtained by dividing its
wt by its valence. Equiv. combining wt of salt is obtained by
dividing its MW by product of valence of basic element and
number of atoms of basic element in the salt.
Procedure in calcg hypothetical combinations by use of equiv.
combining wts and their reciprocals is as follows:
Multiply wts obtained, expressed in mg/L, or, for highly
coned waters, in g/L, for each radical to be combined, by
corresponding reciprocal of equiv. combining wts. If Na and
K are to be detd by calcn, as is frequently the case, subtract
sum of values obtained (reacting values) for basic radicals from
sum of reacting values for acid radicals. Difference represents
reacting value of undetd Na and K.
When all constituents in water have been detd, sums of re-
acting values of acid and basic radicals should be very nearly
equal. In this case, if difference is reasonable and well within
limit of accuracy of methods used, it may be distributed equally
among all radicals detd, or among those believed to be less
accurately detd than others. If difference is unreasonably great,
repeat analysis in whole or in part. Sums of reacting values of
acid and basic radicals must be equal before calcn is made.
Obtain reacting values of the salts by subtracting in succession
reacting values of radicals in specified order. To convert these
values to mg/L of respective salts, multiply each of them by
the equiv. combining wt of respective salt.
Ref.: JAOAC 5, 385(1922).
986.22 TNT, RDX, HMX, and 2,4-DNT
in Wastewater and Groundwater
Liquid Chromatographic Method
First Action 1986
A. Principle
Presence of 2,4,6-trinitrotoluene (TNT), I,3,5-trmitro-l,3,5-
triazacyclohexane (RDX), l,3,5,7-tetranitro-l,3,5,7-tetraaza-
cyclooctane (HMX), and 2,4-dinitrotoluene (DNT) in waste-
water from munitions manufacturing and processing facilities
and in groundwater is identified and measured by comparison
of liq. chromatgc peak areas and retention times. Analytical
detection limits for TNT, RDX, HMX, and 2,4-DNT are estd
to be 14, 22, 26, and 10 |xg/L, resp.
S. Apparatus
(a) Liquid chromato graph. — With high pressure pump and
254 nm fixed wavelength UV detector, or variable wavelength
detector set at 254 nm, strip chart recorder, stand-alone digital
or computer-controlled integrator, and 100 (xL sample loop in-
jector.
AOAC Official Methods of Analysis (1990)
Munitions
333
Table 922.12
Equivalent Combining Weights and Their Reciprocals Based on International Atomic Weights,
1973
Reciprocals
Reciprocals
Equiv.
of Equiv.
Equiv.
of Equiv.
Neg.
Combining
Combining
Pos.
Combining
Combining
Radicals
Wts
Wts
Radicals
Wts
Wts
no 3
62.0049
0.01613
NH 4
18.0383
0.05544
B0 2
42.81
0.02336
Li
6.941
0.14407
As0 4
46.3064
0.02160
K
39.0983
0.02558
I
126.9045
0.00788
Na
22.98977
0.04350
Br
79.904
0.01252
Mg
12.153
0.08228
PCX
31.6571
0.03159
Ca
20.04
0.04990
HS
33.07
0.03024
Sr
43.81
0.02283
S
16.03
0.06238
Ba
68.67
0.01456
Si0 3
38.042
0.02629
Mn
27.4690
0.03640
7.9997
0.12500
Fe ++
27.924
0.03581
CI
35.453
0.02821
Fe ++ +
18.616
0.05372
S0 4
48.03
0.02082
Ai
8.9938
0.11119
C0 3
30.005
0.03333
Cu
31.773
0.03147
HCO3
61.017
0.01639
Reciprocals
Reciprocals
Equiv.
of Equiv.
Equiv.
of Equiv.
Combining
Combining
Combining
Combining
Salts
Wts
Wts
Salts
Wts
Wts
NH4CI
53.491
0.01869
MgCI 2
47.606
0.02101
LiCI
42.394
0.02359
MgS0 4
60.18
0.01662
Li 2 S0 4
54.97
0.01819
MgC0 3
42.157
0.02372
Li 2 C0 3
36.946
0.02707
Mg(HC0 3 ) 2
73.170
0.01367
UHCO3
67.958
0.01471
Mg(N0 3 ) 2
74.157
0.01348
KCI
74.551
0.01341
CaCI 2
55.49
0.01802
K 2 S0 4
87.13
0.01148
CaS0 4
68.07
0.01469
K 2 C0 3
69.103
0.01447
CaC0 3
50.04
0.01998
KHCO3
100.115
0.00999
Ca(HC0 3 ) 2
81.06
0.01234
Kl
166.003
0.00602
CaSi0 3
58.08
0.01722
KBr
119.002
0.00840
Ca 3 (P0 4 ) 2
51.70
0.01934
NaCI
58.443
0.01711
SrS0 4
91.84
0.01089
NaBr
102.894
0.00972
SrC0 3
73.81
0.01355
Nal
149.8942
0.00667
Sr(HC0 3 ) 2
104.83
0.00954
Na 2 S0 4
71,02
0.01408
BaS0 4
116.70
0.00857
Na 2 C0 3
52.994
0.01887
Ba(HC0 3 ) 2
129.69
0.00771
NaHC0 3
84.007
0.01190
MnS0 4
75.50
0.01325
NaN0 2
68.9952
0.01449
MnC0 3
57.474
0.01740
NaN0 3
84.9946
0.01177
Mn(HC0 3 ) 2
88.486
0.01130
NaB0 2
65.80
0.01520
FeS0 4
75.95
0.01317
Na 3 As0 4
69.2961
0.01443
Fe 2 (S0 4 ) 3
66.64
0.01501
NaF
41.9881
0.02382
FeC0 3
57.928
0.01726
NaHS
56.06
0.01784
Fe(HC0 3 ) 2
88.941
0.01124
Na 3 P0 4
54.6488
0.01830
Fe 2 3
26.615
0.03757
Na 2 S
39.02
0.02563
AI 2 (S0 4 ) 3
57.02
0.01754
Na 2 Si0 3
61.032
0.01638
Al 2 3
16.9935
0.05885
Operating conditions: 25 cm x 4.6 mm (5 \xm) LC8 reverse
phase column (Supelco), operated at room temp. Mobile phase
H 2 0-MeOH-CH 3 CN (50 + 38 + 12, v/v), prepd in gradu-
ates, not vol. flask. Prep. 750-1000 mL, vac. -filter thru solv.-
washed Whatman glass microfiber filter to remove particulate
matter, and degas. Prep, fresh sol v. daily. Flow rate 1.5 mL/
min. Set integrator threshold low enough to avoid neg. inter-
cept in working curve and high enough to avoid pos. intercept.
Set chart speed at 0.2 in. /min.
(b) LC syringe. — Liq. -tight syringe of 0.5—1 .0 mL capac-
ity (Hamilton 750, or equiv.).
(c) Filters. — (I) Nuclepore syringe filter, 25 mm diam. (2)
0.4 [xm Nuclepore polycarbonate, 25 mm diam., or Millex-
SR 0.5 iJim filter unit.
(d) Filtration syringe. — 25 mL, glass or polyethylene (e.g.,
Plastipak, Becton, Dickinson and Co., Stanley St, Rutherford,
NJ 07070, or equiv.)
(e) Scintillation vials. — 20 mL glass with polyethylene, not
AI, cap insert. Use as received.
Soak all vol. glassware overnight in detergent, scrub briefly,
rinse well with hot tap H 2 0, rinse with acetone, rinse with
deionized H 2 0, oven-dry at 105°, and rinse with appropriate
so In before filling.
C. Reagents
(a) LC solvents.— H 2 0, MeOH, CH 3 CN, all LC grade.
(b) Methanol-acetonitrile diluent. — MeOH-CH 3 CN (76 +
24). Use throughout as diluent for all H 2 samples. Prep, us-
ing graduates, not vol. flasks. (Diln with this mixt., rather than
MeOH alone, eliminates neg. peak which elutes just before
HMX and may affect integration.)
D. Calibration Standards
(a) Reference stds.—DNT, TNT, RDX, and HMX ref stds
available from U.S. Army Toxic and Hazardous Materials
Agency, Aberdeen Proving Ground, MD 21010, for federal
contract work only. Reference stds for TNT and DNT avail-
able from Chem Service, Inc., 660 Tower La, PO Box 3108,
West Chester, PA 19381-3108.
(b) Individual stock std solns. — Vac. -dry anal. ref. stds of
DNT, TNT, RDX, and HMX at room temp, to const wt (±1
mg). Use vac. desiccator or vac. oven attached to H 2 aspi-
rator or vac. pump. For RDX and HMX, remove most of iso-
propanol by Pasteur pipet, air- dry for 3-4 h, then vac. -dry.
Store std anal. ref. materials in desiccator over dry CaCl 2 or
Drierite and place in dark when not in use.
Accurately weigh ca 0.1 g dried std onto weighing paper,
334
Waters; and Salt
AOAC Official Methods of Analysis (1990)
transfer carefully to 250 mL vol. flask, and reweigh weighing
paper. Record mass to 1 mg.
For DNT and TNT, dissolve and dil. to vol. with MeOH.
For HMX and RDX, add 100 mL CH 3 CN to dissolve, then
dil. to vol. with MeOH.
Wrap stoppered joint with Parafilm to protect against evapn.
Calc. concn exactly in mg/L and label flasks. Store at ca 4°
(not <0°).
(c) Combined analyte stock soln. — Remove stock std solns
from refrigerator and let warm to room temp. (>30 min but
not overnight). Invert flasks several times to mix. Into 1 L vol.
flask, pipet 10.0 mL each of DNT and TNT stock std solns
and 25.0 mL each of RDX and HMX stock std solns. Dil. to
vol. with MeOH. This std soln contains ca 4.0 |xg DNT and
TNT/mL and ca 10.0 |xg RDX and HMX/mL.
Calc. concns exactly in fig/mL; label and date flask. Wrap
stoppered joint with Parafilm and store flask in refrigerator
when not in use. Combined stock std soln can be used 1 week.
(d) Working std solns. — Prep, fresh each day as needed.
Remove combined analyte stock std soln from refrigerator and
let warm to room temp. (>30 min but not overnight). Invert
flask several times to mix. Transfer 2.00, 5.00, 10.00, and
20,00 mL by pipet to four 250 mL vol. flasks, resp. Fill to
mark with MeOH-CH 3 CN diluent. Stopper and invert 10 times
to mix. Calc. concns exactly in |xg/L; label and date flasks.
(e) Injection std solns. — For each working std soln, pipet
10.0 mL into scintillation vial. Add 10.0 mL H 2 by pipet,
cap, and shake to mix. Prep, blank by combining 10.0 mL
MeOH-CH 3 CN diluent with 10.0 mL LC grade H 2 in vial.
Cap and mix. Label all vials appropriately.
Solns contain following concns in 10 mL aliquot:
N
5.54 X (t r /t , 5 ) 2
Aliquot vol.
combined
std, mL
Approx. concns, ^g/L
For
DNT
TNT
and
For RDX and HMX
2
5
10
20
32
80
160
320
80
200
400
800
These values represent concns before addn of H 2 0. Actual
concns are half as large. Solns are treated similarly: 1-to-l diln
is made by adding 10.0 mL MeOH-CH 3 CN diluent to 10.0
mL aq. sample. Thus, anal, results derived from working curve
do not need to be corrected for this extra diln.
Make 10.0 mL MeOH-CH 3 CN/H 2 mixts in scintillation
vials rather than in vol. flasks because of slight vol. contrac-
tion which would cause systematic error. Because std solns
would be dild. with H 2 to vol., and samples would be dild
with org. solv. to vol., vol. contraction would lead to samples
richer in org. solv. than are std solns. Take care at this step
to pipet these 10.0 mL vols accurately because significant er-
ror at this stage is compounded when peak areas are measured,
£ Liquid Chromatography
Initial conditioning — Follow procedure below for instru-
ment warmup, except pass >30 void vols (ca 60 mL) mobile
phase thru column. Continue until UV detector baseline is level
when set to greatest sensitivity.
Calc. plate no. as follows:
Take 1 mL aliquot from combined working stock std soln
and dil. to 100 mL in vol. flask with MeOH-CH 3 CN. Use
proper sample prepn and injection procedure described to ob-
tain chromatogram . All 4 analytes should elute within 10 min.
Use conditions described in Apparatus, but select chart speed
that spreads peaks abnormally wide (such that widths at half
ht are ^2.0 cm). Est. peak width at half ht to nearest 0.1 mm.
Calc. no. of theoretical plates (N) on column from each peak.
where t r — retention time and f 0i5 = width of peak at half ht,
both in min.
Average results for all 4 analytes. If av. value is <3000
plates, carefully recheck calcn. If no error is apparent, let an-
other 30 void vols of mobile phase wash thru column and re-
peat experiment. If calcd value of W still does not exceed 3000,
column is not performing to specification and should be re-
placed.
Warmup procedure. — Turn on all electronic equipment and
let warm up >30 min. Pass >15 void vols of mobile phase
thru column (20 min at 1.5 mL/min) and continue until UV
detector baseline is level when set to greatest sensitivity. Make
sure pumps are not experiencing vapor lock as indicated by
large pressure fluctuations. Check system thoroly for leaks.
Sample injection procedure. — Fill analytical syringe with
MeOH-CH 3 CN and discharge to waste. Repeat twice more to
remove traces of previous sample. Rinse syringe 3 times with
sample. Fill syringe with sample to >500 (jlL and pass most
of this thru sample loop, avoiding introducing air bubbles.
Overfilling loop in this manner assures that sample injected is
not dild by solv. in loop.
F. Preparation of Working Curve
Obtain chromatogram s of 4 working stds and blank in du-
plicate (10 injections total). Sequence injections randomly. Plot
peak area vs concn for each of the 4 analytes. Do not average
duplicates before plotting. Inspect plot for gross deviations from
linearity. Analytical response should be linear from 10 |xg/L
to 20 mg/L for DNT and TNT and from 25 (xg/L to 50 mg/
L for RDX and HMX. Significant deviation from linearity is
evidence for systematic bias. Calc. regression line for each
analyte.
G. Analysis of Water Samples
Remove combined analyte working stock std solns from re-
frigerator and let warm to room temp. (>30 min but not over-
night). Warm up instrument and condition LC column. Pipet
10.0 mL sample into scintillation vial. Add 10.0 mL MeOH-
CH 3 CN diluent by pipet. Attach cap tightly. Shake vigorously.
Let stand >15 min before filtration.
Load new Nuclepore (or Millex-SR) filter into filter holder.
Rinse 25 mL filtration syringe with MeOH~CH 3 CN diluent,
then fill to ca 10 mL with sample. Filter sample and discard
filtrate. Fill syringe with remaining sample. Filter into new
scintillation vial. Label vial appropriately.
Using proper procedure, inject these solns into liq. chro-
matograph. Typical retention times for HMX, RDX, TNT, and
2,4-DNT are 3.2, 4.1, 7.0, and 7.8 min, resp.
H. Calculations
Prep, working curve for each analyte in the form, y ~ b
+ b\x, where x = analyte concn in fig/L and y = peak area.
Det. concn of each analyte in H 2 sample by substituting mea-
sured peak area into calcn.
Refs.: U.S. Army Cold Regions Research and Engineering
Laboratory (1984) CRREL Report 84-29, National
Technical Information Service, Springfield, VA. Anal.
Chern. 58, 170(1986); 58, 176(1986).
925.55 SALT
A. Preparation of Sample — Procedure
If sample is coarser than "20 mesh," grind so that all will
pass No. 20 sieve, but avoid undue grinding so that as much
AOAC Official Methods of Analysis (1990)
Salt 335
as possible will be retained on No. 80 sieve. Mix sample by
quartering and weigh all needed portions as nearly at same
time as possible.
Ref.: JAOAC 5, 384(1922).
B. Moisture—First Action
Place ca 10 g sample in dry, weighed 200 mL erlenmeyer.
Weigh flask and sample. Spread sample evenly over bottom
of flask by shaking gently and insert small funnel in neck.
Heat flask and sample for periods of 1 hr each at ca 250° until
2 consecutive weighings agree within 5 mg. Occasionally shake
flask so that sample will dry evenly. Report loss of wt as H 2 0.
C. Matters insoiuble in Water — First Action
Place 10 g sample in 250 mL beaker, add 200 mL H 2 at
room temp., and let stand 30 min, stirring frequently. Filter
thru weighed gooch with asbestos mat dried at 110°. Transfer
residue to gooch with aid of policeman, using total of <50 mL
H 2 0. Wash residue with ca ten 10 mL portions H 2 0, until 10
mL filtrate shows only faint opalescence upon addn of few
drops AgN0 3 soln. Dry crucible and contents to const wt at
1 10°. Report increase in wt gooch as "matters insol. in H2O"
and report results in % on H 2 0-free basis. If matters insol. in
H 2 are >0.1%, det. their nature.
D. Matters Insoluble in Acid — First Action
Treat 10 g sample with 200 mL HC1 (I + 19), boil 2-3
min, and let stand 30 min, stirring frequently. Filter thru gooch
with mat dried at 110°. Wash, dry at 110°, coot, and weigh.
Express results in %.
Refs.: JAOAC 5, 385(1922); 6, 129(1923).
E. Preparation of Solution for Sulfate, Calcium, and
Magnesium — Procedure
Weigh ca 20 g sample, transfer to 400 mL beaker, and dis-
solve in 200 mL HO (1 + 3). Cover beaker, heat to bp, and
continue boiling gently 10 min. Filter thru paper and wash
residue with small amts of hot H 2 until filtrate is Cl-free.
Unite filtrate and washings, cool, and dil. to 500 mL (Soln
X).
F. Sulfate— First Action
Place 250 mL Soln X, 925.55E, in 400 mL beaker, heat to
bp, and add slight excess hot 10% BaCl 2 soln dropwise while
stirring. Cone, by heating gently and finally evap. to dryness
on steam bath. Facilitate removal of free acid by stirring partly
dried residue. Wash ppt by decantation with small amts of hot
H 2 0, finally transferring ppt to close -grain filter paper with
aid of policeman and stream of hot H 2 0. Test filtrate for pres-
ence of Ba. Wash ppt on paper until filtrate is Cl-free. Dry
and ignite paper contg ppt over Bunsen flame. Report % S0 4
in sample on H 2 0-l 7 ree basis.
G. Calcium — First Action
Place remainder of Soln X in 400 mL beaker. Add excess
of 10% H 2 C 2 4 .2H 2 soln (10 mL usually is enough). Add
few drops Me orange; neutze while hot by adding NH 4 OH
dropwise, stirring constantly. Add ca 1 mL excess NH 4 OH,
stir, and let stand in warm place 3 hr. Decant supernate thru
filter, reserving filtrate for detn of Mg. Test filtrate for Ca with
(NH 4 )2C 2 04 soln. Wash ppt in beaker once with 10 mL 1%
(NH 4 ) 2 C 2 4 soln, decanting thru filter paper. Combine filtrate
and washings. Dissolve ppt on paper with hot HCI (1 + 1),
using same beaker; dil. to 100 mL, add little more H 2 C 2 4
soln, and ppt as before. Let stand 3 hr, filter, and wash with
1% (NH4) 2 C 2 04 soln as before, reserving filtrate and washings.
Transfer ppt to crucible, dry, ignite, and heat over blast lamp
to const wt (CaO). Report as % Ca on H 2 0-free basis.
H. Magnesium— First Action
Combine filtrates and washings from Ca detn, cone, if nec-
essary by boiling gently to ca 150 mL, and proceed as in
920.200. Report as % Mg on H 2 0-free basis.
/. Lead
See 934.07 A and B.
925.56
Bodine in Iodized Salt
Titrimetric Method
Final Action
A. Reagents
(a) Bromine water. — (Caution: See safety notes on bro-
mine.) For alternative method, 925.56C(b), det. approx. concn
(mg Br/mL) by adding measured vol. from buret to flask contg
50 mL H 2 0, 5 mL 10% KI soln, and 5 mL H 2 S0 4 (1 + 9),
and titrg liberated I with 0. l/V Na 2 S 2 3 .
(b) Sodium thiosulfate. — 0.005 N. Prep, daily by dilg 0. 1/V
soln, 942.27.
(c) Starch soln. — 1% (freshly prepd). Mix ca I g sol. starch
with enough cold H 2 to make thin paste, add 100 mL boiling
H 2 0, and boil ca 1 min with stirring.
(d) Potassium iodide control soln. — 0.3270 g KI/250 mL.
Dil. 50 mL to 250 mL, and use 5 mL (= 1 .0 mg I and 1.308
mg KI) for control.
B. Preparation of Sample
Dissolve 50 g sample in FLO and dil. to 250 mL in vol.
flask. Take 25, for 925.56C(a), or 50 mL, for 925.56C(b),
aliquot for analysis.
C. Determination
(a) Application when Na 2 S 2 3 content is <0.5%.— Place
sample aliquot in 600 mL beaker and dil. to ca 300 mL. Neutze
to Me orange with H 3 P0 4 and add 1 mL excess. Proceed as
in 935.14, third par.
(b) Alternative method. Not applicable in presence of
Na 2 S 2 0j. — Pipet 50 mL sample soln into 200 mL erlenmeyer.
Neutze to Me orange with 2N H 2 S0 4 . Add Br-H 2 dropwise
from buret in amt equiv. to 20 mg Br. After few min destroy
greater portion of remaining free Br by adding 1% Na 2 S0 3
soln dropwise while mixing. Wash down neck and sides of
flask with H 2 and complete removal of Br by adding 1 or 2
drops 5% phenol soln. Add 1 mL IN H 2 S0 4 and 5 mL 10%
KI soln, and titr. liberated I with Na 2 S 2 3 soln, adding 1 mL
starch indicator near end of titrn. Correct detn for blank on
reagents and make 1 or more control detns, using 50 mL 20%
reagent grade NaCl soln to which has been added appropriate
amts of dil. control KI soln. 1 mL 0.005 N Na 2 S 2 3 - 0.1058
mg I and 0.1384 mg KI.
Refs.: Biochem. Z. 138, 383(1923); 174, 364(1926). JAOAC
26, 440(1943).
CAS-7553-56-2 (iodine)
925.57 Constituents in Salt
Method of Reporting Results
Procedure
(In absence of added drying agents such as MgC0 3 , Ca phos-
phate, etc.)
336 Waters; and Salt AOAC Official Methods of Analysis (1990)
Convert sulfate to CaS0 4 and unused Ca to CaCl 2 , unless port on H 2 0-free basis % of matter insol. in H 2 ? of S0 4 , of
sulfate in sample exceeds amt necessary to combine with Ca, Ca, of Mg, of CaS0 4 , of CaCl 2 , and of MgCl 2 . Also report
in which case convert Ca to CaS0 4 and unused sulfate first to results of qual. examination of matters insol. in H 2 0, if amt
MgS0 4 and remaining sulfate, if any, to Na 2 S0 4 . Convert un- is >0.1% on H 2 0-free basis.
used Mg to MgCl 2 . Add percentages of CaCl 2 and MgCl 2 . Re-
12. MIcrochemfcal Methods
969.47 Molecular Weight (MW)
Thermoelectric-Vapor Pressure Method
First Action 1969
Final Action 1975
(Applicable to materials with MW <500)
A. Apparatus and Reagents
(a) Molecular weight apparatus. — Vapor pressure osmom-
eter, Hewlett-Packard; isothermal distn app., Thomas Scien-
tific; or equiv. equipment using vapor pressure equilibrium
technic. Instrument must use sensitive bridge system to mea-
sure temp, difference between sol v. and test soln drops sus-
pended on thermistors in const temp, cell whose atm. is satd
with solv. vapor.
(b) Standards. — Benzi.1, MW 210.23, mp 94.5-95.5°, and
C and H analyses within 0.2% of theoretical values (C, 79.99;
H, 4.79). Recrystallize from EtOAc, acetone, or CHC1 3 , if
necessary .
If sample is ionizable salt sol. in H 2 0, use reagent grade
KC1 as std. If sample is not a salt and sol. only in H 2 0, use
sucrose as std.
(c) Solvents. — Use reagent grade solv. from same lot and
preferably from same bottle to sat. cell and to prep, sample
and std solns. Solv. must completely dissolve sample, pref-
erably without heating. (Proper choice of solv. and std is crit-
ical.) Preferred solvs are (number indicates order of choice):
Natu
re of Sample
Solvent
Unknown
Neutral
Acidic Basic
Salt
Acetone
Ethyl acetate
Chloroform
Water
2
1
3
1
2
3
1
2
1
2
1
For samples not sol. in solvs listed, test solubility in H 2
(if thermistor wiring is completely encased in glass or plastic),
n-heptane, and benzene. Other solvs that may be used are:
alcohol, CCl 4 , methylethyl ketone, dioxane, cyclohexane,
CH 2 C1 2 , dimethyl formamide, toluene, and CH 3 CN. Use solvs
such as esters, ketones, or alcohols for samples which tend to
form dimers thru H bonding, e.g., org. acids.
B. Determination
Follow manufacturer's instructions, including recommended
concn range for solns, instrument operation, and reading of
AR response.
Adjust cell temp, so vapor pressure of solv. is 150-350 mm,
preferably 200-300 mm. If instrument is not equipped to cool
cell, cell temp, must be enough above ambient (ca 5°) so ther-
mostatic control maintains const cell temp.
Construct calibration curve with std and solv. to be used in
analysis. Det. AR response at 4 std concns in recommended
range and plot AR against mole fraction (MF). Prep, sample
soln in recommended range and obtain 3 AR readings. Use
median AR value to calc. MW. If calibration curve is straight
line, calc. MW of sample by:
MF std - [(g std)/(MW std)]/[(g std/MW std)
4- (g solv./MW solv.)]
If AR-MF plot yields curved line, interpolate MF of sample
from calibration curve and calc. MW by:
MW = (g solute)(MW solv.)(K - AR)/(AR)(g solv.)
where K = (AR std)/(MF std) and
MF std = [(g std)/(MW std)]/[(g std/MW std)
+ (g solv./MW solv.)]
If AR-MF plot yields curved line, interpolate MF of sample
from calibration curve and calc. MW by:
MW = (g solute)(MW solv.)(l - MF)/(MF)(g solv.)
Refs.: JAOAC51, 992, 1231(1968).
952.24 Microchemical Determination
of Bromine, Chlorine, or Iodine
Carius Combustion Method
Final Action
(Do not alter combustion conditions such as temp., size of
sample, vol. of acid, etc. Variations from specified conditions
present dangerous explosion hazard.)
A. Reagents
(a) Fuming nitric acid. — Reagent grade, halogen-free, sp
grl.50.
(b) Silver nitrate. — Reagent grade, powd.
B. Apparatus
(a) Combustion tubes. — Fig. 952.24. Use clean, heavy- or
std-wall Pyrex tubes, free from flaws, with round seal at bot-
tom, and with following specifications. (Vol. HNO3 and temp,
depend on combustion tube used.)
Dimensions
Heavy-Wall
Std-Wall
Wall thickness, mm
2.3±0.3
1.2±0.2
Outside diam., mm
13±0.8
13±07
Length, mm
210+10
240±10
Length of sealed tube between
bottom and start of taper at
shoulder, mm
150-175
180-210
Vol. HNO3 (sp gr 60°F, ca 1.5), mL
0.5
0.3
Temp., °C
250
300
MW = (g solute)(MW solv.)(K
where K = (AR std)/(MF std) and
AR)/(AR)(g solv.)
(b) Furnace. — Elec, to hold ^4 tubes at ca 45° angle. Must
maintain temp, of 250 ± 10° or 300 ± 10° for >5 hr, with
^5° difference between any 2 points on a tube or <5° differ-
ence between similar points on any 2 tubes. Must have vari-
able resistor or other device to adjust furnace to desired temp.
Open end of furnace wells must have safety device to retain
glass in furnace in case tube explodes, and device must be
provided for removing individual tubes from wells.
(c) Filter tubes. — Micro 3 mL filter tube with medium-coarse
porosity fritted disk (av. pore diam. 15-25 fxm).
(d) Siphon. — Make from 3 mm od glass tubing, with par-
allel arms, one 50 and other 250 mm long, and with 110 mm
connecting section rising with 13° slope to longer arm.
337
338
MlCROCHEMICAL METHODS
AOAC Official Methods of Analysis (1990)
.Glazed
Wall Thickness
Must Be Same
In Perfectly
Round Bottom
As In Side Walls
FIG. 952.24— Combustion tube
C. Sample
Using microchem. balance, weigh 5-20 mg sample contg
min. of 1 .5 mg CI, 2.5 mg Br, or 3.2 mg I; or using semimi-
crochem. balance, weigh 10-20 mg sample contg min. of 2.5
mg CI, 4.5 mg Br, or 5.7 mg I.
(a) Solid samples. — Weigh by difference in weighing tube.
(b) Viscous liquids or gummy solids. — Weigh in porcelain
boat.
(c) Volatile liquids. — Weigh in 5 cm sealed glass tube,
1-2 mm id with capillary tip. Break off tip of capillary before
placing in combustion tube, sealed end down.
D. Determination
Place weighed sample in combustion tube, add powd AgN0 3
100% in excess of amt estd to be necessary, and add 0.5 ±
0.05 or 0.3 ± 0.03 mL fuming HN0 3 , depending on type of
combustion tube, 952.246(a). Using blast lamp and holding
at 30-40° angle, slowly rotate tube in flame until wall thick-
ens, pull out, and seal off narrow neck of tube. Wall of seal
should be > 3 / 4 of thickness of tube wall and sealed tube should
have length shown in table. (If sample and HN0 3 react at room
temp., immediately cool bottom of tube in ice-H 2 or solid
C0 2 -acetone bath, remove, and seal at once.) Immediately place
tube in furnace and heat 5 hr at 250 or 300 ± 10°, according
to tube size.
Observe following precautions before and during opening of
combustion tubes: {a) Place asbestos glove on hand used to
hold small burner or hand torch; (b) protect face by transparent
face mask or work behind safety shield; (c) be certain tube has
cooled to room temp.; (d) force tip of tube 2-5 cm out of
furnace well; (e) gently flame end to drive all acid from tip
and upper walls; and (/) soften tip with small hot flame until
pressure in tube is released by blowing out softened glass.
Remove vented tube from furnace and cut off constricted
end by scratching tube with file ca 1 cm from shoulder of open
end, moistening scratch, and touching with tip of very hot glass
rod. Remove end of tube with care and fire polish to avoid
contaminating ppt with glass splinters.
Rinse walls of tube with H 2 until tube is ca 3 / 4 full, place
in steam or boiling H 2 bath, protected from light, and digest
until ppt coagulates (ca 30 min). Longer digestion is required
for I than for Br or CI since eutectic mixt. of AgN0 3 and Agl
is formed, which melts below temp, of steam bath and persists
as heavy yellow oil on bottom of tube. Stirring with glass rod
speeds up soln of AgN0 3 and greatly reduces digestion time,
which must be continued until ppt is in form of fine powder.
If excessive amts of AgN0 3 have been used, greater dilns than
specified are required for complete pptn. Therefore, after
digestion appears complete, pi pet few drops of clear supemate
aq. soln into test tube contg several mL H 2 0. If turbidity oc-
curs, entire supemate must be dild with H 2 until pptn stops,
and digestion to coagulate ppt must be repeated. If no turbidity
occurs on diln, pipetted portion may be discarded.
Place previously washed, dried, and weighed filter tube in
1-hole stopper in suction flask, connect short arm of siphon
tube to filter tube thru small rubber stopper, and adjust tube
so that long arm of siphon almost touches ppt. Transfer ppt to
filter tube by suction. Rinse tube and ppt alternately with 1%
HN0 3 and alcohol, using 2 or 3 mL portions for each rinse.
Remove siphon, rinse tip and stopper v/ith alcohol, and rinse
filter tube and ppt first with the acid, then with alcohol. Wipe
outside of filter tube with moist chamois (or cheesecloth) and
dry 30 min at 125° in air oven or 30 min at 80° in vac. oven;
cool to room temp, (ca 30 min) and weigh. Handle dry tube
with chamois finger cots or tweezers. Make blank detn and
subtract any correction from wt sample ppt.
% CI = (wt ppt - blank) x
CI
100
AgCl wt sample
Br 100
% Br = (wt ppt - blank) x x
AgBr wt sample
I 100
% I = (wt ppt - blank) x x
Agl wt sample
Refs.: JAOAC 35, 291(1952); 36, 91, 319(1953); 40, 381
(1957); 41, 297(1958). Anal. Chem. 21, 1555(1949);
23, 1689(1951).
CAS-7726-95-6 (bromine)
CAS-7782-50-5 (chlorine)
CAS-7553-56-2 (iodine)
974.36 Microchemical Determination
of Bromine, Chlorine, or Bodine
Oxygen Flask Combustion Method
First Action 1974
Final Action 1975
A. Apparatus and Reagents
(a) s-Diphenylcarbazone indicator. — 1.5% ale. soln. Heat
to dissolve, if necessary. Prep, fresh daily.
(b) Bromophenol blue indicator. — 0.05%. Dil. 5 mL 1%
ale. soln to 100 mL with alcohol.
(c) Mercuric nitrate stdsoln. — Dissolve 1.7 g Hg(N0 3 ) 2 .H 2
in 500 mL H 2 contg 2 mL HN0 3 and dil. to 1 L. Adjust to
pH 1.7, using pH meter, by adding HN0 3 dropwise. Stdze as
follows: Accurately weigh 4-6 mg KC1 and transfer to 250
mL erlenmeyer. Add 20 mL H 2 and 80 mL alcohol, and stir
mag. at moderate speed. Add 5 drops bromophenol blue in-
dicator and 0.5N HN0 3 to yellow end point; then add 3 drops
excess. Add 5 drops s-diphenylcarbazone indicator and titr. at
<5 mL/min with Hg-(N0 3 ) 2 std soln to orchid-pink. Subtract
reagent blank. Repeat stdzn >3 times.
Normality = mg KC1/[74.551 X mL Hg(N0 3 ) 2 ]
(d) Hydrogen peroxide soln. — 30%.
(e) Hydrazine sulfate soln. — Satd aq. soln.
(f) Buret. — Graduated to 0.01 mL. Tip should be fine enough
that 1 drop is ca 0.015 mL.
AOAC Official Methods of Analysis (1990)
Carbon, Hydrogen 339
B. Determination
Accurately weigh sample contg 1.5-3 mg Cl, 3-6 mg Br,
or 6-9 mg I and fold in paper carrier. Insert carrier in Pt holder
in stopper of 500 mL Schoniger flask, 975.53B(b). Add 2.0
mL 0.5N KOH, 4 drops satd aq. hydrazine sulfate soln, and
10 mL H 2 to flask. Flush flask >3 min with rapid stream of
O. Add 1 drop long-chain alcohol (e.g., dodecanol) to carrier
in basket (not on tail) just before combustion. Ignite carrier
and immediately insert into flask. {Caution: Use safety barrier
and reinforced gloves. Remote control igniting device is avail-
able.) After combustion is complete, shake stoppered flask 10
min or until all visible cloudiness disappears. Let stoppered
flask stand 5 min at room temp. Add ca 3 mL H 2 at funnel
portion of stoppered flask as H 2 seal and stopper wash. Re-
move stopper, and rinse stopper, Pt holder, and flask walls
with ca 15 mL H 2 0. Add 8 drops 30% H 2 2 to flask and boil
until small bubbles no longer evolve (ca 10 min). (Do not let
contents go to dryness. Add H 2 if necessary.) Cool to room
temp, and proceed within 5 min.
Rinse walls of flask with enough H 2 to bring to ca 75 mL;
then add 150 mL alcohol. Stir mag. at moderate speed. Add
15 drops bromophenol blue indicator, and proceed as in stdzn,
(c), beginning ". . . and 0.5N HN0 3 . . .", taking as end point
change in color from faint yellow to orchid-pink. Subtract pa-
per blank from vol. used. Typical blank is 0. 15 mL Hg(N0 3 ) 2 .
% Halogen - [mL Hg(N0 3 ) 2 x atomic wt of halogen
x normality x 100]/mg sample
Refs.: J AOAC 56,
1973); 57, 26(1974).
CAS-7726-95-6 (bromine)
CAS-7782-50-5 (chlorine)
CAS-7553-56-2 (iodine)
949.12 Microchemical Determination
of Carbon and Hydrogen
Combustion Method
Final Action
A. Reagents
(a) Copper oxide. — Wire form, ca 1 mm diam. and 3-4
mm long; discard material finer than "20-mesh." Ignite 1 hr
at 800-900° before placing in combustion tube.
(b) Platinum gauze, 52 mesh. — From three 3x5 cm sec-
tions, make 3 rolls, each 30 mm long x 7 mm od. Boil 15
min in HN0 3 (1 + 1) and ignite in nonluminous Bunsen flame.
(c) Asbestos. — Gooch asbestos; ignite 30 min at 800-900°
and store in wide-mouth bottle. (Caution: See safety notes on
asbestos.)
(d) Silver. — Fine wire or ribbon; if tarnished, reduce in
stream of H at 350-450°. (Caution: H ignites explosively in
O. Flush reduction app. with C0 2 or N before and after H use.
Vent exhaust gas into effective fume removal device. Perform
reduction behind safety barrier.)
(e) Lead dioxide. — Pellets, 1-2 mm diam., special grade
for microanalysis; or prep, by digesting com. grade powder 2
hr in HN0 3 , let stand 1 hr, decant, wash with H 2 until acid-
free, evap. to dryness, and cut into 2 mm cubes. Roll cubes
in jar to round corners and sieve out powder.
(f) Glass wool. — Pyrex, pliable.
(g) Dehydrite or Anhydrone. — (Mg(C10 4 ) 2 , anhyd.) Break
pieces to <3 mm long; discard portion passing thru No. 40
sieve.
(h) Ascarite. — (NaOH on asbestos.) Use com. prepn of "8-
20 mesh/'
B. Apparatus (See Fig. 949. 12)
(a) Oxygen. — Cylinder with pressure regulator adjustable
from to 10 lb pressure (69 kPa) on low-pressure side and
with needle-valve control.
(b) Preheater. — Specifications as recommended by Com-
mittee on Microchemical Apparatus, Div. Anal. Chem. (Anal.
Chem. 21, 1555(1949)), except with 12/2 ball joint. Rubber
connectors may be used.
(c) Bubble counter and U-tube. — According to recom-
mended specifications, except with ball joints. Rubber con-
nectors may be used.
(d) Combustion tube. — Fused quartz (or Vycor), dimen-
sions according to recommended specifications but with 12/2
ball joint on side arm and 5/12 or 7/15 inner joint on exit
end. Rubber connectors may be used. Pyrex glass tubes may
be used, but furnace temps should be <725°.
(e) Absorption tubes. — Pregl-type, according to recom-
mended specifications but with 5/12 joints; alternatively, Pra-
ter-type, semimicro size with 7/15 joints. Rubber connectors
may be used.
(f) Bubble counter or flowmeter. — Any convenient ar-
rangement to measure 10-30 mL/min gas flow from exit end
of second absorption tube.
(g) Preheater furnace . — Elec, 12-14 mm id x 13 cm (5")
long, maintained at 600 ± 25°. Gas heaters may be used for
all furnaces but specified temps should be maintained . Temps
of furnaces are measured at center of furnace inside empty
combustion tube with one end stoppered.
(h) Burning furnace. — Elec, 13-14 mm id X 10 cm (4")
long. Furnace should reach 600-700° in 5 min, ca 800° in 15
min, with max. of 850° in 30 min. See (g).
(i) Long furnace. — Elec, 13—14 mm id x 20 cm (8") long;
maintained at 775-800°. See (g).
(j) Constant temperature mortar. — Elec, 13—14 mm id x
8 cm (3") long, thermostatically controlled at 177 ± 2°. See
(g).
C. Preparation of Apparatus
(a) Preheater. — Place CuO in preheater tube, connect spi-
ral cooling coil, immerse coil in beaker of H 2 0, and support
assembly by suitable clamps and stand. Place furnace over pre-
heater tube and maintain at ca 600°. Connect side arm of com-
bustion unit to needle valve of O pressure regulator by suitable
tubing, rubber or Tygon.
(b) Bubble counter-U -tube . — Fill bubble counter and U-tube
by placing glass wool plug at bottom of U, fill side next to
bubble counter with Dehydrite to within 12 mm of side arm,
and cap with another glass wool plug, Place Ascarite layer in
other side to within 38 mm of side arm; then insert glass wool
plug, ca 25 mm of Dehydrite, and finally second plug. Cement
in stoppers with glass cement or paraffin; then with medicine
dropper add H 2 S0 4 to bubbler until level is 3-4 mm above
bubbler tip. Connect to preheater with pressure clamp.
(c) Combustion tube. — Clean and dry combustion tube. Place
10 mm roll of Ag in exit end with 1 or 2 strands reaching to
open end of ground joint. Insert loose asbestos plug (not chok-
ing plug), 40 mm Pb0 2 , asbestos plug, and second Ag roll 25
mm long, which should extend into long furnace ca 12 mm.
Insert asbestos plug, 60 mm CuO, asbestos plug, 30 mm Pt
gauze roll, asbestos plug, 60 mm CuO, asbestos plug, and
finally 30 mm Pt gauze, which should extend about 10 mm
beyond end of long furnace. Place prepd tube in furnaces with
exit end protruding far enough beyond const temp, mortar to
permit connecting absorption tubes. Connect side arm to bub-
ble counter-U-tube.
(d) Absorption tubes.— 'Place glass wool plug in end of H 2
absorption tube, fill tube to within 12 mm of other end with
340
MlCROCHEMICAL METHODS
AOAC Official Methods of Analysis (1990)
COMBUSTION TUBE
10-1 1 rnm od -
PREHEATER
To oxygen source -
7/25$
COOLER
8 to 12 turns'"
5 mm ± 0.5 mm od
1 1.25 ± 0.5 mm od
8 ±0.25 mm id
1.5 mm min. wall
12/3 ball & socket
^
4 mm ± 0.5 mm od
BUBBLE COUNTER
$ No. 9 solid stoppers
4
1.5-2.0 mm id
5 ± 0.5 mm od
3-4 mm od
1.2-1.5 m
9
7 E 30-3530-35V_y
o E
I
mm mm
30-40
mm
COMBUSTION TUBE
Pt "l Pt boat Pt " ~ Cub
mm ■ 12/2 ball & socket
-13 ±0.5 mm od
5/12 JforPreglor
7/1 5$ tor Prater
absorption tubes
mm 10mm
14/20 joints $ ■
Glass
wool
2.25 ± 0.25 mm id
3.25 ± 0.25 mm id
0.40 ± 0.10 mm openings
\ 30-32
X0.40 ± 0.1 mm openings mm
\. J Dehydrite_\ h*— *\
^$ 4 \ 4 Glass Jl I30-
5/12 1 10/10 frZT m W H '
l~8-9 mm id
ABSORPTION TUBES
4-5 mm LENGTH
To flow meter
or bubble counter
5 mm od /
Wall thickness 0.55 ± 0.10 mm
11-12 mm id
-^-9-10 mm od
0.55 ± 0.10 mm wall
PREGL TUBES
Dehydrite ~
PRATER TUBES
FIG. 949.12— Carbon and hydrogen apparatus
Dehydrite or Anhy drone, and cap with second glass wool plug.
If Pregl tubes are used, seal ground-glass joint with enough
glass cement to give clear seal, and remove any excess on
outer surface of tube with cotton dipped in benzene or other
solv. If Prater tubes are used, lubricate lower 2 /3 of inner joint
with min. of light stopcock grease and insert in outer tube.
Prep. C0 2 absorption tube by placing glass wool plug in end
and fill tube to within ca 38 mm of other end with Ascarite.
Insert 6 mm glass wool plug, add 20 mm layer of Dehydrite,
and cap filling with another glass wool plug. Complete assem-
bly of absorption tube as for H 2 absorption tube. Connect
absorption tubes to combustion tube with ground joints (use
no lubricant) or with special impregnated rubber tubing.
Attach calibrated bubble counter or flowmeter to exit end of
C0 2 absorption tube.
D. Determination
(a) Conditioning apparatus. — Condition prepd and assem-
bled app. by heating combustion tube 3-4 hr with long furnace
at 775-800° and with O flowing thru app. at rate of 15-20
mL/min. Use 3-4 lb (21-28 kPa) O pressure on low pressure
side of regulator. At the same time, make 2 simulated sample
burnings, without sample, with burning furnace at 825-850°.
(Temp, must be ca 100° lower if Pyrex combustion tubes are
used.)
Burn un weighed 10—15 mg sample to condition combustion
and absorption tubes. With absorption tubes connected, adjust
needle valve on regulator so that O flow is 15-20 mL/min
and place burning furnace ca 75 mm from long furnace. Place
micro Pt boat contg sample in combustion tube ca 50 mm from
long furnace. Insert third Ptroll 25 mm from boat, and stopper
tube. Turn on burning furnace and let it reach ca 600° before
starting sample combustion by moving furnace over sample at
rate of 25 mm in 6-8 min. Move burning furnace across sam-
ple only once, taking 18-24 min for full travel of furnace.
Turn off burning furnace 5 min after it reaches long furnace
but continue to sweep O thru tube for addnl 15 min before
disconnecting absorption tubes.
Remove absorption tubes and place by balance to equili-
brate. Handle tubes only with clean chamois finger cots. If
Prater tubes are used, turn joints 'At turn to seal. If rubber con-
nections are used, wipe only tips of tubes with moist, then dry,
chamois before placing them by balance. Wait 10 min if ground
joints were used or 15 min if rubber connections were made;
then weigh C0 2 absorption tube first and H 2 absorption tube
next, using glass tare with vol. and surface approx. equal to
that of absorption tubes. Record wts of tubes and reconnect to
combustion tube for subsequent analysis.
(b) Proving the apparatus. — Replace boat with one contg
10-15 mg sample of std compd such as NIST microchem. std,
weighed to nearest 0.01 mg. Repeat combustion and weighing
as in (a). Calc. % C and H in std sample from increase in wt
AOAC Official Methods of Analysis (1990)
Carbon, Hydrogen
341
of C0 2 and H 2 absorption tubes. Repeat analysis until results
from 2 consecutive runs are within 0.30% of theoretical values
and means of C and H results are within 0.20% of theoretical
value for the std compd. (Humidity conditions of room may
make it necessary to correct apparent wt of H 2 by subtracting
a blank value.)
When app. meets this test, analyze samples as above.
%C = wt CO, x 0.2729 X 100/wt sample
%H = wt H 2 x 0. 1 1 19 x 100/wt sample
Refs.: JAOAC 32, 561(1949); 34, 94, 607(1951). Anal. Chem.
23, 911(1951).
CAS-7440-44-0 (carbon)
CAS- 1333-74-0 (hydrogen)
972.43 Microchemical Determination
of Carbon, Hydrogen, and Nitrogen
Automated Method
First Action 1972
Final Action 1975
A. Apparatus
(See instrument instruction manuals)
(a) Automatic carbon-hydrogen-nitrogen (C-H-N) ana-
lyzer. — Model 185 (FM) (current model 185B, Hewlett-Pack-
ard), Perkin-Elmer 240 (PE) (current model 240B, or 240C
2400 CHN Elemental Analyzer, Perkin-Elmer Corp.), or equiv.
(b) Helium. — Cylinder with pressure regulator and needle
valve control. Preheater and purifier optional.
(c) Oxygen. — For PE only. Cylinder with pressure regula-
tor and needle valve control.
(d) Line voltage regulator. — Optional; 50 amp, output 115
v ± 0.25%.
(e) On-line computer or integrator. — Mandatory for FM app.
but optional for PE app.
B. Reagents
(See instrument instruction manuals.)
(a) Catalyst. — Solid oxidn catalyst (oxides of Co, W, Mn,
or Ag) required for FM; optional for PE if time and temp, meet
conditions specified.
(b) Std compounds. — N1ST Acetanilide, or equiv.
C. Preparation of Apparatus
Prep, and assemble app. as in manual. Adjust preliminary
settings and regulate He flow (He and O for PE). Set and let
temp, systems equilibrate until const. Use combustion temp.
> 1080° for FM and 980- 1000° for PE. Use specified 500° and
650° reduction temps, resp., for FM and PE. Maintain detector
column suboven within 5-15° of main oven. Adjust bridge
current to value specified. After sweeping air "slug" from
combustion chamber, use 20-50 sec range combustion period
(gas flow diverted) for FM (40-50 sec for samples difficult to
burn). Use extended "Hold 30 sec" combustion period for PE.
Add C03O4 + Ag 2 W0 4 , Ag 2 + Ag 2 W0 4 , or CoO + W0 3
to combustion tube filling of PE. However, if PE is in opti-
mum condition, only 2 of 3 required conditions (temp., time,
and catalyst) need be adhered to.
D. Determination
Burn 2 unweighed samples ca 2 mg (PE) or 0.6 mg (FM)
to condition app. Make >2 blank runs (simulated sample runs
without sample) to check and adjust timing of each phase where
necessary, to check pattern of final measurements, and to ob-
tain blank factors if required in calcns. Then run std and sam-
ple compds, weighed to nearest 0.001 mg or better for PE and
0.0001 mg for FM. Calc. factors as suggested in manual. Re-
run std to check factors. Different type std may be used for
this rerun. Initially check factors until 2 of 3 detns are within
0.3% of theoretical value. Calc. % C, H, and N, using factors
obtained from std compds.
E. Special Precautions for Voiatiie Samples
Weigh volatile samples in capillaries, Al capsules, or Al
weighing pans. During sweeping period, volatile samples must
be in cooler portion of combustion tube, as near orifice as pos-
sible.
Refs.: JAOAC 54, 808(1971); 55, 676(1972).
CAS-7440-44-0 (carbon)
CAS-1333-74-0 (hydrogen)
CAS-7727-37-9 (nitrogen)
961.16 Microchemical Determination
of Fluorine
Titrimetric Method
First Action 1961
Final Action 1969
A. Reagents
(a) Sodium alizarin sulfonate indicator. — (Alizarin Red S)
0.035% aq. soln.
(b) Sodium fluoride std soln.—0.0\N. Dissolve 0.4200 g
NaF in H 2 and dil. to 1 L.
(c) Thorium nitrate std soln.—0.0\N. Dissolve 1.38 g
Th(N0 3 ) 4 .4H 2 in H 2 and dil. to 1 L. Stdze by titrg against
O.OUVNaF, using 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mL portions
and plotting curve.
0. Apparatus
(a) Schoniger combustion flask. — 500 mL with filter paper
carriers.
(b) Distillation apparatus. — See Fig. 961.16. Attach to steam
generator. Steam enters thru joint, J u passes thru 2 concentric
tubes, IT y and ET U and enters distn flask, D, thru 2 openings.
Vapors enter condenser, C, which consists of 3 concentric tubes.
In JT 2 and ET 2 , vapors are condensed; in £T 5 , cooling H 2 is
circulated. Distillate drains off on right thru descending tube.
Ground joint, J 2 , serves as opening for addn of soln and as
seat for thermometer which records temp, of liq., L. Elec.
heating jacket, //, surrounds section of distn flask contg liq.
and is prepd from 600 cm Nichrome wire, W, of 2. 120 ohms/
foot, 420 cm of which is wound on 48 mm diam. glass cyl-
inder covered with Al foil and asbestos, then covered with
insulating cement, asbestos, and another layer of cement. Jacket
is held in place with ring, R, and temp, is controlled with 7.5
amp variable transformer.
C. Determination
(a) In absence of arsenic, mercury, and phosphorus. — Place
sample contg 0.5-0.7 mg F on filter paper carrier. (Weigh liq.
samples in gelatin or Me cellulose capsules and place closed
capsule on paper carrier.) Add ca 15-20 mg Na 2 G 2 , wrap mixt.
in filter paper, and place in Pt basket carrier in stopper of
Schoniger flask. Place 20 mL H 2 in flask, introduce O sev-
eral min, ignite sample, and immediately insert into flask.
(Caution: Use safety barrier and reinforced gloves. Remote
control igniting device is available.) After combustion is com-
plete, shake vigorously until cloudiness disappears, and let flask
stand undisturbed ca \5 min to ensure complete absorption of
oxidn products. (If enough I is present to give yellow soln,
342
MlCROCHEMICAL METHODS
AOAC Official Methods of Analysis (1990)
warm on steam bath to dispel color.) Wash soln into titrg ves-
sel, adjust to pH 3.0 ± 0.05 with IN and 0. IN HC1 and 0.17V
NaOH, using pH meter, and add 2 mL Na alizarin sulfonate
indicator. (pH adjustment is critical, since alizarin sulfonate is
also acid-base indicator.) Titr. with std Th(N0 3 ) 4 soln to pink
end point, preferably using photoelec. photometer with 520
nm filter. Use entire soln rather than aliquot. If visual titrn is
used, compare color with controls in fluorescent light. Det.
mg F from std curve.
% F = mg F X 100/mg sample
(b) In presence of arsenic, mercury, and phosphorus . — Burn
sample as in (a) and transfer soln to distn app. thru joint J 2
with as little H 2 as possible. Add 20 mL 70-72% HC10 4 , 1
mL 25% AgCl0 4 soln, and ca 12 glass beads. Heat mixt. by
means of jacket, and as temp, rises, start steam generation.
Maintain temp, of mixt. at 135 ± 2° after raising temp, to this
point as quickly as possible by adjusting transformer. Collect
distillate in 250 mL vol. flask. (Practice is required for suc-
cessful manipulation of distn. To avoid sucking back of soln,
keep vol. in flask at min. and keep steam generation const.
Addn of phthln and small amt of 0.1/V NaOH to generator
provides means of detg if suck-back has occurred.) (Clean distn
app. between detns by replacing steam generator with bottle
or flask connected to suction and immersing distillate delivery
tube in F-free H 2 0, which is sucked thru entire system.)
Transfer distillate to titrg vessel, adjust to pH 3.0 ± 0.05,
add 2 mL Na alizarin sulfonate indicator, and titr. as in (a).
Ref.: J AOAC 44, 258(1961).
CAS-7782-41-4 (fluorine)
mm id
ET 3 26 mm id
ET2 16 mm id
IT 2 10 mm id
7 mm id
*\- H 49 mm id
N — W
69 mm
Ji § 24/40
FIG. 961.16— Upper section of distilling apparatus. From Anal.
Chem. 29, 141(1957)
960.52 Microchemical Determination
of Nitrogen
Micro-Kjeldah! Method
First Action 1960
Final Action 1961
(Not applicable to material contg N-N or N-O linkages)
A. Reagents
(a) Sulfuric acid. — Sp gr 1.84, N-free.
(b) Mercuric oxide. — N-free.
(c) Potassium sulfate. — N-free.
(d) Sodium hydroxide-sodium thiosulfate soln. — Dissolve
60 g NaOH and 5 g Na,S,Ov5H 2 in LLO and dil, to 100 mL
or add 25 mL 25% Na 2 S 2 6 3 .5H 2 6 to 100 mL 50% NaOH soln.
(e) Boric acid soln. — Satd soln.
(f) Indicator soln. — (/) Methyl red-methylene blue. — Mix
2 parts 0.2% ale. Me red soln with 1 part 0.2% ale. methylene
blue soln; or (2) Methyl red-bromocresol green soln. — Mix 1
part 0.2% ale. Me red soln with 5 parts 0.2% ale. bromocresol
green soln.
(g) Hydrochloric acid. — 0.027V. Prep, as in 936. ISA and
stdze as in 936.15E-G.
B. Apparatus
(a) Digestion rack. — With either gas or elec. heaters which
will supply enough heat to 30 mL flask to cause 15 mL H 2
at 25° to come to rolling boil in >2 but <3 min.
(b) Distillation apparatus. — One-piece or Parnas- Wagner
distn app. recommended by Committee on Microchemical Ap-
paratus, ACS.
(c) Digestion flasks . — Use 30 mL regular Kjeldahl or Sol-
tys-type flasks (Ref.: Anal. Chem. 23, 523(1951)). For small
samples, 10 mL Kjeldahl flasks may be used.
C. Determination
Weigh sample requiring 3-10 mL 0.01 or 0.02N HC1 and
transfer to 30 mL digestion flask. If sample wt is <10 mg,
use microchem. balance (max. wt 100 mg dry org. matter).
Use charging tube for dry solids, porcelain boat for sticky sol-
ids or nonvolatile liqs, and capillary or capsule for volatile
liqs. Add 1.9 ± 0.1 g K 2 S0 4 , 40 ± 10 mg HgO, and 2.0 ±
0.1 mL H 2 S0 4 . If sample wt is >15 mg, add addnl 0.1 mL
H 2 S0 4 for each 10 mg dry org. matter > 15 mg. Make certain
that acid has sp gr ^ I .84 if sample contains nitriles. (10 mL
flasks and V2 quantities of reagents may be used for samples
<7 mg.) Add boiling chips which pass No. 10 sieve, if boiling
time for digestion rack heaters is 2-2.5 min, digest 1 hr after
all H 2 is distilled and acid comes to true boil; if boiling time
is 2.5-3 min, digest 1.5 hr. (Digest 0.5 hr if sample is known
to contain no refractory ring N.)
Cool, add min. vol. of H 2 to dissolve solids, cool, and
place thin film of Vaseline on rim of flask. Transfer digest and
boiling chips to distn app. and rinse flask 5 or 6 times with
1-2 mL portions H 2 0. Place 125 mL Phillips beaker or er-
lenmeyer contg 5 mL satd H 3 B0 3 soln and 2-4 drops indicator
under condenser with tip extending below surface of soln. Add
8-10 mL NaOH-Na 2 S 2 3 soln to still, collect ca 15 mL dis-
tillate, and dil. to ca 50 mL. (Use 2.5 mL H 3 B0 3 and 1-2
drops indicator, and dil. to ca 25 mL if 0.01 N HC1 is to be
used.) Titr to end point. Make blank detn and calc.
%N = [(mL HC1 - mL blank) X normality
x 14,007 x 100]/mg sample
Refs.: JAOAC 32, 561(1949); 33, 179(1950); 43, 689(1960).
CAS-7727-37-9 (nitrogen)
AOAC Official Methods of Analysis (1990)
Oxygen
343
963.29 Microchemlcal Determination
of Oxygen
Gravimetric Method
First Action 1963
Final Action 1965
A. Principle
Org. O compds are thermally decomposed in inert atm. to
H 2 0, CO, and C0 2 . At 1120°, following reactions are com-
plete: C + C0 2 -> 2CO; H 2 + C -» H 2 + CO. The CO is
converted to C0 2 by reaction with CuO and C0 2 is detd gravi-
metrically.
B. Reagents
(a) Copper oxide. — See 949.12A(a).'
(b) Ascarite.—See 949.12A(h)
(c) Dehydrite or Anhydrone. — See 949.12A(g).
(d) Nitrogen. — Purify high purity N by passing thru series
of scrubbing bottles contg different solid desiccants (CaCl 2 ,
anhyd. Mg(C10 4 ) 2 , and P 2 5 ), then thru tube contg closely
packed reduced Cu turnings at 600°, thru Anhydrone, and fi-
nally thru bubble counter and U-tube.
(e) Carbon. — Fisher, C-198, lampblack, orequiv. Pelletize
by swirling constantly while adding CCU dropwise; dry in oven
at 100-120°. Purify by digesting with HC1. Add large vol.
H 2 0, stir mech., let C settle, and decant H 2 0. Repeat until
H 2 wash is Cl-free. Dry C, place in quartz tube, and heat in
slow stream of N, (d), several hr, increasing temp, gradually
to 550°.
(f) Quartz chips.— Clean chips with HF, rinse with H 2 0,
and dry in oven.
C. Apparatus {See Figs. 963.29A and B)
(a) Long stationary furnace, 675°. — 949.12B(i), except
maintained at 675 ± 5°; (A).
(b) Long stationary furnace , 1120°. — See (c); (B).
(c) Short movable furnace, 1120°. — This furnace (C) and
that in (b) may be available as unit.
(d) Nitrogen purification train. — Preheater furnace (D),
949.12B(g), with section of combustion tubing, quartz, Vy-
cor, or Pyrex glass No. 1720, or equiv., packed with ca 10
cm reduced Cu turnings.
(e) Bubble counter and U-tube.— See 949.12B(c) and
949.12C(b).
(f) Cap. — J 14/35 cap with 2 mm stopcock; (F).
(g) Thermal decomposition tube. — Clear fused quartz, so-
lar radiation grade, 7.5-8 mm id, 10-11 mm od, and 60 cm
long, exclusive of capillary tube; (G). At one end is ¥ 14/35
male joint. About 1 cm from joint is bent side arm, 6-7 mm
od and 2-4 mm id. At other end is 15 cm capillary tube 6-7
mm od and 1-2 mm id. Wash tube with HF and H 2 0, and
dry.
Fill tube as shown in Fig. 963. 29B, with repeated tapping
to avoid channeling. The C must at all times be in section of
furnace that is at 1120°.
(h) By -pass stopcocks. — 3 way T-type i ball joint on side
arm; (H) and (H f ).
(i) Spiral by-pass tube. — Glass tube (/), contg spiral, for
flexibility, and & socket joints on each end. Length should be
sufficient to connect to 4 ball joints on J stopcocks, (//) and
("')■
(J) Quartz tube. — Filled with reduced Cu; (J).
(k) Short stationary furnace, 900°.-949A2B(h), except
capable of maintaining 900°; (K).
_ High purity dry nitrogen or
helium, further purified by
some solid desiccants
Heavy-walled
impregnated
rubber
Purification tube
copper turnings
Thin-walled
rubber tubing
600"
pre heater
combustion furnace
FIG. 963.29A — Gravimetric setup for oxygen determination. (Note: (1) All rubber connections made of heavy-walled impregnated
rubber except Mariotte bottle. (2) Tubes marked (Rfl) and (L) may be replaced with regulation absorption tubes)
Capillary tubing
6-7 mm od
1-2 mm id
7.5 -8 mm id
10-11 mm od
6-7 mm od
2-4 mm id
Stopcock to
end cap 2 mm
Bore
Glass
hooks
FIG. 963.29B — Quartz reaction tube and filling for oxygen determination
344
MlCROCHEMICAL METHODS
AOAC Official Methods of Analysis (1990)
(1) Scrubber tube. — Any type of drying tube fitted with
crushed KOH pellets or Ascarite, (L), for removing halogens
and S.
(m) Drying tube. — See 949.12B(e), or use U-tube. Fill with
Anhydrone only, as in 949.12C(d); (A/).
(n) Carbon dioxide absorption tube. — Fill with Ascarite and
Anhydrone as in 949.12C(d); (TV).
(o) Oxidation tube. — Tube, 30-40 cm, exclusive of tip,
similar to that in (d) except packed with ca 25 cm CuO wire.
Let 5 cm extend beyond furnace. Maintain at 675 ± 5° to
oxidize CO to C0 2 ; (0).
(p) Guard tube.— Glass tube 110-120 mm x 10-12 mm
od, 1 mm wall, contg Anhydrone; (P).
(q) Mariotte bottle. — Glass 2 L bottle. Place 1-hole rubber
stopper in top of bottle. Insert glass tube to 7-10 cm of bottom
of bottle. Attach 3- way stopcock to top of tube. In bottom
opening insert 1-hole rubber stopper contg drain tube to 1 L
graduate. Fill bottle with H 2 0; (Q),
(r) Safety trap. — Gas washing bottle, 125 mL, Drechsel tall-
form, Kimble Glass, No. 15060 or equiv. Connect T-tube to
top of center tube. Fill bottle ca 1 / 3 with Hg and connect by
T-tube between preheater and N supply. (Gas pressure valve,
Friedrich, or other regulator may also be used.)
(s) Nitrogen tank. — Use tank of high purity N equipped with
pressure reducing valve, and safety valve to blow off at set
pressure.
D. Assembling Apparatus
Assemble as in Fig. 963. 29A, starting with N tank (s), not
shown, and connecting as follows, using ground glass joints
and paraffin-impregnated heavy-wall tubing: Safety trap (r),
not shown; purification train (Z>); bubble counter and U-tube
(£); thermal decomposition tube (G); spiral by-pass tube (/);
quartz tube (J); U-tube (L); oxidn tube (O); drying tube (M);
C0 2 absorption tube (N); guard tube (P); and Mariotte bottle
(0-
E. Conditioning Apparatus
Assemble app. up to C0 2 absorption tube; set various fur-
naces in place. Pass slow stream of N (10 mL/ min) thru sys-
tem 1-2 hr at room temp. Heat all units to specified temps
and continue N stream 2 days. Attach remainder of app. with
arm of Mariotte bottle slightly below horizontal, and adjust ht
of Hg in safety trap (r) to obtain 10 mL N/min thru system
with all parts at operating temp. Record rates of bubble flow
thru bubble counter and Mariotte bottle.
C in thermal decomposition tube (g) must be at 1120°. Best
results are obtained by keeping furnaces at specified temp, at
all times, even when not in use.
F. Determination
Adjust N flow to 10 mL/min with all furnaces heated to
specified temp. Cool short movable furnace (C) to room temp.
Weigh enough sample to produce 1 .0-1 .3 mg O; if solid, weigh
directly in Pt boat; if liq., weigh in capillary tube, preferably
quartz, and insert in Coombs-Alber Pt sleeve or long Pt boat.
Turn stopcocks (H) and (//') and open stopcock on cap (F) to
let N flow in reverse direction thru tube (G). Remove cap (F),
and insert Pt boat contg sample to within ca 7 cm of long
furnace (B) with aid of Pt hook on end of glass rod. Imme-
diately replace cap (F) with its stopcock open and let reverse
flow of N continue ca 20 min to expel all air.
Weigh C0 2 absorption tube (N) as in 949.12D(a) and attach
in position. Close stopcock on cap (F) and turn stopcocks (H)
and (//') so N flows forward thru decomposition tube (G). Open
stopcock on Mariotte bottle (g) and let N flow thru entire sys-
tem at 10 mL/min. Heat movable furnace (C) to ca 1120°,
move to within ca 3 cm of sample, and turn on automatic drive
to pyrolyze sample slowly. About 25-30 min is required for
furnace (C) to reach furnace (B). Move furnace forward and
heat parts of tube insulated by walls of furnaces 5-10 min to
pyrolyze any material condensed in cooler portions of tube.
Return furnaces to original positions. Let furnace (C) cool.
Continue flow of N until ca 700 mL has passed thru from
beginning of pyrolysis, using exactly same amt in detn and
blank. Remove CO^ absorption tube N, and reweigh as in
949.12D(a).
Perform blank detn with empty Pt boat and subtract wt C0 2
of blank from that of detn. (Well-functioning app. gives zero
blank.)
% O - Wt C0 2 x 0.3635 x 100/wt sample
G. Cleaning Reaction Tube
When visibility becomes poor on thermal decomposition tube
from deposited C, remove as follows: With entire app. assem-
bled (with absorption tube and Mariotte bottle attached), close
stopcock (//) to both (G) and (/). Turn stopcock (//') to con-
nect (G) to (/). Open cap stopcock (F), lower arm of Mariotte
bottle, and suck air thru reaction tube. Heat movable furnace
to 1 120° and move it over against long furnace; C will be burned
off in few min. Close stopcock of cap (F), and turn stopcock
(H) to connect reaction tube (G) with N supply. Pass N thru
system overnight to remove air.
Refs.: Steyermark, A., "Quantitative Organic Microanaly-
sis," 2nd Ed., Academic Press, New York (1961).
J AOAC 46, 559(1963).
CAS-7782-44-7 (oxygen)
957.18 Microchemical Determination
of Phosphorus
Kjeldahl Digestion Method
Final Action
A. Reagents
(a) Nitric- sulfuric acid mixture. — Slowly pour 420 mL HNO3
into 580 mL H 2 0; then slowly add 30 mL H 2 S0 4 .
(b) Ammonium nitrate soln. — 2%. Prep. 2% soln of NH4NO3
in H 2 0, add 2 drops HNO3, and store in g-s bottle. Filter im-
mediately before use.
(c) Molybdate reagent. — Dissolve 150 g powd NH 4 mo-
lybdate in 400 mL H 2 and cool under tap. Place 50 g
(NH 4 ) 2 S0 4 in 1 L vol. flask, dissolve in mixt. of 105 mL H 2
and 395 mL HN0 3 , and cool under tap. Pour cooled molyb-
date soln slowly into (NH 4 ) 2 S0 4 soln with const stirring and
cooling under tap. Dil. soln to 1 L, store in refrigerator 3 days,
filter, and store in paraffin-lined, g-s, brown bottle in refrig-
erator. Filter reagent immediately before use and check by pe-
riodically analyzing std sample.
B. Apparatus
(a) Kjeldahl digestion flasks (30 mL), rack, and mani-
fold. See 960.52B(a) and (c).
(b) Filter tubes and filtration assembly. — See 952.24B(c)
and (d).
(c) Rubber stoppers. — Two or three small, solid rubber
stoppers to loosen ppt from walls of flask.
C. Determination
Weigh 3-20 mg sample, depending on P content and whether
microchem. or semimicrochem. balance is used (max. wt ppt
= 50 mg). Weigh in charging tube, if possible, and transfer
to Kjeldahl flask. Use porcelain boat for sticky solids and vis-
cous liqs, and glass capillary for volatile liqs.
AOAC Official Methods of Analysis (1990)
Sulfur
345
Add 0.5 mL H 2 S0 4 followed by 4-5 drops HN0 3 . Heat on
digestion rack to white S0 3 fumes and cool under tap. Add 4-
5 drops HN0 3 , repeat digestion, and cool under tap. Add 4-
5 drops HNO3 and again digest to SO3 fumes. Cool to room
temp.; add 2 mL acid mixt., (a), and 12.5 ml H 2 0, rinsing
down neck of flask. (If porcelain boat was used to add sample,
remove boat with Pt wire; if glass capillary was used, filter
digestion mixt. to remove capillary. Rinse filter and boat or
capillary with 12.5 mL H 2 used to dil. sample.)
Place flask on steam bath 15 min to convert P to H 3 P0 4 .
Remove from steam bath and pipet 15 mL molybdate reagent,
(c), into center of digest, not down walls of flask. Let stand
2-3 min; then gently swirl to mix contents, being careful to
prevent reagents from splashing on neck of flask. Cover flask
and set in dark place overnight.
Condition filter tube as described below and weigh empty
tube. Connect tared filter tube to filtration assembly and trans-
fer ppt to filter thru siphon tube. Wash flask alternately with
1-2 mL portions of the NH 4 N0 3 soln and alcohol. Add 2-3
small rubber stoppers to digestion flask, shake to loosen any
ppt, and transfer with the NH4NO3 soln and alcohol. Discon-
nect siphon tube; rinse ppt from tip and stopper into filter tube
with the NH4NO3 soln and alcohol. Wash ppt with more
NH4NO3 soln, alcohol, and finally with acetone, and suck dry.
Wipe filter tube with chamois skin, place in vertical position
in vac. desiccator contg no desiccant, and evacuate to 1 mm
for 30 min with mech. vac. pump in continuous operation.
Release vac. and weigh immediately to nearest 0. 1 mg. (Rapid
weighing is essential because of hygroscopic nature of ppt.)
% P - mg ppt x 0,014524 X 100/mg sample
Ref.: JAOAC 40, 386(1957).
CAS-7723-14-0 (phosphorus)
952.25 Microchemicai Determination
of Sulfur
Titrimetric Carius Combustion Method
Final Action
(Not applicable in presence of P)
A. Reagents
(a) Fuming nitric acid. — Reagent grade, sp gr 1.50.
(b) Sodium chloride. — Reagent grade, fine crystals.
(c) Barium chloride soln. — Approx. 0.02N. Stdze by titrg
5-7 mg freshly dried K 2 S0 4 , ACS (weighed to nearest 0.01
mg), by method used for sample titrn, 952. 25D. Correct titrn
for indicator error by blank detn.
Normality
= mg K 2 S0 4 /174.258 (mL BaCl 2 soln - mL blank)
(d) Potassium sulfate. — ACS, powd and dried.
(e) Phenolphthalein soln. — 0.5% soln in 50% alcohol.
(f) Sulfate indicator.-— Tetrahydroxyquinone (THQ) sulfate
indicator (No. 215, Betz Laboratories, Inc., 4636 Somerton
Rd, Trevose, PA 19047-6783) or mix 0.1 g K rhodizonate
with 15 g sucrose by grinding in mortar.
B. Apparatus
(a) Combustion tubes and furnace. — See 952.24B(a) and
(b).
(b) Titration assembly. — 5 mL buret graduated in 0.01 mL;
rectangular titrn cell ca 2 X 4 X 5 cm with min. capacity of
50 mL; and std orange-red glass color filter (Corning Glass
Works No. 3482, CS 3-67) selected to have 37% T at 550 nm.
(Alternatively, use ref. titrn cell contg 30 mL of soln of 20 g
Na 2 Cr 2 7 /L H 2 0.) Place cell and filter side by side on milk
glass window illuminated from below, preferably by fluores-
cent light. Mask light source so that only cells and filter are
illuminated. For best results use no overhead illumination. Place
microscope slides (1-3) with ground glass surface (prepd by
grinding with H 2 suspension of fine SiC) over glass filter to
compensate for increased turbidity.
C. Sample
Using microchem. balance, weigh 5-20 mg sample contg
>0.75 mg S, or using semimicrochem. balance, weigh 10—20
mg sample contg ^0.75 mg S (1.5 mg for gravimetric detn).
Weigh samples as in 952. 24C.
D. Determination
Place weighed sample in combustion tube, add NaCl 100%
in excess of amt equiv. to S in sample, and proceed as in
952. 24D, beginning "... and add . . . fuming HN0 3 , ..."
thru end of third par. "... with glass splinters. "
Transfer contents of tube to 50 mL beaker, rinsing tube
4-6 times with 3-5 mL portions H 2 0. Evap. to dryness on
steam bath.
Dissolve residue in 10 mL H 2 0, pour soln into titrn cell,
add 1 drop phthln, and make just alk. with ca 0AN NaOH,
then acid with ca 0.02N HC1, adding 1 drop excess. Add ca
0.15 g of the sulfate indicator, stir to dissolve, and rinse beaker
2 or 3 times, using enough alcohol so that final soln contains
ca 50%. Titr. with std BaCl 2 soln from 5 mL buret until stable
color of soln immediately after stirring matches std glass color
filter. Make certain end point taken is real and not pseudo end
point which fades on standing 1-2 min. (Addn of 1-2 drops
BaCl 2 soln develops definite red.) Det. bJank on reagents and
correct titrn.
% S = (mL BaCL - mL blank)
x normality X 16.032 x 100/mg sample
Refs.: JAOAC 35, 305(1952); 36, 94, 335(1953).
CAS-7704-34-9 (sulfur)
955.48 Microchemicai Determination
of Sulfur
Gravimetric Carius Combustion Method
Final Action
(Applicable in presence of P)
A. Apparatus
Crucible and filter stick. — Porcelain crucible, ca 15 mL ca-
pacity, with black inside glaze, wt ca 10 g; with porcelain
filter stick, with unglazed bottom, wt ca 2 g. (Altho filter stick
is weighed with crucible, it is removed before addn of soln of
residue.) (Anal. Chem. 21, 1555(1949).)
B. Determination
Dissolve residue, 952. 25D, in 3 mL H 2 0, pour into previ-
ously ignited and weighed (with filter stick) porcelain crucible,
and rinse beaker with four 2 mL portions H 2 0. Place crucible
on steam bath until soln is near bp. If vol. exceeds 10-11 mL,
evap. to this vol. Add drop wise 0.5 mL 10% BaCl 2 soln
(1 mL for samples contg >5 mg S), digest >15 min, and cool
15 min.
Connect porcelain filter, previously ignited and weighed with
crucible, to arm of siphon with rubber tubing. Connect other
arm of siphon to suction flask thru rubber stopper. Lower filter
into crucible, slowly draw off soln, and rinse ppt, walls of
crucible, and filter with five or six 3 mL portions HO (1 +
346
MlCROCHEMICAL METHODS
AOAC Official Methods of Analysis (1990)
300), drawing off as much liq, as possible. Carefully detach
filter, place in crucible, wipe outside of crucible and end of
filter with moist chamois or cheesecloth, and handle thereafter
with crucible tongs. Place crucible and filter in larger crucible
and dry in oven 10 min at ca 110°. Ignite in furnace 10 min
at 700-750° (ppt may also be ignited by heating larger crucible
contg crucible and filter to dull red heat with Meker burner),
cool on metal block 30 min or in desiccator 1 hr, and weigh.
Det. blank on reagents.
% S = (wt BaS0 4 - blank) x 0.1374 x 100/wt sample
Ref.: JAOAC 38, 377(1955).
CAS-7704-34-9 (sulfur)
on steam bath. Dissolve acid -free residue in ca 5 mL H 2 and
transfer to titrn cell. Rinse beaker with ca 5 mL H 2 0. Add 1
drop phthln and make just alk. with ca 0. \N NaOH; then acid-
ify with ca 0.027V HN0 3 . Add ca 0.15 g THQ indicator, stir
to dissolve, and rinse beaker 2-3 times with enough alcohol
so that final soln in cell contains ca 50% alcohol. Titr. with
std BaCl 2 soln from 5 mL buret until stable color of soln im-
mediately after stirring matches std glass color filter as in
952.25D.
% S = [(mL BaCl 2 - mL blank) x normality
x 16.032 x 100|/mg sample
ReL: JAOAC 58, 146(1975).
CAS-7704-34-9 (sulfur)
975.53 Microchemicai Determination
of Sulfur
Oxygen Flask Combustion Method
First Action 1975
(Not applicable in presence of P)
A. Reagents
(a) Barium chloride soln. — Approx. 0.02N. Stdze as fol-
lows: Accurately weigh 3.5-5.5 mg K 2 S0 4 into titrn cell, dis-
solve in 15 mL H 2 0, add 0.15 g sulfate indicator, (f), and
dissolve; dil. soln to 30 mL with alcohol. Titr. to end point
(same color as ref. glass) with BaCl 2 soln, making certain end
point taken is real; see 975. 53C. Titr. blank. Calc. normality
as in 952.25A(c).
(b) Hydrogen peroxide soln. — 30% (Fisher Scientific Co.
No. H-325, or equiv. purity). (Caution: See safety notes on
peroxides.)
(c) Oxygen cylinder. — With regulator and connections for
filling combustion flask.
(d) Potassium sulfate. — ACS, powd and dried.
(e) Phenolphthalein soln. — 0.5% soln in 50% alcohol.
(f) Sulfate indicator. — Tetrahydroxyquinone (THQ) sulfate
indicator (Betz Laboratories, Inc., 4636 Somerton Rd, Tre-
vose, PA 19047-6783) or mix 0.1 g K rhodizonate with 15 g
sucrose by grinding in mortar. Vac. -dry overnight at room temp.
B. Apparatus
(a) Mechanical shaker. — Optional.
(b) Oxygen flask combustion apparatus .—Thomas-Ogg in-
frared igniter (Thomas Scientific No. 6516-G10) and 500 mL
thick wall combustion flask (No. 6514-FlO), black sample
wrappers (No. 65I4-F70), Pt sample carrier, stopper, and clamp
to avoid loss of sample during pressure changes which occur
during combustion. App. is completely shielded within hinged
cabinet. Precautions used when employing other manually op-
erated elec. units should include proper safety shielding and
reinforced gloves. Flasks must be free of org. solvs to avoid
explosion .
(c) Titration assembly. —See 952.25B(b).
C. Determination
Weigh sample contg ca 0.75 mg S and fold into paper car-
rier. Add 5 mL Q.IN NaOH and 3 drops H 2 2 to 500 mL
combustion flask. Flush flask with O ^2 min. Place paper
carrier contg sample in Pt basket, hang on hook of stopper,
and insert stopper in flask. Ignite. Shake 30 min. (If gas phase
has not cleared, let stand 10 min.) Open flask, and rinse stop-
per and Pt sample basket with H 2 0. Transfer soln to 100 mL
beaker, rinsing flask with min. vol. H 2 0. Acidify with 2 mL
0.5N HN0 3 (S:l mL in excess of base) and evap. to dryness
976.29 Microchemical Determination
of Sulfur
Alternative Oxygen Flask Combustion Method
First Action 1976
(Not applicable in presence of P. All reagents must be as pure
as possible, since high concns of CI, F, N0 3 , P0 4 , K, and Na
interfere. Useful in absence of titrn assembly necessary for
THQ titrn.)
A. Reagents
(a) Barium perchlorate std soln. — Approx. 0.017V. Accu-
rately weigh ca 6 g Ba(Ci0 4 ) 2 .3H 2 and dissolve in 200 mL
H 2 0. Add 2 drops HC1 and dil. to 1 L. Stdze as follows: Ac-
curately weigh ca 3.8 mg 5-benzylisothiourea.HCl, (e), and
proceed as in 976. 29C, beginning "... and fold into paper
carrier." and continuing to ". . . and correct titrn values." Calc.
factor F, mg S/mL Ba(CJ0 4 ) 2 .
F = (mg 5-benzyJisothiourea.HCJ X 0.1582)/
[mL Ba(Ci0 4 ) 2 ~ mL blank]
where 0.1582 is fraction S in S-benzylisothiourea.HCl.
(b) Hydrogen peroxide soln.— 6%. Dil. 20 mL 30% H 2 2
(Fisher Scientific Co., No. H-325, or equiv. purity) to 100 mL
with H 2 0. (Caution: See safety notes on peroxides.)
(c) Methylene blue indicator soln. — Approx. 0.0125%.
Dissolve 12.5 mg methylene blue (J. T. Baker Chemical Co.,
or equiv.) in 100 mL H 2 0.
(d) Oxygen cylinder. — See 975. 53 A (c).
(e) S-Benzylisothiourea.HCL — Purity equiv. to NIST spec-
ifications.
(f) Thorin indicator soln. — Approx. 0.2%. Dissolve 200
mg thorin (J. T. Baker Chemical Co., or equiv.) in 100 mL
H 2 0.
B. Apparatus
(a) Mechanical shaker. — Optional.
(b) Oxygen flask combustion apparatus. — See 975.53B(b).
C. Determination
Weigh sample contg ca 0.60 mg S and fold into paper car-
rier. Add 10 mL 6% H 2 2 soln to 500 mL combustion flask.
Flush flask with O >2 min. Place paper carrier contg sample
in Pt basket, hang on hook of stopper, and insert stopper in
flask. Ignite. Shake 30 min. (If gas phase has not cleared, let
stand addnl 10 min.) Open flask, and rinse stopper and Pt sam-
ple basket with alcohol. Transfer soln to 200 mL beaker, rins-
ing flask with alcohol. Place mag. stirring bar in beaker. Add
alcohol to 100 mL mark. Add 2 drops thorin indicator soln
and 2 drops methylene blue indicator soln, and titr. with
AOAC Official Methods of Analysis (1990)
Alkoxyl Groups 347
Ba(C10 4 ) 2 soln to faint pink end point, stirring mag. Det. blank
on reagents and correct titrn values. Calc. % S, using factor
F from 976.29A(a).
% S - [(mL Ba(C10 4 ) 2 - mL blank)
X F x 100]/mg sample
Ref.: JAOAC 59, 1135(1976).
CAS-7704-34-9 (sulfur)
952.26* SVIicrochemical Determination
of Sulfur
Titrimetric Catalytic Combustion Method
Final Action
Surplus 1970
See 38.031-38.034, 10th ed.
955.49* EVAicrochemical Determination
of Sulfur
Gravimetric Catalytic Combustion Method
Final Action
Surplus 1970
See 38.035, 10th ed.
956.07 Microchemical Determination
of Alkoxyl Groups
Titrimetric Method
Final Action
4. Reagents
(a) Acetic acid-potassium acetate-bromine soln. — Dis-
solve 10 g KOAc in enough HO Ac to make 100 mL, and add
3 mL Br. Prep, fresh.
(b) Sodium acetate soln. — Dissolve 25 g NaOAc.3H 2 in
enough H 2 to make 100 mL.
(c) Starch indicator. — Mix ca 2 g finely powd potato starch
with cold H 2 to thin paste; add ca 200 mL boiling H 2 0, stir-
ring constantly. Add ca 1 mL Hg, shake, and let soln stand
over the Hg.
(d) Sodium thiosulfate std soln. — 0.02/V. Prep, daily by dilg
O.liV soln, 942.27.
(e) Hydr iodic acid. — Place 250 mL constant boiling HI
(57%, sp gr 1.7) in 500 mL r-b flask connected by ¥ joint to
air condenser, and reflux 2 hr while stream of C0 2 or N bub-
bles thru from glass tube extending to bottom. Do not let acid
vapors come in contact with org. material. As soon as reflux-
ing stops, discontinue gas flow. Cool, and store in g-s bottle.
B. Apparatus
Use modified Clark app., Figs. 956. 07 A and B.
C. Determination
Fill scrubber halfway with NaOAc soln, and fill receiver 2 / 3
full with freshly prepd KOAc-Br soln. Weigh enough sample
(?) Flask— capacity of
bulb 50 mL appro*.
©Receiver, volumeti
-$ 14/20
-*■"■*- 4.5-5.0 mm od
Must fit
into spiral (f9
©lr
5.5
Mu
i
-6,0 mm id
t ti; over
nlei tube (3>
<§
'
|rod 2 mm appro
-H h-9.5-
-10.0 mm od
Must fit into volumetric
receiver ©at "x"
©Spiral, volum
etric
FIG. 956.07A— Details of modified Clark apparatus
348
MlCROCHEMICAL METHODS
AOAC Official Methods of Analysis (1990)
RG. 956.07B— Modified Clark apparatus
in Pt boat to require ca 8 mL Na 2 S 2 3 soln in detn, and place
in bottom of boiling flask. Add 2.5 mL melted phenol from
wide-tip pipet and 5 mL of the HI, and connect boiling flask.
Pass C0 2 thru app. from side arm of flask at uniform rate of
15 niL/min. Let reaction mixt. remain at room temp. 30 min.
With manteled microburner. boil liq. at such rate that vapors
of boiling liq. rise into condenser, but not more than halfway;
continue boiling 60 min (first 30 min with H 2 circulating thru
condenser and last 30 min with H 2 drained from condenser).
Disconnect flask, remove receiver, and rinse delivery tube and
contents of receiver into 125 mL erlenmeyer contg 5 mL NaOAc
soln. Adjust vol. to ca 50 mL and add formic acid dropwise
until excess Br is destroyed.
Remove any Br vapors by blowing air over liq.; then add
0.5 g KI and 5 mL 10% H 2 S0 4 . Swirl soln to dissolve KI and
mix contents; then titr. liberated I with the Na 2 S 2 3 soln, using
starch indicator as in stdzn.
Det. blank on all reagents by making detn without sample.
% Alkoxyl group - (mL in detn — mL in blank)
X normality x equiv. wt x 100/mg sample
Equiv. wt: methoxyl = 5.173; ethoxyl — 7.510.
Ref.: JAOAC 39, 108, 401(1956).
13. Radioactivity
Edmond J. Baratta, Associate Chapter Editor
Food and Drug Administration
930.39* Radioactivity of Substances
Qualitative Method
Final Action
Surplus 1965
(Applicable to solids)
See 40.001, 10th ed.
933.09* Radioactivity of Substances
Quantitative Methods
Final Action
Surplus 1965
A. Emanation or Radon Method
(Applicable only to Ra in amts <10~ 9 g. Limit is arbitrary,
depending on particular equipment used and accuracy
required.)
See 40.002-40.005, 10th ed.
B. Gamma Ray Method Using Electroscope
(Applicable only to Ra in amts >10~ 5 g. Limit is arbitrary,
depending on particular equipment used and accuracy
required.)
See 40.006-40.010, 10th ed.
C. Gamma Ray Method Using Geiger-Mulier Counter
(Applicable only to Ra in amts >10~ 7 g. Limit is arbitrary,
depending on particular equipment used and accuracy
required.)
See 40.011^40.015, 10th ed.
969.48 Tritium in Water
Scintillation Spectrometric Method
First Action 1965
Final Action 1969
A. Principle
Sample is distd to remove quenching materials and nonvol-
atile radioactive materials. Distn is to dryness to ensure com-
plete transfer of 3 H to distillate. Aliquot of distillate is mixed
with scintillation soln and counted in liq. scintillation spec-
trometer (coincidence-type). Std 3 H and background samples
are prepd and counted alternately to nullify errors produced by
aging of scintillation medium or instrument drift.
B. Apparatus
(a) Liquid scintillation spectrometer. — Coincidence-type.
Available from Packard Instrument Co. , 2200 Warrenville Rd,
Downers Grove, 1L 60515; and others.
(b) Liquid scintillation vial. — 20 mL; low-K glass, poly-
ethylene, nylon, or equiv. bottles, available from manufactur-
ers under (a).
C. Reagents
(a) Scintillation soln. — Thoroly mix 4 g PPO (2,5-diphen-
yloxazole), 0.05 g POPOP (l,4-bis(5-phenyloxazol-2-yl) ben-
zene), and 120 g solid naphthalene in 1 L spectral grade 1,4-
dioxane. (Available from Aldrich Chemical Co., Inc.). Store
in dark bottles. Soln is stable 2 months.
(b) Tritium std soln. — Pipet 4 mL H 2 of known 3 H activity
and 16 mL scintillation soln into scintillation vial, tightly cover
vial with screw cap, and mix thoroly by shaking.
(c) Background soln. — Mix 4 mL distd H 2 (free of 3 H ac-
tivity to be measured in samples) with 16 mL scintillation soln
as in (b).
0. Preparation of Sample
Distil 20-30 mL sample to dryness. Mix 4 mL sample dis-
tillate with 16 mL scintillation soln as in 969.48C(b).
E. Determination
Prior to counting, dark-adapt and cool sample, background,
and std solns ca 3 hr in instrument freezer at >2° (to prevent
solidification of soln with time), or at ambient temp, if am-
bient temp. liq. scintillation spectrometer is used. Count solns
for total of 200,000 counts or 100 min, whichever is sooner.
F. Calculation
Counting efficiency, E = (S — B)/D
3 H, pCi (picoCuries)/mL - (C - B)i(E x 4 x 2.22)
where S = gross cpm (counts/min) of std, B — cpm back-
ground, D — dpm (disintegrations/min) of 3 H activity in std,
and C = gross cpm for sample.
Ref.: JAOAC52, 90(1969).
CAS- 10028- 17-8 (tritium)
973.66 Strontium-90 in Water
Beta Particle Counting Method
First Action 1973
Final Action 1974
A. Principle
Applicable to H 2 0, and to sewage and industrial waste if
org. matter is destroyed and interfering ions are eliminated.
Added carrier Sr along with radionuclides are sepd from other
radioactive elements and inactive sample solids by pptn as
Sr(N0 3 ) 2 from fuming HN0 3 . Sr is finally pptd as SrC0 3 , which
is dried, weighed, and set aside ca 2 weeks for ingrowth of
90 Y. Ppt is then dissolved and 90 Y is prepd for counting by (a)
extn by tributyl phosphate and evapg on planchet, or (b) addn
of Y carrier and pptg as oxalate.
Radioactive Ba and Ra which interfere are removed by addn
of Ba carrier. Ca interferes with Sr pptn, but is removed by
HN0 3 pptn and acetone treatment.
B. Apparatus
(a) Counting pans-
7 mm deep.
-Stainless steel, ca 50 mm diam. and
349
350
Radioactivity
AOAC Official Methods of Analysis (1990)
(b) Filtration assembly. — For mounting ppts for counting.
Consists of (/) 2- piece filtering app. for 2.4 cm filter such as
stainless steel filter holder (Interex Corp., 3 Strathmore Rd,
Natick, MA 01760, No. 12-103; ICN Pharmaceuticals, Inc.,
Life Sciences Group, No. 83012), Teflon filter holder, or equiv.
(2) Nylon (Zytel 101) disk with ring for mounting ppt.
(c) Film, Mylar. —To cover ppts during counting and stor-
age, ca 1 mil (0.025") thick. Available in rolls 1.5" (3.8 cm)
wide as manufacturer's No. 92A, E. I. DuPont Co., Inc.,
Electronics Dept, Barley Mill Plaza, Kirk Mill: Mylar Product
Information, Wilmington, DE 19805.
(d) Glass fiber filter paper. — No. 934-AH, 2.4 cm diam.,
available from Whatman, Inc.
(e) Centrifuge tubes.— 40 mL, heavy duty with short cone
bottom and pour-out lip.
(f) Beta particle counter, — Low background, shielded, an-
ticoincidence counter. Det. counter efficiency for 90 Y as ox-
alate and 89 Sr as carbonate for specific counter and geometry.
C. Reagents
(a) Dilute acetic acid. — 67V. Add 345 mL HO Ac to H 2
and dil. to 1 L.
(b) Ammonium acetate buffer. — pH 5.5. Dissolve 154 g
NH 4 OAc in 700 mL H 2 0, add 57 mL HO Ac, adjust pH to 5.5
with dropwise addn of either HOAc or 6N NH 4 OH, as nec-
essary, and dil. to 1 L.
(c) Dilute ammonium hydroxide. — 6N. Dil. 400 mL NH 4 OH
to 1 L with H 2 0.
(d) Barium carrier soln. — 10 mg Ba/mL. Dissolve 19.0 g
Ba(N0 3 ) 2 in H 2 and dil. to 1 L.
(e) Dilute hydrochloric acid. — 67V. Add 500 mL HC1 to H 2
and dil. to 1 L.
(f) Fuming nitric acid.—2\N. Sp gr 1.48, 90% HN0 3 .
(g) Dilute nitric acid.— (I) 14N.— Add 875 mL HNO, to
H 2 and dil. to 1 L. (2) 67V.— Add 384 mL HN0 3 to H 2 and
dil. to 1 L. (3) 0. IN.— Add 6.25 mL HN0 3 to H 2 and dil.
to 1 L.
(h) Oxalic acid soln. — Satd. Approx. 11 g H 2 C 2 4 .2H 2
in 100 mL H 2 0.
(i) Mixed rare earth carrier soln. — Dissolve 12.8 g
Ce(N0 3 ) 3 .6H 2 0, 1.4 g Zr0Cl 2 .8H 2 Q, and 25 g FeC] 3 .6H 2 in
600 mL H 2 contg 10 mL HCI, and dil. to 1 L. (Caution:
Ce(N0 3 ) 3 is toxic. Wear resistant rubber or plastic gloves.)
(j) Sodium carbonate soln. — 2/V. Dissolve 142 g
Na 2 C0 3 .H 2 in H 2 and dil. to 1 L.
(k) Sodium chr ornate soln. — 0.5M. Dissolve 117 g
Na 2 Cr0 4 .4H 2 in H 2 and dil. to 1 L.
(1) Sodium hydroxide soln. — 6A^. Dissolve 240 g NaOH in
H 2 and dil. to 1 L.
(m) Strontium carrier soln. — 10 mg Sr/mL. Dissolve 24. 16
g Sr(N0 3 ) 2 in H 2 and dil. to 1 L. Stdze by pipetting (in trip-
licate) 10 mL soln into 40 mL centrf. tubes and adding 15 mL
2N Na 2 C0 3 . Stir and heat in boiling H 2 bath 15 min. Filter
thru weighed, fine porosity, fritted glass, 15 mL crucible. Wash
with three 5 mL portions H 2 and three 5 mL portions absolute
alcohol or acetone, wipe crucible with absorbent tissue, and
dry to const wt at 110° (ca 20 min). Cool in desiccator and
weigh.
mg Sr/mL - mg SrC0 3 x 0.5935/10
(n) Tributy I phosphate (TBP), equilibrated. — Shake TBP
with equal vol. 14iV HN0 3 . Sep. and discard lower acid phase.
(o) Yttrium carrier soln. — 10 mg Y/mL. Dissolve 12.7 g
Y 2 3 in 30 mL HN0 3 by stirring and heating. Add addnl 20
mL HN0 3 and dil. to 1 L with H 2 0. 1 mL = 34 mg
Y^(C 2 4 ) 3 .9H?0. Det. exact equivalence as in 973.66D(f) or
(g).
D. Determination
{Caution: See safety notes on nitric acid and fuming acids.)
(a) Precipitation as carbonate. — To 1 L drinking H 2 (or
less, but contg >25 pCi 90 Sr) or filtered raw H 2 sample in
beaker, add 2.0 mL HN0 3 and mix. Add 2.0 mL each of Ba
and Sr carrier solns and mix well. (Ppt of BaS0 4 will not cause
difficulty.) Heat to bp, and add 20 mL 6N NaOH and 20 mL
2N Na 2 C0 3 . Stir, and let simmer ca 1 hr at 90-95°. Let cool
until ppt has settled (1-3 hr). Decant and discard supernate.
Transfer ppt to 40 mL centrf. tube, centrf., and discard su-
pernate.
(b) Purification as nitrate. — Cautiously add 4 mL HN0 3
dropwise to ppt. Heat to bp, stir, and cool under running H 2 0.
Add 20 mL fuming HN0 3 , cool 5-10 min in ice bath, stir,
and centrf. Discard supernate. Add 4 mL H 2 to residue, stir,
and heat to bp to dissolve Sr(N0 3 ) 2 . Centrf. while hot and
decant supernate into clean centrf. tube. Add 2 mL 6/V HN0 3
to residue, heat to bp, centrf. while hot, and combine super-
nate with aq. supernate. Discard insol. residue of BaS0 4 , Si0 2 ,
etc.
Cool combined supernates, add 20 mL fuming HN0 3 , cool
5-10 min in ice bath, stir, centrf., and discard supernate. Add
4 mL H 2 to ppt and dissolve by heating, cool, add 20 mL
fuming HN0 3 , cool 5-10 min in ice bath, stir, centrf., and
discard supernate. If >200 mg Ca is present in sample, repeat
H 2 soln and fuming HN0 3 pptn.
(c) Removal of rare earths. — After last HN0 3 pptn, invert
tube in beaker ca 10 min to drain off most of excess HN0 3 .
Add 20 mL acetone to ppt. Stir thoroly, cool, centrf., and
discard supernate. Dissolve ppt of Sr and Ba nitrates in 10 mL
H 2 and boil 30 sec to remove any remaining acetone.
Add 0.25 mL mixed rare earth carrier soln and ppt rare earth
hydroxides by making soln basic with 6N NH 4 OH. Digest in
boiling H 2 bath 10 min. Cool in ice bath, centrf., decant
supernate to clean tube, and discard ppt. Repeat addn of rare
earth carrier soln, pptn, and decantation. Note time as begin-
ning of 90 Y ingrowth period.
(d) Removal of barium. — Add 2 drops Me red indicator and
then 6/V HOAc, dropwise with stirring, until soln is red. Add
5 mL acetate buffer soln, heat to bp, and add 2 mL Na 2 Cr0 4
soln dropwise with stirring. Digest in boiling H 2 bath 5 min.
Cool in ice bath, centrf., decant supernate into clean tube, and
discard residue.
(e) Precipitation as strontium carbonate. — Add 2 mL 67V
NaOH to supernate; then add 5 mL 2N Na 2 C0 3 and heat to
bp. If pH is <9, add addnl NaOH soln. Cool in ice bath ca 5
min, centrf., and discard supernate. Add 15 mL H 2 to ppt,
stir, centrf., and discard wash H 2 0. Repeat washing and weigh
SrC0 3 as in (/) or (2):
(V) Slurry ppt with small vol. H 2 0, and transfer to weighed
stainless steel pan. Dry under 1R lamp, cool, and weigh. (2)
Transfer ppt to weighed paper or glass filter mounted in 2 piece
funnel. Let settle by gravity for uniform deposition; then apply
suction. Wash ppt with three 5 mL portions H 2 0, three 5 mL
portions alcohol, and three 5 mL portions ether or acetone.
Dry 15-30 min in 110-125° oven, cool, and weigh.
Store ppt >2 weeks to permit ingrowth of 90 Y. Sep. and
count 90 Y by (f) or (g).
(f) Separation by TBP extraction. — If SrC0 3 is weighed in
pan, place pan in small funnel in mouth of 60 mL separator
and carefully add 1 mL 6N HN0 3 dropwise. Tilt pan to empty,
and rinse with two 2 mL portions 6yV HN0 3 .
AOAC Official Methods of Analysis (1 990)
Strontium 351
If SrC0 3 is weighed on filter, dislodge bulk of ppt into small
funnel in mouth of 60 mL separator. Cautiously add 1 mL 6N
HN0 3 dropwise to dissolve remaining ppt. Rinse filter and funnel
with two 2 mL portions of 6/V HN0 3 .
Remove pan or filter and add 10 mL fuming HN0 3 to sep-
arator thru funnel. Remove funnel and add 1 mL Y carrier soln
to separator. Add 5.0 mL TBP, shake thoroly 3-5 min, let
sep., and transfer aq. layer to second 60 mL separator. Add
5.0 mL TBP to second separator, shake 5 min, let sep., and
transfer aq. layer to third 60 mL separator. Combine TBP exts
and wash with two 5 mL portions \4N HN0 3 . Record time as
beginning of ^Y decay. (Combine acid washings with aq. phase
in third separator if second ingrowth of 90 Y is desired.)
Back-ext 90 Y from combined org. phase with 10 mL 0.1 AT
HNO3 5 min. Either (7) repeat TBP extn as above, beginning
'Add 5.0 mL TBP,
and finally back-extg J Y into 10
mL QAN HN0 3 and continue as in (g), line 10 beginning
"Gradually . . ."; or (2) transfer aq. phase to 50 mL beaker
and evap. on hot plate to 5-10 mL. Transfer residual soln to
weighed stainless steel counting pan and evap. Rinse with two
2 mL portions 0. IN HN0 3 , add rinsings to counting pan, evap.
to dryness, and weigh. Count in internal proportional or end
window counter and calc. 90 Sr as in 973. 66E.
(g) Separation by yttrium oxalate precipitation. — Dissolve
SrC0 3 by cautiously adding 2 mL 6jV HN0 3 dropwise, and
transfer to 40 mL centrf. tube, rinsing with 0AN HN0 3 . Add
1 mL Y carrier soln, 2 drops Me red, and NH 4 OH dropwise
to Me red end point. Add addnl 5 mL NH 4 OH and record time
as end of 90 Y ingrowth and beginning of decay. Centrf. and
decant supernate into beaker. (Save supernate and washings
for second ingrowth, if desired.) Wash ppt with two 20 mL
portions hot H 2 0. Add 5-10 drops 6N HN0 3 , stir to dissolve
ppt, add 25 mL H 2 0, and heat in H 2 bath at 90°. Gradually
add 15-20 drops satd oxalic acid soln with stirring, and adjust
pH to 1.5-2.0 (pH meter or indicator paper) with dropwise
addn of NH 4 OH. Digest ppt 5 min; then cool in ice bath with
occasional stirring.
Transfer ppt to weighed glass fiber filter in 2-piece funnel.
Let ppt settle by gravity for uniform deposition and then apply
suction. Wash ppt with 10-15 mL H 2 6, three 5 mL portions
alcohol, and then three 5 mL portions ether. Air dry ppt 2 min
with suction, weigh, mount on nylon disk and ring with Mylar
cover, count, and calc. 90 Sr as in 973. 66E.
£. Calculations
(a) Strontium-90 calculation. —
90 Sr, pCi/L - net cpm/(abcdfg x 2.22)
where a = counting efficiency for 90 Y; b = chem. yield (frac-
tion) of extd or pptd 90 Y; c = mg final Y oxalate ppt/mg Y
oxalate in 1 mL carrier; d = chem. yield (fraction) of Sr detd
as in 973.66D(e) (20 mg Sr equiv. to 33.6 mg SrC0 3 ) or by
flame photometry; f ~ vol., L, original sample; g = 90 Y decay
factor = e~ xt ; e = base of natural logarithms; X = 0.693/T, /2 ;
T]/2 = 64.2 hr for 90 Y; and t = time, hr, between sepn and
counting.
(b) Counting efficiency. — Prep, curve from various wts Y
oxalate ppt spiked with 90 Sr/ 90 Y, pptd as in 973.66D(g).
(c) Correction for carrier recovery. — If sample contains more
than trace stable Sr, it will act as carrier and will result in
>100% yield. In such case det. Sr by flame photometry.
Ref.: JAOAC 56, 208(1973).
CAS-37380-96-4 (strontium-90)
974.37 Strontium-89 and Strontium-90
in Milk
Ion Exchange Method
First Action 1974
Final Action 1976
A. Principle
Fresh milk samples are preserved with HCHO and stored to
obtain 90 Y ingrowth. After storage, Y, Sr, and Ba carriers and
citrate soln are added. Citrate forms Y complex which is ad-
sorbed on anion exchange resin. Y is desorbed and sepd from
radionuclides by tributyl phosphate extn. Y is re-extd into dil.
HNO3 and pptd as oxalate, which is weighed and counted for
90 Y activity to calc. 90 Sr.
Radio-Sr is desorbed along with Ca and radio-Ba; Ca, radio-
Ba, and rare earth radionuclides are sepd by repetitive pptns;
Sr is pptd as SrC0 3 and counted. Total radio-Sr minus 90 Sr by
90 Y measurement yields value for 89 Sr.
Milk contg known increments of 89 Sr and 90 Sr detd in trip-
licate by 11 laboratories showed following results (av. of trip-
licates):
Amt
Std Dev.
Bias
Present, pCi/L
%
pCi/L
%
pCi/L
89 Sr 29.0
197.0
90 Sr 32.4
151.2
10.0
3.4
0.9
2.8
2.9
7.2
0.3
4.2
+ 7.0
+ 1.5
+ 0.3
-0.9
+2.0
+3.0
+ 0.1
-1.3
B. Operating Notes
Radio-Ba and La radionuclides will interfere without puri-
fication. Purification from Ca is important for recovery tests
but need not be as thoro if Sr recovery is detd by 85 Sr tracer
or flame photometry. Thoroly desorb columns before re-use
and test periodically to assure complete desorption.
C. Apparatus
See 973.66B(b)-(f), plus following:
Ion exchange system. — Consists of 1 L graduated separator,
250 mL separator with fritted glass disk as cation exchange
column, and 30 mL separator with fritted glass disk as anion
exchange column (Kontes Glass Co., No. K-427530).
D. Reagents
See 973.66C(c), (e)-(g), plus following:
(a) Ammonium acetate buffer. — -pH 5.0. Dissolve 153 g
NH 4 OAc in 900 mL H 2 0. Adjust pH to 5.0 with HOAc, using
pH meter, and dil. to 1 L.
(b) Anion exchange resin. — Dowex 1-X8 (CI form), anal,
grade, 50-100 mesh size, available from Bio-Rad Laborato-
ries.
(c) Barium carrier soln. — 20 mg Ba/mL. Dissolve 38. 1 g
Ba(N0 3 ) 2 in H 2 0, add 1 mL HN0 3) and dil. to 1 L.
(d) Cation exchange resin. — Dowex 50W-X8 (Na form),
anal, grade, 50-100 mesh size, available from Bio-Rad Lab-
oratories. Convert com. available H form into Na form by
passing 1.5 L 4N NaCl thru 170 mL resin placed in column
and rinsing with ca 500 mL H 2 until wash H 2 is Cl-free
when tested with 1% AgN0 3 .
(e) Citrate soln. — 2M. Dissolve 384 g anhyd. citric acid
(420 g monohydrate) in H 2 0, adjust to pH 6.5 with dil. NaOH
soln, and dil. to 1 L.
(f) Oxalic acid soln.— IN. Dissolve 126 g H 2 C 2 4 H 2 in
warm H 2 0, cool, and dil. to 1 L.
352
Radioactivity
AOAC Official Methods of Analysis (1990)
(g) Silver nitrate soln. — 1%. Dissolve 1 g AgN0 3 in H 2
and dil. to 100 mL. Store in brown bottle.
(h) Sodium chloride soln. — AN, Dissolve 236 g NaCl in H 2
and dil. to 1 L.
(i) Sodium carbonate soln. — 3N. Dissolve 159 g Na 2 C0 3
in H 2 and dil. to 1 L.
(j) Sodium chr ornate soln. — IN. Dissolve 81 g Na 2 Cr0 4 in
H 2 and dil. to 1 L.
(k) Strontium carrier soln. — 20 mg Sr/mL. Dissolve 48.3
g Sr(N0 3 ) 2 in H 2 0, add 1 mL HN0 3 , and dil. to 1 L. Stdze
by pipetting 1 mL portions into six sep. 40 mL centrf. tubes
contg 15 mL H 2 0. Adjust pH (indicator paper or meter) to
8.5-9.0 with 6jV NH 4 OH. Add, with stirring, 3-5 mL 3N
Na 2 C0 3 and digest 5 min in near boiling H 2 bath. Cool to
room temp, and process ppt as in 974.37G(d) or (e).
(1) Tributyl phosphate (TBP), pre- equilibrated. — Add 150
mL H 2 and 30 mL 3N Na 2 C0 3 to 300 mL TBP in 1 L sep-
arator. Shake 2-3 min and let sep. Discard lower aq. phase.
Add 150 mL H 2 to separator, shake 2-3 min, and let sep.
Discard lower aq. phase. Add 150 mL IAN HN0 3 and shake
5 min. Let sep. and discard lower aq. phase. Repeat IAN HN0 3
treatment twice.
(m) Yttrium carrier soln. — 10 mg Y/mL. Dissolve 12.7 g
Y 2 3 in 50 mL HN0 3 by heating (avoid boiling). Dil. to 900
mL with H 2 0, adjust pH to 2.0 with NH 4 OH, and dil. to 1 L
with H 2 0. Stdze by pipetting 1 mL portions into each of six
40 mL centrf. tubes contg 15 mL H 2 0. Add 5 mL 2N oxalic
acid and adjust pH to 1.5 with 6N NH 4 OH, using pH meter.
Digest in hot H 2 bath 10 min, and cool to below room temp.
Centrf. and discard supernate. Process ppt as in 974.37G(d)
or (e).
Use Y 2 3 of 99.999% purity (Morton-Thiokol Inc. , 152 An-
dover St, Danvers, MA 01923). Material of lower purity may
require purification because of radioactive contaminants.
E. Preparation of Sample
Preserve freshly drawn sample with ca 3 mL HCHO soln
for each L milk and refrigerate for known period of time up
to 2 weeks to allow ^Y ingrowth. Thoroly mix preserved, stored
sample. If homogeneous, transfer 1 L to separator, 974. 37C,
If nonhomogeneous, before transfer, filter thru loose bed of
Pyrex glass wool to prevent clogging of resin columns.
F. Removal of Radioelements by Ion Exchange
Combine 1.00 mL each of Y, Sr, and Ba carriers with 10
mL citrate soln, (e) in small beaker or vial. Using distd H 2 0,
transfer quant, to 1 L sample in separator, and mix well.
Add 170 mL Dowex 50W-X8, (d), to 250 mL separator
filled with H 2 0. Add 15 mL Dowex 1-X8, (b), to 30 mL sep-
arator filled with H 2 0. Connect all separators together in order
sample (top), anion column, cation column (bottom), and place
beaker to collect effluent. Open stopcocks of sample, anion,
and cation separators, in that order, and note time. Control
effluent rate at 10 mL/min with anion column stopcock. Check
and adjust effluent flow periodically.
Stop flow when milk level reaches top of each resin bed and
note time. Record as mean time the av. period of effluent flow, i
This time is taken as beginning of 90 Y decay. Do not permit 1
unnecessary delay during elution. Discard eluate.
Connect separator contg 300 mL warm H 2 0, continue elu-
tion at 10 mL/min as above, and discard. Sep. columns.
G. Yttrium-90 Separation, Purification, and Determination
Connect separator contg 100 mL 2N HC1 to top of anion
separator. Open upper stopcock and then lower stopcock, and
control effluent flow at 2 mL/min. Collect 15 mL eluate. Close
both stopcocks and remove top separator. Stir resin thoroly
with glass stirring rod, and rinse into resin column with small
vol. 2N HC1. Reconnect separator, and continue 27V HCJ elu-
tion, collecting total of 70 mL Y eluate. Retain eluate.
Adjust flow rate to 10 mL/min for remaining 30 mL acid
to recharge separator. Discard this eluate. Wash resin with >100
mL H 2 until Cl-free by AgN0 3 test. Separator is ready for
next detn.
Add 5 mL 2/V oxalic acid to retained eluate and adjust pH
to 1 .5 with 67V NH 4 OH, using pH meter. Stir, heat to near bp
in H 2 bath, cool in ice bath, centrf., decant, and discard su-
pernate. Proceed as in (a) or (b), depending on whether 140 Ba-
La is absent or present from gamma analysis of sample.
(a) If fresh fission products are absent. — Dissolve ppt in 1
mL 6N HC1, add 15 mL H 2 0, and filter thru Whatman No.
541 paper into 40 mL centrf. tube. Wash paper, collecting
washings in tube, discard paper, and continue as in (c).
(b) If fresh fission products are present. — Dissolve ppt in
10 mL HN0 3 ; transfer soln to 60 mL separator, washing centrf.
tube with addnl 10 mL HN0 3 . Add 10 mL equilibrated TBP,
(1), shake 2-3 min, let sep., and drain and discard lower acid
phase. Add 15 mL 147V HN0 3 to separator, shake 2-3 min,
let sep., and drain and discard lower acid phase. Repeat 147V
HN0 3 treatment to remove light lanthanide elements, partic-
ularly J40 La. Add 15 mL H 2 to separator and shake 2-3 min.
Let sep., and drain aq. phase contg most of Y into 40 mL
centrf. tube. Repeat wash, using 15 mL 0.1/V HN0 3 , adding
it to centrf. tube,
(c) Preparation of yttrium oxalate. — Add 5 mL 27V oxalic
acid to purified Y soln from (a) or (b), and adjust to pH 1.5
with NH 4 OH, using pH meter. Digest soln in hot H 2 bath 10
min with occasional mixing. Cool in ice bath, centrf., and dis-
card supernate. Sep. and count 90 Y oxalate as in (d) or (e),
stdze carrier by the same technic used for sample, and calc.
90 Sr activity from 9D Y count as in 974.371(a).
(d) Filtration method. — Place 2.8 cm glass fiber filter on
stainless steel planchet and weigh together. Transfer tared fil-
ter to filter holder, 973.66B(b)(7), and assemble.
With H 2 spray, quant, transfer Y oxalate ppt to filter fun-
nel, using min. of suction so that ppt is distributed uniformly
over filter area. Increase suction as necessary after most of ppt
is on filter. Wash ppt with three 10 mL portions warm H 2 0,
three 5 mL portions alcohol, and three 5 mL portions ether.
Continue suction ca 2-3 min. Carefully remove filter, place
on original planchet, and let stand at room temp. 10-15 min.
Weigh and calc. yield Y oxalate (likely Y 2 (C 2 4 ) 3 .9H 2 0) by
dividing this wt by wt obtained on stdzn of carrier, 974.37D(m).
Remove filter from planchet, place on top of nylon disk,
cover with piece of Mylar film, place nylon ring over Mylar
film, and press ring onto nylon disk. Cut off excess film. Count
90 Y activity, without undue delay, in low background anti-
coincidence beta counter. Repeat counting after 3 days to con-
firm purity of ^Y by its rate of decay. Record dates and time
of counting.
(e) Direct dispersion method. — Wash ppt twice with 20 mL
portions warm H 2 0, cool to below room temp., centrf., and
discard supernate. Quant, transfer ppt to tared stainless steel
dish. Uniformly disperse ppt over dish bottom and dry under
IR lamp to const wt. Count in p particle counter.
H. Strontium-89 Separation, Purification, and Determination
(Caution: See safety notes on nitric acid and fuming acids.)
Connect 1 L separator contg 1 L AN NaCl to cation sepa-
rator. Open upper stopcock and then lower stopcock, and con-
trol effluent flow at 10 mL/min. Collect ca 1 L eluate in 2 L
beaker, but leave resin covered with 2-3 mL soln. Retain eluate.
Wash cation separator with 500 mL H 2 from top separator
at rate of 10 mL/min. Discard wash H 2 0. If resin becomes
AOAC Official Methods of Analysis (1 990)
Strontium
353
clogged with milk solids, back- wash separator or transfer resin
to beaker, agitate with H 2 0, and decant.
Dil. retained eluate to 1.5 L with H 2 0, heat to 85-90° on
hot plate, and add 100 mL 3N Na 2 C0 3 with gentle stirring.
Remove from heat and cool to room temp. Decant bulk of
clear supernate. Quant, transfer ppt to 250 mL centrf. bottle
with H 2 and centrf.; discard supernate. Add 50 mL H 2 and
disperse ppt. Centrf., discard supernate, and repeat. Dry ppt
4 hr in oven at 1 10°.
Dissolve ppt with vigorous stirring by adding ca 4 mL 67V
HN0 3 in small amts (mag. stirrer is helpful). Filter thru What-
man No. 541 paper into 40 mL graduated centrf. tube. Rinse
bottle with 4 mL 67V HN0 3 and pour washing thru paper. Dis-
card paper. Add 20 mL 21N HN0 3 to filtrate. Stir and cool
in ice bath; centrf. and discard supernate. (Sr(N0 3 ) 2 pptn is
critical in obtaining good recovery of Sr adequately sepd from
Ca.) Recoveries from single pptn are as follows:
[HNO3]
Sr Rec, %
Ca Rec.,%
14/V
16W
18/V
81 ±4
98±1.4
100±1.7
2.6±0.9
11 ±2
51 ±3
Dissolve ppt in 5 mL H 2 and adjust to p'H 5.0 with NH 4 OH,
using pH meter. Add 5 mL NH 4 OAc buffer. Heat in H. 2 bath,
add 1 mL \N Na 2 Cr0 4 , and mix well. Digest in bath 5 min.
Centrf. and decant supernate into small beaker. Evap. to ca 2
mL, add 2 mL 6N HNO3, and transfer to 40 mL centrf. tube,
using one 3 mL H 2 rinse. Add 20 mL 217V HN0 3 , stir, cool
in ice bath, centrf., and discard supernate. Add 3 mL H 2 and
5 mL 6N HNO3 to dissolve ppt. Add 20 mL 21N HNO3, stir,
cool in ice bath, centrf., and discard supernate. Record time
as beginning of 90 Y ingrowth.
Dissolve ppt in few mL H 2 and adjust pH to 8.5-9.0 with
6N NH4OH. Add 3-5 mL 3N Na 2 C0 3 to ppt SrC0 3 . Centrf.,
and discard supernate. Disperse ppt in 10 mL H 2 0, centrf.,
and discard supernate. Sep. and count SrC0 3 as in (a) or (b):
(a) Filtration method. — Proceed as in 974.37G(d), but wash
ppt with three 5-10 mL portions H 2 0, transfer to original
planchet, and dry 30 min in oven at 110°. Cool in desiccator
and weigh. Count as in 974.37G(d), record time of counting,
and calc. 89 Sr as in 974.371(b).
(b) Direct dispersion method. — Wash ppt twice with ca 10
mL portions H 2 0, dispersing ppt, centrf., and decant and dis-
card supernate. Quant, transfer ppt to tared stainless steel dish.
Uniformly disperse ppt over dish bottom, dry 30 min in 110°
oven. Cool in desiccator and weigh. Count in p particle counter.
Record time of counting and calc. 89 Sr as in 974.371(b)
/. Calculations
(a) For strontium-90 activity. —
90 Sr activity, pCi/L = (cpm ± o)/R s R Y E Y D Y I Y V
where cpm = net beta count rate of 90 Y
f t s t b
— sample count rate
= background count rate
= sample counting time
background counting time
= fraction Sr carrier recovered
R Y = fraction Y carrier recovered
E Y = counter efficiency for 90 '
pCi
V
Y as Y oxalate, cpm/
D Y = decay correction factor (= e ', defined in
973.66E(a)) for 90 Y, where t is time of sepg 90 Y
from ^Sr to time of counting, 974.37G(d) or
(e)
I Y — ingrowth correction factor (— 1 — e M ) for de-
gree of equilibrium attained during 90 Y in-
growth period, where t is time from start of in-
growth period to time of sepg 90 Y from 90 Sr
V = sample vol., L
(b) For strontium-89 activity. —
9 Sr activity, pCi/L
1
N s + cr
E S D S L R S V
- C s (a s E' s + E Y I Y )
where E s = counter efficiency for 89 Sr as SrC0 3 , cpm/pCi
D s = decay correction factor (= e~ xt ) for 89 Sr, where
t is time from sample collection to time of
counting
V t, t b
R s = fraction Sr carrier recovered
N s = net counts /min of observed radio-Sr
V = sample vol., L
C s = ^Sr activity, pCi/L
a s - absorption factor for ^Sr as S1CO3 obtained from
self-absorption calibration curve. (Self-absorp-
tion curves for 89 Sr and 90 Sr derived by pptg
series of carrier SrC0 3 concns over expected re-
covery range in presence of const amt of 89 Sr
and ^Y-free ^Sr, resp. Ordinate is ratio of count
rate for each thickness to count rate at sample
thickness and abcissa is sample wt for given type
of sample mount.)
E' s = counter efficiency for 90 Sr as SrC0 3 , cpm/pCi
E Y = counter efficiency for 90 Y as Y oxalate, cpm/
pCi
1 Y = correction factor (= 1 — e l ) for degree of
equilibrium attained during 90 Y ingrowth pe-
riod, where t is time ^Y was sepd from SrC0 3
to time of counting, 974.37H(a) or (b)
Refs.: JAOAC 56, 213(1973); 57, 37(1974).
CAS-3 1083-24-6 (strontium-89)
CAS-37380-96-4 (strontium-90)
CAS- 10098-9 1-6 (yttrium-90)
973.67 lodine-131, Barium-140,
and Cesium-137 in Milk and Other Foods
Gamma-Ray Spectroscopic Method
( 137 Cs in milk: first action 1973, final action 1974. ,37 Cs in
other foods: first action 1986. 140 Ba in milk: first action 1982,
final action 1983. ,3i l in milk: first action 1982, final action
1983. 131 I in other foods: first action 1986.)
A. Principle
Applicable to
%
140 Ba, and 137 Cs in fluid milk preserved
with HCHO, and I and Cs in foods. Because of the nature of
gamma-emitting radionuclides, attenuation of gamma-rays in
food slurries or mixt. would be similar to that of milk or H 2 0.
Unlike in milk samples, other radionuclides might be present
in foods. Therefore, before performing calcn, gamma-ray en-
ergy spectrum should be inspected for any radionuclides be-
sides K, 131 I, l37 Cs, and 140 Ba. Since cessation of above-ground
354
Radioactivity
AOAC Official Methods of Analysis (1990)
weapons testing in 1960s, no other gamma-ray emitters have
been regularly observed or detected in food. (Should any be
detected, matrix technic should be expanded using std source
for suspected radionuclide to det. matrix coefficient.)
Known vol . is placed in counting vessel positioned over and
around right cylinder scintillation crystal detector Nal (Tl) of
multichannel gamma spectrometer. Gamma radiation is counted
for given time. Accumulated pulses from selected photon en-
ergy range are sepd from other gamma-emitting radionuclides
and background radiation by simultaneous equations. ^K is
always present as natural contaminant and may contribute counts
in 1 or more of photopeak ranges. Mutual interferences among
these 4 photopeaks are eliminated by applying matrix technic
to sep. activities of the 4 nuclides. Measurement of one std
source of each nuclide provides the matrix coefficients.
In special cases, newly formed fission products may be pres-
ent, e.g., 133 I and n5 I, which may interfere either thru direct
overlapping of photopeaks or by contributing Compton-con-
tinuum counts. Such interference may be minimized by wait-
ing for decay of short-lived radionuclides, by addnl counting
following decay, or by chem. sepn.
Milk contg known increments of 131 I, 137 Cs, and 140 Ba, detd
in triplicate by 25 laboratories, and 2nd milk contg known in-
crement of 131 I, detd in triplicate by 40 laboratories, showed
following results (av. of triplicates);
a = 47 mm
95mm
-111 mm •-
254 mm
FIG. 973.67— Cross-section of Marinelli beaker
Amt Nuclide
Std Dev
(CV, %)
Bias ± 95%
Uncertainty
Present,
Within
Between
pCi/L
Labs
Labs
pCi/L
%
131 1
98
6.1 (6.2)
8.2 (8.3)
+ 0.9 ± 3.7
+ 0.9 ± 3.8
633
29.0 (4.6)
30.1 (4.8)
+ 2.3 ± 14.3
+ 0.4 ± 2.3
140 Ba
72
6.5 (9.1)
11.2 (15.6)
+ 4.0 ± 4.8
+ 5.5 ± 6.7
515
19.5 (3.8)
35.8 (7.0)
+ 7.9 ± 15.8
+ 1.5 ± 3.1
137 Cs
52
4.7 (9.1)
4.1 (8.0)
+ 1.3 ± 2.0
+ 2.4 ± 3.8
305
11.4 (3.7)
13.5 (4.4)
-9.8 ± 6.1
-3.2 ± 2.0
131 1
82
5.6 (6.8)
6.8 (8.3)
-0.4 ± 2.4
-0.5 ± 2.9
B. Apparatus
(a) Alignment sources. — Gamma ray energies, at least 1 near
,37 Cs spectrum, with well known energies and abundance of
gamma rays in photopeaks, for alignment. Solid sources, ca
0.1 jxCi, are preferred over liq. sources. 207 Bi is satisfactory
single source with several photopeaks; 137 Cs and 60 Co are good
pair.
(b) Counter, — Low level gamma spectrometer consisting
of shielded Tl-activated Nal scintillation detector, 4x4 in.,
coupled to multichannel pulse-ht analyzer and readout system.
(c) Counting vessel {Marinelli beaker). — Use 3.5 L beaker,
Fig. 973.67, for 4 x 4 in. detector. Beaker and lid available
from plastic laboratory-ware suppliers such as Bel-Art Prod-
ucts, Industrial Rd, Pequannock, NJ 07440-1992, No. H26852
for beaker and No. H26587 for lid.
C. Reagents
(Caution: See safety notes on radioactive chemicals.)
(a) Carrier solns. — lOmg/mL. Prep, solns of CsCI (1.267
g/100 mL), Nal (1.181 g/100 mL), and BaCJ 2 .2H 2 (1.779
g/100 mL). Store in polyethylene or glass bottles.
(b) Stock std solns. — 10 000 pCi/mL. Dil. calibrated solns
of 131 I, 140 Ba, and 137 Cs to approx. indicated strength.
(c) Potassium-40 stock std soln. — 1.89 dpm (disintegra-
tions/mm) 40 K/mg K. Dissolve 240 g KC1 (equiv. to 126 g
K) in 3 L H 2 in Marinelli beaker and dil. to 3.5 L.
(d) Calibrating solns.— For l37 Cs and 140 Ba, add 3-5 mL
carrier soln, (a), to 3 L H 2 in Marinelli beaker, mix, add
convenient amt of stock std soln, (b), sufficient to reduce
counting error to ca 1% when counted within 10-100 min,
mix, adjust pH to 3.5-4.5, and dil. to 3.5 L. Prep. 131 I soln
similarly, but adjust pH to 8.5.
D. Preparation of Sample
No special preparation is needed for milk samples. For other
foods, do not include inedible material such as bone, apple
cores, nut shells, and egg shells as part of sample. Homoge-
nize sample in blender or mech. homogenizer. Dietary sam-
ples prepd for consumption do not require blending, but must
be sufficiently mixed to ensure representative sample.
E. Determination
Using alignment sources centered on detector, adjust spec-
trometer to cover range at least between and 2 meV, in in-
tervals (channels) of 10 or 20 keV. Adjust voltage or gain con-
trol so that the 2 gamma photopeaks of std fall in their
appropriate channels. Check and adjust alignment daily.
Place Marinelli beaker contg 3.5 L calibrating soln, (d), over
detector, and count std for time (10-100 min) sufficient to
reduce counting error to ca 1%. Repeat with each calibrating
soln and with H 2 0. Recalibrate spectrometer yearly or more
frequently if gamma ray resolution changes.
Transfer 3.5 L well mixed sample at room temp, into
Marinelli beaker, place over detector, and count 100 min or
time sufficient to give desired counting statistics.
F. Calculations
(a) Counter efficiency. — Total individual counts observed
in channels of photopeak range for each calibrating soln. Sub-
tract total background count for same photopeak range. Divide
net count by counting time in min and amt of radionuclide in
pCi, and record cpm/pCi for each,
(b) Interference coefficients. — When counting std soln of
each radionuclide, m I, 137 Cs, 140 Ba, 40 K, ratio of net counting
rate in energy range of each of the other radionuclides to net
counting rate in its own photon energy range gives its frac-
tional interfering coefficient for each of the other energy ranges,
e.g., 131 I ratio of net counting rate in 137 Cs energy range to net
AOAC Official Methods of Analysis (1990)
Contamination
355
counting rate in l3l I photon energy range gives its fractional
interfering coefficient for 137 Cs energy range.
Designate counting rate for 131 I, 140 Ba, ]37 Cs, and 40 K with
symbols 1, B, C, and K, resp. Designate net counting rates
(observed — background) in their resp. photon energy ranges
as Ni, N b , N c , and N k , resp. Then, f, fractional coefficients or
contributions of nuclide in particular range, is designated by
2 lower case subscripts; first one indicates nuclide contributing
counts to energy range (column) and second, nuclide photon
energy range (row). The following 4 equations:
N; = 1 + f bi B + f ci C + f kl K
N b = f ib I + B + f cb C + f kb K
N c = f ic I + f bc B + C + f kc K
N k = f ik I + f bk B + f ck C + K
(1)
(2)
(3)
(4)
can be solved simultaneously by matrix algebra, using inver-
sions to provide numerical consts W, X, Y, and Z in equations
5, 6, 7, and 8. These consts are used to solve for concn of
each of these 4 nuclides in sample. Net counting rate for each
nuclide is:
131 I - I - W,Ni + W 2 N b + W 3 N C + W 4 N k (5)
l40 Ba = B = X^i + X 2 N b + X 3 N C + X 4 N k (6)
I37 Cs - C - Y,Ns + Y 2 N b + Y 3 N C + Y 4 N k (7)
40 K = K = Z|N'i + Z 2 N b + Z 3 N C + Z 4 N k (8)
Calibration to derive values for consts in equations 5, 6, 7,
and 8 is applicable as long as instrument alignment and mode
of operation remain const and gamma-emitting nuclides are
limited to the 4 elements in matrix. Long- hand inversion of 4
x 4 matrix is tedious and subject to mistakes. Use of computer
is recommended to provide numerical consts for equations 5—
8. Thereafter, desk calcns can det. concns of 131 I, ,40 Ba, 137 Cs,
and 40 K in samples in absence of computer by summing counts
in each photopeak, subtracting background, and applying
equations 5-8.
(c) Iodine-131 , barium-140, cesium- 1 37 y potassium-40 ac-
tivities. — From spectral gamma counts of sample, substitute
net value from equations 5 thru 8 and convert net counts /min
for each nuclide to pCi/L sample at time of counting:
131 I(pCi/L) = (net cpm)i /(E; x V)
140 Ba(pCi/L) = (netcpm) b /(E b X V)
l37 Cs(pCi/L) = (net cpm) c /(E c x V)
40 K(pCi/L) = (net cpm) k /(E k x V),
where E { , E b , E c , E k = counting efficiency /pCi from std solns
for ,31 I, 140 Ba, 137 Cs, 40 K, resp., and V = sample vol., L.
Refs.: JAOAC 56, 204(1973); 65, 1039(1982); 69, 270,
301(1986); 72, 667(1989).
CAS-10045-97-3 (cesium-137)
CAS- 10043-66-0 (iodine-131)
955.50 Radioactive Contamination
Emergency Level Measures
Procedure
A. Apparatus
(a) Portable count-rate meter. — Consists of: (/) Self-
quenching glass Geiger-Muller tube, side wall <32 mg/cm 2 ,
mounted in slide opening metal shield; threshold ca 800 v,
operated at ca midpoint of voltage plateau, slope of which is
<10%, connected with coaxial cable to (2) Suitable power
supply and electronic amplifier unit with meter calibrated in
milliroentgens (mr)/hr, connected thru sensitivity switch pro-
viding 3 ranges of scale reading, e.g., 0-20, 0-2, and 0-0.2
mr/hr; linear response within each range.
(b) Comparison std. — Induces meter response identical to
that from surface of H 2 contaminated with fission products
decaying at rate of 2 X 10 5 dpm/mL (emergency tolerance
level for H 2 to be consumed for <10 day period). Construct
such std as follows: Uniformly suspend suitable amt of "60-
mesh" U0 2 (OAc) 2 .2H 2 (ca 3 g, adjusted by trial) {Caution:
See safety notes on uranyl acetate and toxic dusts.) in 5 g liq.
casting plastic, level, and solidify in shallow container, such
as lid of ointment tin, ca 80 mm diam. and side wall 15 mm
deeper than layer of plastic. Base of ointment tin, fitted with
indented ring 15 mm below its edge, serves as container for
liqs and finely divided solids to be tested, and to protect com-
parison std when not in use. Supplementary std of '/2 this ac-
tivity may be prepd similarly for monitoring supplies to be
consumed over 30 day period.
B. Determination
With selectivity switch set for highest range (e.g., 0-20 mr/
hr), and with shield open, place G-M tube diametrically across
std in contact with edge of container at 2 points. Adjust meter
pointer to convenient value ca midway of scale with calibra-
tion screw and record reading as av. of fluctuations over 1-2
min. Duplicate reading should check within ±5%. Avoid ex-
traneous radiation, such as that from luminous dial watch.
Fill sample container with liq. or finely divided solid to level
of indented ring and obtain duplicate readings. Sample read-
ings within ± 100% of std reading are of practical quant, sig-
nificance for monitoring under emergency conditions.
Ref.: JAOAC 38, 678(1955).
14. Veterinary Analytical Toxicology
P. Frank Ross, Associate Chapter Editor
National Veterinary Service Laboratory
Methods have been included in this chapter because they
provide data which may be used to support a clinical diag-
nosis. Other methods in this book may also be used for this
purpose provided the method has been adopted for the analyte/
substrate combination involved.
Arsenic in Feeds
See 957.22.
986.30 Arsenic in Liver Tissue
Spectrophotometric Method
First Action 1986
Final Action 1989
A. Principle
Liver tissue is dry-ashed overnight at 500°, ash is dissolved,
and portion is reacted with Zn metal to evolve arsine gas. Ar-
sine is trapped and As is detd spectrophtric .
B. Apparatus and Reagents
(a) Hydrochloric acid. — 3N.
(b) Cupric sulfate. — Anhyd., powd (J.T. Baker, Inc., No.
1850, or equiv.).
(c) Magnesium oxide-magnesium nitrate slurry. — Suspend
7.5 g MgO and 10.5 g Mg(N0 3 ) 2 .6H 2 in enough H 2 to
make 100 mL. Agitate vigorously before adding to sample.
(d) Stannous chloride soln, 20% (w/v). — Dissolve 20 g As-
free SnCl 2 .2H 2 in HC1 and dil. to 100 mL with HC1.
(e) Silver diethyldithiocarbamate (AgDDC) soln. — Dis-
solve 0.50 g AgDDC salt in pyridine and dil. to 100 mL with
pyridine. Mix and store in amber bottle. Reagent is stable sev-
eral months at room temp. (Fisher Scientific Co., Cat. No. S-
666, or equiv.).
(f) Arsenic std so Ins, — (I ) Stock soln. — 500 (xg/mL. Ac-
curately weigh 0.660 mg NIST Ref. Std As 2 3 , or equiv.,
dissolve in 25 mL 2N NaOH, and dil. to 1 L with H 2 0. (2)
Intermediate soln. — 10 jxg/mL. Transfer 2 mL stock soln to
100 mL vol. flask, and dil. to vol. with H 2 0. (3) Working
soln. — 2 fjLg/mL. Transfer 10 mL intermediate soln to 50 mL
vol. flask and dil. to vol. with H 2 0.
(g) External control.— MST Std Ref. Material (SRM) 1566
Oyster Tissue (13.4 ± 1.9 mg As/kg or equiv.
(h) Potassium iodide soln, 15% (w/v). — Dissolve 15 g KI
in H 2 and dil. to 100 mL.
(i) Zinc— Shot contg <0. 00001% As (Fisher Scientific Co.,
No. Z-12).
(j) Distillation apparatus. — See 973.78A(e). Use 125 mL
erlenmeyer instead of 250 mL. Use narrow test tube as re-
ceiver and submerge delivery tube in AgDDC soln.
C. Preparation of Standard Curve
Transfer 0.5, 1.0, 3.0, 6.0, and 10.0 mL aliquots of work-
ing soln corresponding to 1, 2, 6, 12, and 20 |xg As to sep.
125 mL erlenmeyers. Dil. to 50 mL with 3N HCL Carry these
solns thru distn procedure. Plot A at 540 nm on ordinate vs
|ig As on abscissa. Det. best fitting straight line, using all 5
points, by method of least squares.
D. Preparation of Sample
Blend tissue in high-speed blender until completely homo-
geneous. Accurately weigh 2.00 g tissue into 30 mL Coors
crucible. Analyze one external control with each set of 10 sam-
ples or fraction thereof. Add 5mL well mixed MgO/
Mg(N0 3 ) 2 .H 2 slurry and mix thoroly with stirring rod. Prep,
blank by adding 5 mL well mixed slurry to sep. crucible and
carrying it thru subsequent steps in procedure. Dry samples,
controls, and blank to apparent dryness on hot plate or in drying
oven at <100°. Cover each crucible with watch glass and place
in cold muffle furnace. Set furnace temp, at 250° for 3 h; then
gradually increase temp, to 500° and leave overnight.
Cool crucibles to room temp., moisten residue with 5 mL
H 2 0, and transfer quant, to 50 mL vol. flask with 3N HCL
Dil. to vol. with 3N HO and mix well. Transfer 25 mL aliquot
to 125 mL erlenmeyer and dil. to 50 mL with 3N HCL
E. Distillation
Add 2 mL 15% KI soln and swirl. Add .1 mL SnCl 2 soln
and swirl. Cool flasks in freezer or ice bath 45 min or until
samples reach 4°. Pipet 6 mL AgDDC soln into narrow re-
ceiver test tube, one for each std, external control, sample,
and blank. Have all parts of distn app. ready for immediate
assembly. Quickly add 10 g Zn shot and pinch of Cu 2 S0 4 to
erlenmeyer, assemble app., and distil 1 h at room temp. Det.
A at 540 nm for blank, external control, sample, and std AgDDC
solns in suitable spectrphtr. Subtract blank reading from sam-
ple and control, and det. trig As /kg directly from std curve.
External control results must fall within accepted range (95%
confidence limit) for all results to be valid.
Ref.: JAOAC 69, 493(1986).
985.40 Copper in Liver
Atomic Absorption Spectrophotometric Method
First Action 1985
Final Action 1987
A. Principle
One g liver tissue is digested overnight at 60° in 5 mL HN0 3 ,
then dild to 25 mL with H 2 and analyzed by A AS.
B. Apparatus and Reagents
(a) Atomic absorption spectrophotometer (AAS). — Equipped
with nebulizer and 10 cm, air-C 2 H 2 burner head. Monitor per-
formance by assuring that 4.0 mg Cu/L std produces >0.200
absorbance unit.
(b) External control. — Standard Reference Material (SRM
1577) Bovine Liver (193 ± 10 mg Cu/kg; NIST, Washington,
DC 20243) or equiv.
(c) Nitric acid. — Coned and dild (1 +4).
(d) Teflon screw-cap bottles. — 30 mL wide mouth (Cole-
Parmer, K-6103-30) or equiv.
(e) Copper std solns. — (J) Stock std soln. — 1000 mg Cu/
L. Dissolve 1.000 g Cu metal in 10 mL HN0 3 -H 2 (1 + 1).
356
AOAC Official Methods of Analysis (1990)
Copper
357
Dil. to 1000 mL with 1% HN0 3 . (2) intermediate std soln. —
100 mg Cu/L. Dil. 10 mL stock std soln to 100 mL with H 2 0.
(3) Working std solns.—DW. 0.0, 0.25, 0.5, 1.0, 2.0, and 4.0
mL intermediate std soln to 100 mL with HN0 3 -H 2 (1 +4)
to give Cu stds contg 0.0, 0.25, 0.5, 1.0, 2.0, and 4.0 mg
Cu/L, resp.
C. Sample Preparation
Rinse all glassware with 2N HCL Mix samples thoroly be-
fore weighing. Into sep. Teflon screw-cap bottles, accurately
weigh 1.0 g liver tissue and 0.25 g external control for each
10 samples or fraction thereof. (Note: Complete digestion will
not occur for >0.5 g dry wt of samples or controls.) Add 5
mL coned HN0 3 to each bottle, tighten cap, and place bottles
overnight in 60°, ventilated oven.
Remove bottles from oven and cool to room temp. Using
H 2 to rinse bottles, transfer sample digests to 25 mL vol.
flasks, allowing any fat to remain adhering to digestion bot-
tles. Dil. flasks to vol. with H 2 0.
D Determination
Analyze by AAS using following conditions: wavelength
324.7 nm; slit 0.7 nm; flame air-C 2 H 2 (lean-blue). Aspirate
series of working std solns, external control solns, and sample
dilns. Prep, std curve of concn (mg Cu/L) vs A and det. sam-
ple soln conens. Dil. with HN0 3 (1+4) any samples above
range of working stds. Repeat analysis if external control Cu
value is not within accepted range. Calc. mg Cu/kg tissue (X):
X = (C x 25 x D)/W
where C = sample soln concn (mg Cu/L); D — addnl. sample
diln, if necessary; and W - tissue wt (g).
Ref.: JAOAC 68, 44(1985).
CAS-7440-50-8 (copper)
shake 10 s. Use 1 external control for each 10 samples or frac-
tion thereof.
D. Determination
Analyze by AAS using the following conditions: wavelength
324,7 nm; slit 0.7 nm; flame air-C 2 H 2 (lean-blue). Aspirate
series of working std solns, external control soln, and sample
dilns. Repeat analysis if Cu value in external control soln is
not within accepted range. Prep, std curve of concn, mg Cu/
L, vs A, and det. concn of sample. Multiply result by 200 to
account for sample diln and to convert result to |xg Cu/100
mL.
Ref.: JAOAC 66, 1140(1983).
CAS-7440-50-8 (copper)
Drugs in Feeds
See chapter on drugs in feeds.
Drug Residues in Animal Tissues
See chapter on drug residues in animal tissues.
Metals
See chapter on metals and other elements at trace levels.
Mycotoxins
See chapter on natural poisons.
983.24 Copper in Serum
Atomic Absorption Spectrophotometry Method
First Action 1983
Final Action 1986
A. Principle
Samples are dild 1 + 1 with H 2 0, and Cu is detd by AAS
using std solns prepd in 10% glycerol.
B. Apparatus and Reagents
(a) Atomic absorption spectrophotometer. — Equipped with
nebulizer and air-C 2 H 2 burner head. Monitor performance by
assuring that 4.0 mg/L std produces response 5:0.200 absor-
bance unit.
(b) External control.— Precilip, Cat. No. 125067 (Bio-Dy-
namics/bmc, 9115 Hague Rd, Indianapolis, IN 46250), or
equiv. with established value for Cu. Dil. according to label.
(c) Glycerol USP.~ 10% (v/v) aq. soln.
(d) Copper std solns. — (J) Stock std soln. — 1000 mg/L.
Dissolve 1.000 g Cu metal in min. vol. of HN0 3 -H 2 (1 +
1). Dil. to 1000 mL with 1 % HN0 3 . (2) Intermediate std soln. —
100 mg/L. Dil. 10 mL stock std soln to 100 mL with H 2 0.
(3) Working std solns.— -Dil. 0.0, 0.25, 0.5, 1.0, 2.0, and 4.0
mL intermediate std soln to 100 mL with 10% glycerol to give
std solns contg 0.0, 0.25, 0.5, 1.0, 2.0, and 4.0 mg Cu/L.
C. Sample Preparation
Rinse all glassware used with 2N HC1. Mix samples thoroly
before pipetting. Using Mohr pipet, transfer 1.0 mL serum and
1.0 mL Precilip (external control) to sep. test tubes. Add 1 .0
mL H 2 to each and mix 5 s on vortex mixer or cap tubes and
984.32 Pesticide Residues
A. General Methods
See 970.52.
B. Specific Methods
See chapter on pesticide residues.
984.33 Urea in Feeds
See 941.04 and 967.07.
986.31 Nitrate in Forages
Potentiometric Method
First Action 1986
Final Action 1989
A. Principle
Nitrate is extd from sample into aq. Al 2 (S0 4 ) 3 soln and detd
potentiometrically. Lower limit of detection is 0.50% KN0 3 .
B. Apparatus
(a) Drying oven. — Forced air, capable of heating to 100°.
(h) Nitrate -specific ion electrode. — Model 93-07, Orion
Research, Inc., or equiv. Monitor performance by assuring ab-
solute value of std curve per decade slope >54 mV.
(c) Reference electrode. — Double junction (Model 90-02,
Orion Research, Inc., or equiv.). Use extg soln as outer filling
soln.
358
Veterinary Analytical Toxicology
AOAC Official Methods of Analysis (1990)
Table 986.31 Conversion Factors for Units of Nitrate and Nitrite Concentrations 9
From
To
NO3-N
(14.01)
N0 3
(62.01)
NaN0 3
(85.01)
KNO3
(101.11)
N0 2 -N
(14.01)
N0 2
(46.01)
NaN0 2
(69.01)
KN0 2
(85.11)
N0 3 -N
(14.01)
1.000
0.2259
0.1648
0.1386
1.000
0.3045
0.2030
0.1646
N0 3
(62.01)
4.426
1.000
0.7294
0.6133
4.426
1.348
0.8986
0.7286
NaN0 3
(85.01)
6.068
1.371
1.000
0.8408
6.068
1.848
1.232
0.9988
KNO3
(101.11)
7.217
1.631
1.189
1.000
7,217
2.198
1.465
1.188
N0 2 -N
(14.01)
1.000
0.2259
0.1648
0.1386
1.000
0.3045
0.2030
0.1646
N0 2
(46.01)
3.284
0.7420
0.5412
0.4550
3.284
1.000
0.6667
0.5406
NaN0 2
(69.01)
4.926
1.113
0.8118
0.6825
4.926
1.500
1.000
0.8108
KN0 2
(85.11)
6.075
1.373
1.001
0.8418
6.075
1.850
1.233
1.000
a Find current unit of concentration on top row. Find desired unit of concentration in left column. Obtain conversion factor at intersection of row and column.
Desired concentration - current concentration x conversion factor. Numbers in parentheses below units of concentration are formula masses. Atomic masses:
N: 14.01; O: 16.00; Na: 23.00; K: 39.10. 1% = 10 000 ppm.
(d) pH meter. — Capable of measuring electrode potentials
to nearest mV.
C. Reagents
(a) Preservation soln. — Dissolve 0.1 g phenylmercuric
acetate in 20 mL dioxane (Caution: May form dangerous per-
oxides; see safety notes on peroxides.) and dil. to 100 mL with
H 2 0.
(b) Extracting soln.— Dissolve 15.76 g A1 2 (S0 4 ) 3 .18 H 2 <3,
70.0 mg oven-dried KNO3 (dry 2 h at 100°), and 1.0 mL pres-
ervation soln, (a), in 500 mL H 2 0. Dil. to 1 L with H 2 0.
(c) Nitrate std solns. — (!) Stock std soln. — 100 000 mg
KNO3/L. Weigh 20.00 g KNO3 (dried 2 h at 100°) into 200
mL vol. flask, dissolve in 100 mL H 2 0, and dil. to vol. with
H 2 0. (2) Intermediate std soln. — 10 000 mg KNO3/L. Dil.
20.0 mL soln (J) to 200 mL with extg soln. (3) Working std
solns.— Dil. 1.00, 2.00, 4.00, and 10.0 mL soln (2) to 200
mL with extg soln to make 120, 170, 270, and 570 mg KNO3/
L solns. (Note: Prep, all std solns from same lot of extg soln.)
D. Preparation of Standard Curve
Det. potential of blank (use extg soln as blank, equiv. to 70
mg KNO3/L) and each working std soln while mag. stirring.
Plot potential against nitrate concn (mg KNO3/L) on semilog
paper with concn on log scale. Det. std curve per decade slope.
Per decade slope specification of electrode manuf . should be
met or exceeded. (Note: Measure potential only after sample
and stds are at same temp.)
E. Preparation of Sample
Dry sample in 60° forced air oven to const wt. Grind dried
sample to pass 2 mm screen and thoroly mix. Ext 1 .00 g mixed
ground sample with 100 mL extg soln by shaking 15 min.
Keep ext sealed in container until potential is measured.
F. Quality Control
Analyze external control samples of known nitrate concn or
spiked samples (at least one for every 10 samples or fraction
thereof). Results should indicate acceptable accuracy.
Hold suspect any analyses with unacceptable external con-
trol results and unacceptable std curve per decade slopes.
G. Determination
Det. potential of unfiltered ext at same temp, that std po-
tentials are measured, stirring ext at same rate used for stds.
Record potential after reading becomes stable or 1 min after
insertion, whichever comes first.
H. Calculation
Calc. forage nitrate concn (% KNO3 on dry wt basis):
% KNO3 - C u = (C g - 70) x 0.010
where C u = forage nitrate concn and C g = nitrate concn (mg
KNO3/L) of ext obtained from std curve. If desired, convert
nitrate concn expressed as % KNO3 to other units as shown in
table 986.31.
Ref.: JAOAC69, 196,283(1986).
15. Cosmetics
Anthony D. Hitchins, Associate Chapter Editor
Food and Drug Administration
GENERAL METHODS
966.22 Water and Ethyl Alcohol
in Cosmetics
Gas Chromatographic Method
First Action 1966
A. Principle
Sample is dissolved or dispersed in ethylene glycol mono-
methyl ether, which also serves as internal std, and H 2 and
alcohol are detd by GC, using relatively inert column, per-
il uorocarbon substrate coated with high MW polyethylene gly-
col, to minimize tailing.
B. Apparatus
Gas chromato graph. — With thermal conductivity detector
operated at following temps (°): Detector ca 250, injection port
ca 260, oven, ca 100; He flow rate, 50 mL/min; bridge cur-
rent, as directed by manufacturer.
C. Standard Solutions
Prep. 3 std solns in 125 mL g-s flasks contg H 2 and ab-
solute alcohol, weighed to 0.1 mg, in ethylene glycol mono-
methyl ether weighed to 10 mg, as follows:
H 2 0, mg
Alcohol, mg
Ethylene Glycol
Monomethyl
Ether g
125
250
375
375
250
125
24.50
24.50
24.50
H 2 content of ethylene glycol monomethyl ether should be
<0.05%. Use same batch for stds and samples. Com. product
is usually satisfactory, but because of hygroscopicity, expose
solns to air as little as possible.
D. Preparation of Column
Weigh 10 g polyethylene glycol 20000 (Carbowax 20M) into
800 mL beaker, dissolve completely in ca 400 ml warm CH 2 C1 2 ,
and cool to ca 0°. Slurry cold soln with 190 g precooled (ca
0°) Fluoropak 80, 40-60 mesh (Applied Science). Transfer to
15 cm crystg dish, place in hood, and evap. to dryness at room
temp, with occasional stirring. Recool to ca 0° and screen thru
No. 40 on No. 60 precooled screen. Pack fraction remaining
on No. 60 in 4.6 m (15') X y / 4 " precooled Cu column, using
vibrator. (Use of cooled column packer (Press-Pak, available
from Alltech Associates, 2051 Waukegan Rd, Deerfield, IL
60015) at 35 Ib/sq in. (240 kPa) N pressure allows packing
of cooled, precoiled column.)
E. Standardization
Inject 3 (xL of one of std solns with 10 jxL syringe and det.
elution time of ethylene glycol monomethyl ether. (Order is
alcohol, H 2 0, and glycol ether.) Adjust oven temp., if nec-
essary, so latter elutes in 15-20 min. With satisfactory col-
umn, pen will return to within 1% of recorder between al-
cohol and H 2 peaks. Det. sample size for each std soln such
that response for smallest peak is > l U full scale on x 1 atten-
uation. Det. all attenuations necessary to keep all peaks on
chart scale. (Too large samples will overload column and skew
glycol ether peak.)
With some gas chromatographs, alcohol peak response will
vary with time interval between emergence of glycol ether of
previous injection and injection of sample. Thus, inject all
samples (std curves and actual detns) at same time interval
after emergence of glycol ether. This requires use of prelim-
inary sample. If time sequence is broken, inject another pre-
liminary sample to re-establish sequence.
Obtain chromatograms, in duplicate, and on same day, for
each std soln, using sample sizes and attenuations detd above.
Duplicate sample sizes to 0.1 (xL and use same technic for
injecting and withdrawing syringe needle. Det. peak ht of each
component, correcting for attenuation.
Calc. ratios: peak ht H 2 0/peak ht ethylene glycol mono-
methyl ether (/?p W )> an d wt H 2 0/wt ethylene glycol mono-
methyl ether (/? ww )- Average the 2 R PW values for each std
soln and plot av. R PW values against corresponding /? ww val-
ues. Draw best straight line thru 3 points. Make same calcns
for alcohol stds, and plot corresponding R PA and R WA values,
(Curves should be straight lines intersecting x or y axis near
origin.)
F. Determination
Accurately weigh sample contg ca 100-400 mg H 2 and/
or alcohol into 125 mL g-s flask, add 24.50 ± 0.1 g ethylene
glycol monomethyl ether, and mix thoroly. (Complete soln is
unnecessary but glycol ether phase should contain all the H 2
and alcohol.) Det. proper sample size as in 966. 22E, recording
necessary attenuations. From good chromatogram, calc. /? PW
and R PA values. Read tf ww and R WA values from std curves.
From latter values prep, final std soln of 24.50 g ethylene gly-
col monomethyl ether plus H 2 and /or alcohol which ap-
proximates (within 10%) curve of sample. Det., in sequence,
(a) curve of proper size sample, (b) curve of adjusted final std
soln, and repeat (a) and (b) y in that order. Det. av. /? PW and
R PA values of unknown and final std solns, then /? ww and /? WA
of std soln.
For H 2 0: /? ww (sample) - R PVV (sample) X /? ww (std)//? PW
(std); wt H 2 (sample) = wt ethylene glycol monomethyl ether
(sample) x /? ww (sample); and % H 2 (sample) = (wt H 2 0/
wt sample) x 100.
For alcohol: 7? WA (sample) = R PA (sample) x /? WA (std)//? PA
(std); wt alcohol (sample) = wt ethylene glycol monomethyl
ether (sample) x /? WA (sample); and % alcohol (sample) = (wt
alcohol/ wt sample) x 100.
Ref.: JAOAC 49, 718(1966).
359
360
Cosmetics
AOAC Official Methods of Analysis (1990)
970.61 Propylene Glycol in Cosmetics
Gas Chromatographic Method
First Action 1970
Final Action 1971
(Applicable to all types of cosmetics)
A. Apparatus
(a) Distillation apparatus. — All-glass, with "£20/ 40 joints:
250 mL r-b flask, elec. heating mantle, 20 mL Barrett H 2
trap with 1 stopper, and driptip condenser.
(b) Gas chromato > graph. — With H flame detector and ca-
pable of operating at ca 200°.
(c) GC column. — 1.8 m (6') X l / 4 " od Cu or Al column
packed with 80-100 mesh Chromosorb 101. Pack resin in col-
umn, using vibrator and column packer, 966. 22D, operated at
25-35 lb (170-240 kPa) pressure. Heat column overnight at
240° with He flow rate ca 100 mL/min. Condition column
with propylene glycol as in 970. 61D. Inject enough aq. soln
of propylene glycol-trimethylene glycol (1 + 1) to give > l / 2
full-scale response. If column is satisfactory, 2 symmetrical
peaks will be obtained. Reject batches of Chromosorb 101 which
give unsymmetrical peaks.
B. Reagents
(a) Propylene glycol. — Eastman Kodak Co. No. 1321, or
equiv.; assay by periodate oxidn: Place aliquot of aq. soln contg
<45 mg propylene glycol in g-s flask, add 35 mL 0.02M KI0 4
soln, dil. to ca 100 mL with H 2 0, and Jet stand I hr. Add ca
1.0 g NaHCO^, 0.5 g KI, and 2.5 mL starch indicator (mix
ca 1 g sol. starch with enough cold H 2 to make thin paste,
add 100 mL boiling H 2 0, and boil ca 1 min while stirring).
Titr. with 0.02/V KAs0 2 soln to disappearance of blue. Stdze
25 mL 0.02M KI0 4 soln by same titrn, using H 2 for sample,
and calc. amt of K10 4 reduced by sample. 1 mL 0.02N KAs0 2
= 0.76 mg propylene glycol.
(b) Trimethylene glycol. — Propylene glycol-free.
(c) lsooctane (2 ,2 ,4-trimethylpentane). — Bp 99-100°.
(d) Propylene glycol and trimethylene glycol std solns. —
10 mg/mL. Prep. sep. std solns. Accurately weigh ca 1 .0 g
std, dissolve in H 2 0, transfer to 100 mL vol. flask, and dil.
to vol.
C. Separation of Propylene Glycol by Co-distillation
Accurately weigh sample contg ca 2-40 mg propylene gly-
col into 250 mL r-b flask. Add 8-10 mL H 2 and few boiling
chips. Connect flask to distn app. and add, thru condenser,
enough isooctane to fill H 2 trap and provide 25-40 mL
isooctane in distn flask. Adjust voltage on heating mantle so
that isooctane distils at 5—10 mL/min. Continue distn 30 min
after all H 2 appears to be collected in trap. Drain as much
H 2 as possible (leave ca 0.25 mL in H 2 6 trap) into small
g-s container. (Stoppered 25 mL graduate is convenient.) Re-
move heat from distn flask and, when boiling stops, discon-
nect flask from app. and add 5 mL H 2 0. Reconnect to app.
and distil as before. Drain distillate into container contg first
H 2 distillate. Repeat with second 5 mL portion H 2 0. Mix
combined distillates.
D. Preparation of instrument
With Chromosorb 10 J column in gas chromatograph, set
column temp, at ca 180°, injection port and detector temps at
ca 300°, and He flow rate near 70 mL/min. If necessary, ad-
just column temp, to elute propylene glycol in ca 6 min. Con-
dition column by initial 0.5 |ulL injection of propylene glycol.
Column must be conditioned in this manner once a day before
use for detg propylene glycol. Use 5 jjlL aq. test soln contg
ca 1 mg propylene glycol/mL and adjust H and air flow to
flame detector until max. response is obtained. (See manufac-
turer's directions.) Note range and attenuation settings needed
to keep peak on scale.
Establish rough calibration curve of peak ht response against
wt propylene glycol by injecting known amts propylene glycol
and observing response.
E. Determination
Det. approx. propylene glycol content of aq. soln, obtained
by codistn of sample with isooctane, by injecting known amt
distillate into chromatograph. To sample soln add known ali-
quot of std trimethylene glycol soln (preferably sample soln
should contain approx. equal wts propylene glycol and tri-
methylene glycol). Prep., from accurately measured aliquots
of stds, soln contg approx. same wt propylene glycol and tri-
methylene glycol as prepd sample and dil. to approx. same
vol. as sample soln.
Det., by trial injections, resp. vols of sample and std solns
required to give nearly equal responses of ca 3 / 4 full-scale for
propylene glycol. Alternately inject these vols sample and known
solns, making >:2 injections of each soln. Det. sample and its
corresponding std at same range and attenuation settings.
F. Calculations
From chromatograms, calc. following peak ht ratios for
sample, R, and std, R\ resp.:
R = peak ht propylene glycol /peak ht trimethylene glycol
R r — peak ht propylene giycol/peak ht trimethylene glycol
Using av. values of R and R' , calc. amt propylene glycol in
sample.
mg Propylene glycol — (R/R f )x mg propylene glycol (std)
x [mg trimethylene glycol (sample)]/
[mg trimethylene glycol (std) J
(If same aliquot of trimethylene glycol is used for prepd
sample and std, last factor - 1 .)
Ref.: J AOAC 53, 82(1970).
CAS-57-55-6 (propylene glycol)
973.59 Eye Irritants
in Cosmetic Constituents
Bioassay
First Action 1973
Use 6 albino rabbits of either sex, weighing 2.0-2.5 kg,
randomly selected, for each substance. Facilities must be de-
signed and maintained so as to exclude extraneous materials
that might produce eye irritation. Examine both eyes of each
animal before testing. Use only animals without defects or eye
irritation.
Hold animal firmly but gently until quiet. Instill 0. 1 mL test
material onto cornea so that it flows into conjunctival sac of
1 eye of each animal by gently pulling lower lid away from
eyeball to form cup into which test substance is dropped. Hold
lids gently together 1 sec and release animal. Other eye, un-
treated, serves as control.
Examine eyes grossly or microscopically and record grade
of ocular reaction of cornea, iris, and conjunctiva at 24, 48,
and 72 hr, and 7 days. After 24 hr observation, further ex-
amine eyes after applying fluorescein- impregnated paper strips
(Barnes-Hind, Ay erst, etc.). Eyes may be washed with sterile
isotonic NaCl soln after 24 hr reading.
Independently grade each portion of eye (cornea, iris, and
conjunctiva) without reference to total score, using definitions
AOAC Official Methods of Analysis (1990)
Deodorants 361
and color photographs in "Illustrated Guide for Grading Eye
Irritation by Hazardous Substances," Superintendent of Doc-
uments, Government Printing Office, Washington, DC 20402.
Consider animal as exhibiting pos. reaction if test substance
produces, at any of the readings, ulceration of cornea (other
than fine stippling), or opacity of cornea (other than slight dulling
of normal luster), or inflammation of iris (other than slight
deepening of folds (or rugae) or slight circumcorneal injection
of blood vessels), or if substance produces in conjunctivae (ex-
cluding cornea and iris) obvious swelling with partial eversion
of lids or diffuse crimson-red with individual vessels not easily
discernible. Basis for irritation is grade >1 for cornea and iris
and >2 for redness and chemosis.
Sample is eye irritant if >4 of 6 rabbits have irritation. Sam-
ple is not eye irritant if or 1 of 6 rabbits has irritation. For
combined parameters, eye is considered irritated if >1 param-
eter has a grade considered to be eye irritant. If 2 or 3 animals
exhibit pos. reaction, repeat test with 6 different animals. Sec-
ond test is considered pos. if >3 animals exhibit pos. reaction.
If only 1 or 2 animals in second test exhibit pos. reaction,
repeat test with 6 different animals. In third test, substance is
considered irritant if any animal exhibits pos. reaction.
Ref.: J AOAC 56, 905(1973).
DEODORANTS AND ANTIPERSPSRANTS
938.11 Aluminum and Zinc in Deodorants
Gravimetric Method
Final Action
A. Reagents
(a) 8 -Hydroxy quinoline soln. — Dissolve 5.0 g 8-hy-
droxyquinoline in 12 mL HO Ac, dil. to 100 mL with H 2 6,
and filter if not clear. Prep, fresh soln :<2 weeks.
(b) Ammonium acetate soln. — Approx. 2N '. Dissolve 150—
160 g NH 4 OAc in 1 L H 2 and filter if not clear.
(c) Hydrochloric acid. — Approx. 2N (1 + 5).
(d) Ammonium hydroxide. — Approx. 27V. Vol. of NH 4 OH
required to neutze 20 mL 2N HC1, (c), should be known to
within ±2 mL.
B. Preparation of Sample
(a) Liquids. — Dil. 5 mL sample to 250 mL with H 2 in
vol. flask. If perfume oils sep., filter before taking aliquot for
analysis.
(b) Creams and pastes. — Accurately weigh 2-3 g sample
into 250 mL beaker. Add 5 mL HC1 (HN0 3 if chlorides are
to be detd) and ca 50 mL H 2 0, and heat until oils liquefy and
sep.; cool until oils solidify, and decant aq. layer thru fluted
paper into 250 mL vol. flask. Return filter to original beaker
and macerate thoroly. Repeat above extn twice, decant as be-
fore, and finally thoroly wash residue and paper with H 2 0. (It
is unnecessary to return filter paper to beaker after these extns.)
Cool combined exts to room temp., dil. to vol. with H 2 0, and
mix.
(c) Solids. — Accurately weigh 2-3 g sample into 250 mL
beaker, add 5 mL HO (HN0 3 if chlorides are to be detd) and
ca 50 mL H 2 0, and heat to bp. Cool, and filter thru fluted
paper into 250 mL vol. flask. If filtrate is cloudy, refilter thru
fine quant, paper. Thoroly wash beaker and paper with H 2 0.
Cool flask and contents to room temp., dil. to vol. with H 2 0,
and mix.
C. Determination
(a) Interfering metals absent. — Take aliquot of sample soln
contg 12-25 mg AJ or 20-60 mg Zn. Add 1-2 drops phthln,
and then add 2N NH 4 0H until neut. or until faint permanent
turbidity results. Add 5 mL HOAc (1 + 9), dil. to ca 100 mL,
and heat to 70-90°. Add 10 mL 8-hydroxyquinoline soln and
then slowly add NH 4 OAc soln until 20 mL (see Note) in excess
of vol. required to produce permanent ppt has been added. If
permanent ppt forms on addn of 8-hydroxyquinoline, add only
20 mL NH 4 OAc soln. Heat below bp 2-5 min and set aside
30-60 min. (Moderate excess of 8-hydroxyquinoline is re-
quired for complete pptn. If enough reagent has been added,
soln will be yellow at this point; if it is not, repeat detn, using
larger vol. Of 8-hydroxyquinoline soln.) Filter thru tared gooch,
wash thoroly with H 2 0, dry 1-2 hr at 130-140°, cool, and
weigh. Dry again 30 min, cool, and weigh. Repeat to const
wt (±0.3 mg). (Alternatively, ppt may be dried overnight.)
Wtppt x 0.05871 - Al
Wtppt x 0.1848 - Zn
Note: Final pH of soln from which metals are pptd should
be 4.9-5. 1 . Vol. of NH 4 OAc soln required to produce this pH
should be detd experimentally each time new set of reagents
is prepd. If NH 4 OAc is of usual purity, ca 20 mL soln will be
required.
(b) In presence of magnesium. — Ppt as in (a) and set aside
ca 30 min. Decant most of liq. thru quant, paper (part or all
of ppt may be transferred to paper if necessary) and discard
filtrate. Place beaker used for pptn under funnel and dissolve
ppt on paper in hot 2/V HO (20 mL is usually enough if added
in several small portions). Wash paper and funnel with 20-30
mL H 2 0. Add 2 mL 8-hy droxyquinoline soln, 5 mL HOAc
(1 + 9), and vol. of 2N NH 4 OH equiv. to 2N HO used to
dissolve ppt (do not use excess). Dil. to ca 100 mL, heat to
70-90°, and proceed as in (a), beginning "... slowly add
NH 4 OAc soln . . ."
Refs.: Ind. Eng. Chem. Anal. Ed. 10, 212(1938). J AOAC
28, 734(1945).
CAS-7429-90-5 (aluminum)
CAS-7440-66-6 (zinc)
944.11
Zinc in Deodorants
Gravimetric Method
Final Action
A. Reagent
8-Hydroxyquinaldine soln. — Dissolve 5.0 g 8-hydroxyquin-
aldine in 12 mL HOAc, dil. to 100 mL with H 2 0, and filter
if soln is not clear. (Soln is stable ca 1 week. If only tech.
grade base is available, purify by recrystn from alcohol (2 +
1), using 6 mL sol v. for each g base, before prepg soln.)
B. Determination
Pipet aliquot of sample soln, 938.1 IB, contg 20-50 mg Zn,
into 400 mL beaker. Adjust soln to slight acidity, add 1 g NH 4
tartrate if Al is present, and then add 2 mL 8-hydroxyquin-
aldine soln for each 10 mg Zn present; dil. to 200 mL and heat
to 60-80°. Neutze excess acid by adding NH 4 OH (1 +4) until
Zn complex salt that forms on addn of each drop just redis-
solves on stirring. Slowly add, with stirring, 45 mL NH 4 OAc
soln, 938.11A(b), and let mixt. come to room temp.
Det. pH of soln; if pH is not 5.7-5.9, adjust with the NH 4 OH
soln, and let mixt. stand 10-20 min to achieve equilibrium.
Decant thru tared gooch and wash ppt in beaker twice with
hot H 2 0, decanting each wash into crucible. Finally transfer
ppt to crucible and again wash with hot H 2 0. (Total vol. wash-
ings should be >200 mL.) Dry crucible and ppt 2 hr at 130-
140°, cool, and weigh. Reheat 30 min at 130-140°; cool, re-
362
Cosmetics
AOAC Official Methods of Analysis (1990)
weigh, and repeat heating, cooJing, and weighing to const wt.
Wt ppt x 0.1712 = wtZn.
Rets.: Ind. Eng. Chem. Anal. Ed. 16, 387(1944). JAOAC
33, 371(1950).
CAS-7440-66-6 (zinc)
950.84 Aluminum in Deodorants
Gravimetric Method
Final Action
Multiply wt Zn found, 944. 11B, by 5.411 to obtain equiv.
wt 8-hydroxyquinoline salt, multiply by appropriate factor for
aliquot taken, and subtract from wt combined Al and Zn salts,
938.11C. Difference x 0.05871 = wt Al.
976.24 Zirconium (Soluble)
in Antiperspirant Aerosols
Colorimetric Method
First Action
A. Reagents
(a) Alizarin red S (sodium alizarin sulfonate) soln. — Dis-
solve .1.5 g alizarin red S indicator (Allied Chemical Corp.,
or equiv.) in 300 mL hot H 2 0. Cool and filter thru double
layer of rapid, medium porosity paper (Whatman No. 12, 24
cm folded, or equiv.). Dil. filtrate to 1 L with H 2 0, and re-
filter. Soln is stable ^1 month.
(b) Zirconyl chloride octahydrate. — Eisner No. Z-80, or
equiv. Assay as in 976. 24B.
B. Assay of Standard
Accurately weigh 500-600 mg ZrOCl 2 .8H 2 into 400 mL
beaker and dissolve in 50 mL H 2 0. Add 4 g NH 4 N0 3 and
warm on steam bath to ca 50°. Slowly add, with stirring, 100
mL NH 4 OH and continue heating 20 min. Filter while hot thru
15 cm Whatman No. 42, or equiv., paper. Complete transfer
of ppt with 2-3 portions 2% NH4NO3 in NH 4 OH (2 + 98).
Carefully fold paper and place in ca 50 mL Pt crucible. Dry
in oven at 105°. Partially cover crucible and gently heat with
Meker burner until paper is well charred. Continue heating at
max. burner temp, to const wt.
% Zr - ZrO residue x 74.03 /wt ZrOCl 2 .8H 2
C. Preparation of Standard Curve
Dissolve ZrOCl 2 .8H 2 contg 200 mg Zr in 70 mL H 2 in
200 mL vol. flask. Add 110 mL HC1, cool, and dil. to vol.
with H 2 0. Prep, this 1 mg/mL stock soln fresh weekly. Pipet
2, 5, 10, and 15 mL stock soln into 100 mL vol. flasks. Dil.
each to vol. with HC1 (55 + 45), and mix. Pipet 5 mL each
dil. std and, as blank, 5 mL HC1 (55 4- 45) into sep. 100 mL
vol. flasks for color development (0, 100, 250, 500, and 750
jag Zr/100 mL). Add to each flask 10.0 mL alizarin red S
soln, (a), and 8 mL H 2 0. Swirl and place in 75 ± 3° H 2
bath. Monitor temp, of solns with thermometer in 100 mL vol.
flask contg 23 mL H 2 added at room temp, and placed si-
multaneously in bath. Swirl flasks occasionally while heating.
After solns reach 70°, keep flasks in bath addnl 6.5 ± 1 min.
Remove and let cool 20 min at room temp. Dil. each to vol.
with H 2 and mix. Measure A in 2 cm cells against blank at
525 nm, scanning on recording spectrophtr from 700-460 nm.
Plot A against jxg Zr/100 mL.
(If 750 (jug Zr/100 mL cannot be read on A scale, use higher
range of spectrophtr or 14.0 mL aliquot (700 (xg Zr/100 mL).)
D. Preparation of Sample
Remove cap and any paper wrapping from aerosol can. Re-
cord wt of can to nearest 0.01 g. Replace cap and freeze con-
tents by placing inverted can in beaker contg mixt. of solid
C0 2 and acetone and cooling >1 hr. Transfer can to smaller
beaker in exhaust hood and cautiously open bottom end with
can opener, keeping end partially attached to can. Let volatile
gases escape at room temp. Remove dip tube after initial thaw-
ing when gas evolution has subsided, but keep tube with can
in beaker. Place beaker on steam bath, and heat gently to evolve
higher boiling gases. Increase heat slowly and maintain until
bubbling subsides. Place beaker contg can and dip tube in 70°
forced-draft oven 45 min. Raise temp, to 115° and maintain
2.5 hr, stirring occasionally with stainless steel spatula. Re-
move from oven and let cool to room temp. Thoroly mix con-
tents, including any portion clinging to sides, to form homo-
geneous cone. Weigh can plus contents, spatula, and dip tube.
Stir contents ca 1 min and weigh again. Repeat stirring and
weighing until wt is const to within 0.01 g. Record wt and
immediately transfer contents to g-s weighing bottle (quant,
transfer unnecessary). Stopper bottle and protect from further
wt loss by opening only when necessary.
Remove cap from can and thoroly remove remaining con-
tents from can, spatula, and dip tube with H 2 and alcohol.
Dry cap, can, spatula, and dip tube to const wt at 1 10°. Obtain
wt of cone, by subtracting latter wt from wt previously re-
corded after drying contents. Obtain wt of intact can contents
by subtracting wt of clean, dry can (without cap) and dip tube
from gross wt initially recorded.
E. Determination
Record gross wt of prepd sample in weighing bottle. Mix
sample briefly and remove ca 1 g with spatula. Calc. wt re-
moved as difference in gross wt.
Transfer sample as completely as possible from spatula to
lower half of 600 mL beaker. Thoroly wipe spatula with small
piece of filter paper and add paper to beaker. Add 5 mL al-
cohol and break up sample with glass rod. Slowly add 200 mL
HC1 while stirring vigorously. Heat to bp on steam bath. Boil
2-3 min and immediately transfer thru funnel to 1 L vol. flask.
Complete transfer with 200 mL and 150 mL portions hot HO.
Warm flask on steam bath and shake vigorously 3-4 min. Rinse
beaker with 450 mL H 2 0, add to flask, and mix thoroly. Cool
to room temp., dil. to vol. with HO, and mix thoroly. Let
undissolved material settle and coagulate. Filter portion thru
double layer of 24 cm Whatman No. 12, or equiv., folded
paper, discarding first 50 mL. Make appropriate dilns with
HO (55 + 45) to obtain Zr concn of 40-100 jjLg/mL. Pipet
5 mL into 100 mL vol. flask, and proceed as in 976. 24C,
beginning "Add to each flask 10.0 mL alizarin red ..."
% Zr in intact can contents - (C/W s ) X (WjW { ) X (F/10 4 )
where C = u>g Zr/100 mL read from std curve; W s , W £? and
W-, = g sample, prepd cone, and intact can contents, resp.;
and F — appropriate diln factor.
Refs.: JAOAC 59, 830, 1421(1976); 60, 663(1977).
CAS-7440-67-7 (zirconium)
952.15 Boric Acid in Deodorants
and Antiperspirants
Ion Exchange Method
Final Action
A. Preparation of ton Exchange Column
Provide glass tube 58 cm long x 2 cm diam. (23 x 0.75")
with stopcock and outlet tube. Tamp 3 cm glass wool plug
AOAC Official Methods of Analysis (1990)
Deodorants 363
into bottom of tube, fill tube with H 2 0, and add Amberlite 1R-
120(H) ion exchange resin slowly to form 20 cm column. Wash
with HC1 (1 + 9) and then with 50 mL portions H 2 until
effluent gives neg. CI test.
Regenerate after use by transferring accumulated resin from
number of detns to large glass tube and washing with HCl (1
+ 9) until effluent gives neg. test for adsorbed cations, e.g.,
Zn, Al. Then remove HC1 from resin by washing with H 2
until effluent gives neg. CI test.
B. Determination
Place sample contg 50-200 mg H 3 B0 3 in 250 mL casserole,
add 2 drops phthln, and make alk. with 10% NaOH soln. Evap.
to dryness on steam bath under gentle air current, dry residue
1 hr at 140° in oven, and ash 1 hr at 550°. Cool to room temp.,
add ca 50 mL hot H 2 0, acidify cautiously with HO, and filter
hot soln thru quant, paper into 250 mL beaker. Wash paper
with little hot H 2 and reserve filtrate (may be slightly cloudy).
Transfer paper to same casserole and make alk. by wetting
with c a 10 mL H 2 and few drops 10% NaOH soln. Evap. to
dryness on steam bath, dry 1 hr at 140°, and ash 2 hr at 550°.
Cool, add ca 50 mL hot H 2 0, acidify with HO, and filter into
reserved filtrate. Wash casserole and paper thoroly with hot
H 2 0, and discard paper. (Total vol. soln should be ca 200
mL.)
Cool soln; add NH 4 OH until barely alk. to litmus paper or
until flocculent ppt appears. Reacidify with HO until slightly
acid to litmus paper or until ppt just redissolves. Pass soln thru
ion exchange column into 1 L flask at rate requiring 10-15
min for passage. Follow sample soln with several 50 mL por-
tions H 2 until effluent is only slightly acid to pH test paper.
Add 5 drops Me red, 936.15D(a), make alk. with freshly prepd
10% NaOH soln, and then barely acid with HCl.
Connect flask to H 2 0-cooled reflux condenser and boil 5
min. Wash down condenser with little H 2 and cool soln to
room temp, under running H 2 0. Neutze to Me red with 0.17V
NaOH, 936. 16C; add 4-5 g mannitol and ca 0.5 mL phthln.
Titr. with 0. 17V NaOH to pink color, add more mannitol, and
if pink disappears, continue titrn until it reappears. Repeat addn
of mannitol until there is no further change in color.
Det. blank as follows: To ca 350 mL H 2 add vol. of freshly
prepd 10% NaOH soln equal to that required to neutze sample
after passing thru column. Barely acidify with HO and pro-
ceed as above, beginning "Connect flask to H 2 0-cooled reflux
condenser ..." Subtract blank titrn from sample titrn and
calc. H3BO3 content of sample. 1 mL 0. 1/V NaOH - 0.00618
g H3BO3.
Refs.: Anal. Chem. 24, 182(1952). JAOAC 36, 791(1953).
CAS-10043-35-3 (boric acid)
951.04
Chlorides in Deodorants
Gravimetric Method
Final Action
Pipet aliquot of sample soln, 938. 1IB, contg ca 100 mg CI
into 250 mL beaker. Oil. to 150 mL with H 2 0, neutze to lit-
mus with NH4OH (1 + 1), and acidify with 1 mL HN0 3 (1
+ 1). If any undissolved ppt remains, add more HNO3 (1 +
1) until clear soln is obtained. Add dropwise, stirring con-
stantly, slight excess of 0.1W AgN0 3 . (Excess should be <5
mL.) Pptn and succeeding operations must be carried out in
subdued light. Heat mixt. to 90-95° and stir until ppt coag-
ulates. Let ppt settle; add 1-2 drops QAN AgN0 3 to supernate
to ensure presence of excess Ag. Let mixt. stand 1-2 hr in
dark.
Decant thru tared gooch, wash ppt 2-3 times with 0.017V
HN0 3 by decantation, and finally transfer ppt to gooch with
0.01N HN0 3 . Continue washing ppt with 0.0YN HN0 3 until
washing gives neg. test for Ag when 1 drop 0. IN HC1 is added.
Complete washing by removing most of the HN0 3 with two
10 mL portions H 2 0. Dry crucible 2 hr at 120-130° and weigh.
Repeat drying to const wt (0.2 mg). Wt AgCI X 0.2474 = wt
CI.
Ref.: JAOAC 34, 298, 299(1951).
951.05
Sulfates in Deodorants
Gravimetric Method
Final Action
938. 11B, contg ca 100 mg
to 350 mL with H 2 0, neutze
and acidify with 2 mL HC1.
add more HO until soln is
Pipet aliquot of sample soln,
sulfate into 600 mL beaker. Dil.
to litmus with NH4OH (1 + 1),
If any undissolved ppt remains,
clear.
Heat 50 mL 1% BaCl 2 soln almost to bp and add rapidly
with stirring to sulfate soln which has also been heated to near
bp. Let ppt settle, and add little BaCI 2 soln to ensure excess
of Ba. Let mixt. stand 1-2 hr on steam bath. Decant thru tared
gooch, wash ppt 4-5 times with small portions of warm H 2
by decantation, and finally transfer ppt to gooch with warm
H 2 0. Continue washing ppt with warm H 2 until washing gives
neg. test for CI. Dry crucible 2 hr at 110-120° and weigh.
Repeat drying to const wt (0.2 mg). Wt BaS0 4 x 0.4116 =
wt sulfate.
Ref.: JAOAC 34, 298, 299(1951).
974.28 Hexachlorophene in Deodorants
Spectrophotometric Method
First Action 1974
Final Action 1978
A. Apparatus
(a) Chromatographic equipment. — Insert 2 cm plug of glass
wool in bottom of tube 55 cm long x 2.5 cm od with con-
stricted tip 2,5 cm long and 8 mm od. Provide with brass tamper
of diam. slightly smaller than id of tube, and fittings, including
pressure gage, for applying pressure to top of column.
(b) Spectrophotometer. — Capable of isolating band <5 nm
in region 220-360 nm.
B. Reagents
(a) Silanized C elite.— Weigh ca 700 g Celite 545 into 4 L
beaker, add 3 L HCl (1 + 4), and stir thoroly. Heat on steam
bath several hr, stirring occasionally. Filter slurry thru buchner
under vac. and wash with H 2 until washings are Fe- and Cl-
free. Suck dry, transfer to beaker, and dry ca 15 hr at 135°.
Transfer ca 150 g dried Celite to crystg dish, and let stand in
air 30 min. In well ventilated hood, pour 25 mL GE SC-77
Dri-Film (General Electric Co., 1 River Rd, Schenectady, NY
12305) into bottom of large glass desiccator. Place dish contg
Celite on porcelain support in desiccator, and let stand in closed
desiccator 4 hr. Remove dish, and let stand in hood until re-
sidual HCl dissipates.
(b) Immobile solvent. — Mix equal vols CC1 4 and n-hep-
tane.
364
Cosmetics
AOAC Official Methods of Analysis (1990)
(c) E Luting solns. — In sep. 250 mL vol. flasks, add 1 mL
HCI to 25, 87.5, and 150 mL alcohol and dil. each to vol.
with H 2 (10, 35, and 60% alcohol, resp.).
(d) Hexachlorophene std solns. — (1) Stock soln. — 0.6 mg/
mL. Accurately weigh ca 60 mg hexachlorophene USP and
dil. to vol. in 100 mL vol, flask with acidified 60% alcohol,
(c). (2) Working std solns. — Dil. 5, 10, and 20 mL aliquots
stock soln to 100 mL with acidified 60% alcohol, (c) (0.03,
0.06, and 0.12 mg/mL).
C. Preparation of Sample
(a) For products containing sulfated surface -active agents. —
Accurately weigh, in weighing bottle, ca 1 g sample and trans-
fer quant, to ¥250 mL r-b flask with 75 mL 20% alcohol. Add
10 mL HCI and few boiling chips, attach H 2 0-cooled con-
denser, and reflux 15 min. Cool to room temp, and transfer
to 250 mL separator with ca 25 mL H 2 0. Rinse flask with 30
mL CHC1 3 and transfer to separator. Shake well and drain CHC1 3
into another 250 mL separator. Rinse beaker with two addnl
30 mL portions CHC1 3 , ext aq. soln with each, and combine
CHCI3 exts in 250 mL separator. Wash combined exts with
10 mL H 2 acidified with HCI, and filter thru CHCl r wetted
plug of cotton in powder funnel into 250 mL beaker. Wash
cotton with 20 mL CHC1 3 , and evap. CHC1 3 to ca 10 mL on
steam bath under air current. Complete evapn to dryness at
room temp, under air current.
(b) For products not containing sulfated surface-active
agents. — Accurately weigh, in weighing bottle, ca 1 g sample
and transfer quant, to 250 mL separator with 40 mL warm
H 2 0. Acidify with HCI, and ext with three 30 mL portions
CHCI3. Wash combined CHC1 3 exts with 10 mL H 2 acidified
with HCI, and continue as in (a), beginning, "... and filter
thru CHCI 3 -wetted plug ..."
D. Isolation of Hexachlorophene
Weigh two 12 g portions silane-treated Celite, To 1 portion
in 250 mL beaker, add 7 mL CCl 4 -«~heptane (1 + 1), mix
well, and pack mixt. gently but firmly with tamper into chro-
matgc tube in ca 4 g portions.
To residue from 974.28C(a) or (b), add 7 mL CCl 4 -n-hep-
tane (1 + 1), and stir well to dissolve or disperse residue. Add
the second 12 g portion si lane-treated Celite, stir thoroly, and
pack mixt. into tube as before. Wipe beaker, stirring rod, and
tamper with small piece of glass wool and lightly tamp it on
top of completed column.
Elute column with 100 mL acidified 10% alcohol, then with
200 mL acidified 35% alcohol, and finally with 250 mL acid-
ified 60% alcohol. Maintain flow at ca 2 mL/min with aid of
air pressure and do not permit level of eluting liqs to fall below
glass wool plug. Collect two 100 mL portions and then 50 mL
portions in vol. flasks. Hexachlorophene should elute with
acidified 60% alcohol.
E. Determination
Add 1 drop HCI to each eluate in vol. flasks and obtain
spectra over 220-360 nm in 1 cm cell against corresponding
eluting soln. If necessary, dil. with corresponding eluting soln.
Obtain spectra of working std solns similarly.
From curves, identify eluates contg hexachlorophene. Calc.
hexachlorophene in each eluate by comparing A at 297 nm of
sample with that of std, using straight line background cor-
rection. Add amts in eluates to obtain amt in sample.
Ref.: J AOAC 57, 563(1974).
CAS-70-30-4 (hexachlorophene)
952.16 SVlethenamine in Deodorants
Titrimetric Method
Final Action
A. Reagent
Borax-carbonate soln. — Dissolve 5.0 g Na 2 C0 3 and 4.0 g
Na 2 B 4 O 7 .10H 2 O in 100 mL H 2 0.
B. Determination
Pipet aliquot of sample soln, 938. 1.1 B, contg 150-200 mg
methenamine into 500 mL r-b flask and dil. to 30 mL with
H 2 0. Neutze to litmus with either NaOH soln or dil. H 2 S0 4 ;
then acidify with 1 mL H 2 S0 4 . Connect flask to H 2 0-cooled
condenser and reflux 30 min to hydrolyze methenamine. Dil.
to 175 mL by adding H 2 thru top of condenser, and discon-
nect condenser. Connect flask thru Kjeldahl trap to efficient
straight- wall condenser and distil into 200 mL vol. flask contg
10 mL freshly prepd 10% NaHS0 3 soln. Continue distn until
residual vol. is ca 5 mL, taking care to avoid charring.
Wash down condenser with little H 2 and cool distillate to
room temp. Dil. distillate to vol. with H 2 0, mix well, and let
stand 30 min. Pipet 20 mL aliquot into wide-mouth 250 mL
erlenmeyer, add 3-4 mL starch indicator (mix ca 2 g finely
powd. potato starch with cold H 2 to thin paste; add ca 200
mL boiling H 2 0, stirring const, and immediately discontinue
heating. Add ca 1 mL Hg, shake, and let soln stand over the
Hg), and destroy excess bisulfite with ca \N 1 soln. Carefully
adjust to starch-I end point with 0.5% NaHS0 3 soln and 0.05N
I. Dil. to 50 mL with H 2 0, add 10 mL borax-carbonate soln,
and titr. with 0.05/V I to permanent blue. 1 mL 0.05/V I con-
sumed in alk. titrn = 0.5841 mg methenamine.
Ref.: JA0AC35, 279(1952).
CAS- 100-97-0 (methenamine)
952.17* Phenolsulfonates in Deodorants
Bromination Method
Final Action
Surplus 1970
See 35.025-35.026, 11th ed.
954.12 Phenolsulfonates in Deodorants
Spectrophotometric Method
Final Action
A. Apparatus and Reagents
(a) Spectrophotometer. —See 974.28A(b).
(b) Zinc phenols ulfonate std soln. — 10 mg/L in ca 0. \N
NaOH. Dissolve 100 mg Zn phenolsulfonate, NF XI (equiv.
to 62.67 mg phenolsulfonic acid), in 100 mL H 2 0. Dil. 10
mL aliquot to 100 mL with H 2 0. Pipet 10 mL aliquot into 100
mL vol. flask, add 4 mL freshly prepd 10% NaOH soln, and
dil. to vol. with H 2 0.
B. Determination
(a) In presence of sulfated surface-active agents. — Accu-
rately weigh sample contg 5-10 mg phenolsulfonic acid into
250 mL erlenmeyer. Add 10 mL H 2 and 2 mL HCI, connect
to H 2 0-cooled condenser, and reflux 0.5 hr. Cool to room temp. ,
transfer quant, to 100 mL separator with 20 mL H 2 0, and pro-
ceed as in (b), beginning "... ext with three 30 mL portions
CHCI3."
AOAC Official Methods of Analysis (1990)
Face Powders 365
(b) In absence of sulfated surface-active agents. — Accu-
rately weigh, in weighing bottle, sample contg 5- 10 mg phe-
nolsulfonic acid. Transfer quant, to 100 mL separator with aid
of 30 mL H 2 0. Acidify with HO and ext with three 30 mL
portions CHC1 3 . Discard CHC1 3 exts. Filter aq. soln thru mois-
tened quant, paper into 100 mL vol. flask and dil. to vol. with
H 2 0. Pi pet 10 mL aliquot into 100 mL vol. flask, neutze to
litmus paper with freshly prepd 10% NaOH soln, add 4 mL
excess, and dil. to vol. with FLO. Det. A of sample soln and
A f of std soln at 253 nm in 1 cm cells, using 0.LV NaOH as
blank.
% phenolsulfonic acid = C x A/ [10 A' x (g sample)]
where C — concn phenolsulfonic acid (mg/L) in std soln.
Ret.: JAOAC 37, 798(1954).
951.06
Urea in Deodorants
Titrimetric Method
Final Action
Pipet aliquot of sample soln, 938.11B, contg 50-100 mg
urea into ¥ 100 mL r-b flask. Acidify with HC1, adding 0.5
mL excess. Immerse flask in steam bath and evap. to dryness.
Add 10 g cryst. MgCl 2 .6H 2 and 1 mL HC1, and connect flask
to reflux condenser. Carefully heat mixt. with small flame un-
til MgCl 2 dissolves in its FLO of crystn, and reflux slowly 2
hr so that rate of return of liq. from condenser is 9-14 drops/
min.
Let soln cool, add H 2 thru top of condenser, disconnect
flask, and if necessary, heat to dissolve solids. Transfer soln
to 1 L flat-bottom flask, dil. to ca 400 mL with H 2 0, make
alk. with 10% NaOH soln, and distil ca 275-300 mL into suit-
able portion of 0. IN H 2 S0 4 contg several drops of Me red,
936.15D(a). Titr. excess acid with ca 0. IN NaOH, using more
indicator if necessary. Stdze the 0.1 AT NaOH against the std
0.LV H 2 S0 4 , using Me red as indicator.
Correct for blank by refluxing 10 g cryst. MgCl 2 .6H 2 and
1 mL HO and proceeding as above. 1 mL 0.1 AT H 2 S0 4 -
3.003 mg urea.
Ref.: JAOAC 34, 298, 299(1951).
CAS-57-13-6 (urea)
DEPILATORIES
940.32 Sulfides in Depilatory Powders
Titrimetric Method
Final Action
Pipet 50 mL 0.1/V As 2 3 soln, 939.12B, into 250 mL g-s
vol. flask. Weigh sample contg <0.12 g sulfide calcd as H 2 S
and transfer to flask, washing down any material on sides of
flask with H 2 0. Add 20 mL HC1 (1 + I), stopper immedi-
ately, and shake vigorously until sample decomposes. (If sam-
ple contains CaC0 3 , slowly add the 20 mL acid thru dropping
funnel fitted with rubber stopper to fit flask. Shake gently,
letting liberated C0 2 bubble up thru acid. When reaction sub-
sides, drain remainder of acid into flask, remove funnel, stop-
per flask, and shake vigorously.)
Cool to room temp, and dil. to vol. with H 2 0. Filter thru
dry paper into dry flask. Pipet 100 mL filtrate into 300 mL
erlenmeyer; add 5 mL starch soln, (mix ca 1 g sol. starch with
enough cold FLO to make thin paste, add 100 mL boiling H 2 0,
and boil ca 1 min with stirring), and enough I soln to form
blue soln. Make alk. with NaHC0 3 , adding 1-2 g excess. Titr.
to permanent blue with 0.1/V I, 939.13A. Subtract mL 0. IN 1
consumed in alk. titrn from mL 0. 17V As 2 3 present in aliquot.
1 mL 0.17V As 2 3 = 0.005411 g CaS or 0.01271 g BaS.
Refs.: JAOAC 23, 437(1940); 25, 113(1942); 27, 1 12(1944).
CAS-21 109-95-5 (barium sulfide)
CAS-20548-54-3 (calcium sulfide)
950.85*
FACE POWDERS
Final Action
Surplus 1970
A. Fats and Fatty Acids as Stearic Acid
See 35.032, llthed.
0. Boric Acid
See 35.033, 11th ed.
C. Zinc (Total)
See 35.034-35.035, 11th ed.
D. Caicium (Acid-Soluble)
See 35.036, 11th ed.
E. Magnesium (Acid-Soluble)
Det. Mg in filtrate from acid-sol. Ca as in 973. 54 A. Mg 2 P 2 7
x 0.3622 = MgO.
F. Barium Sulfate
&e 35.038- 35.039, 11th ed.
G. Titanium and Iron (Total)
See 35.040-35.041, llthed.
H. Iron (Total)
See 35.042-35.043, llthed.
/. Titanium (Total)
% total (Ti0 2 + Fe 2 3 ) - % total Fe 2 3 - % total Ti0 2 .
J. Oxides of Iron, Titanium, and Aluminum (Total)
See 35.045, llthed.
K. Aluminum (Total)
% Total A1 2 3 =
% Total (A1 2 3 + Fe 2 3 + Ti0 2 ) - % total (Fe 2 3 + Ti0 2 )
L. Calcium (Acid-Insoluble)
Det. Ca in filtrate from NR^OH ppt, 950.85J*, as in 921.06B,
beginning "... heat to boiling ..."
M. Magnesium (Acid-Insoluble)
Det. Mg in filtrate from acid-insol. Ca as in 920.200. Mg 2 P 2 7
x 0.3622 - MgO.
N. Silica
See 35.049, llthed.
O. Starch
See 35.054, 13th ed.
366
Cosmetics
AOAC Official Methods of Analysis (1990)
HAIR PREPARATIONS
945.70* 2,5-DiaminotoBuene
In Hair Dyes and Rinses
Final Action 1965
Surplus 1970
A. Acetylation Method
See 35.051, 11th ed.
B. Dichlorimide Method
See 35.052-35.053, 11th ed.
942.21* Resorcinol in Hair Lotions
Bromate Titration
Final Action
Surplus 1970
See 35.062-35.063, 11th ed.
945.72* Salicylic Acid in Hair Lotions
Bromate Titration
Final Action
Surplus 1970
945.71 * Paraphenylenediamine
in Hair Dyes and Rinses
Final Action
Surplus 1970
A. Acetylation Method
See 35.054-35.055, 11th ed.
B. Dichlorimide Method (Benzoquinone Method)
See 35.056, 11th ed.
952.18 Potassium Bromate
and Sodium Perborate
in Cold Wave Neutralizers
Qualitative Tests
Final Action
(a) General tests. — KBr0 3 and NaB0 3 are white cryst. salts
sol. in H 2 0. Aq. soln of KBr0 3 is slightly acid; of NaB0 3 ,
slightly alk. In flame test, using Pt wire in slightly darkened
room, KBr0 3 gives reddish violet flame when viewed thru Co
glass; NaB0 3 , typical yellow Na flame. Both compds give fol-
lowing test: Dissolve 0.1 g sample in 10 mL H 2 0, acidify with
HCI, and add 0.5 g KI. Liberation of 1 indicates presence of
oxidizing agent.
(b) Confirmatory test for bromate. — To 1 mL 5% soln of
sample in test tube, slowly add 2 mL H 2 S0 4 with vigorous
shaking. Note odor and color of liberated gas. (Caution.) Cool
test tube, carefully add 2 mL CS 2 , and shake. CS 2 layer be-
comes yellow or red if Br is present.
(c) Confirmatory test for boron. — Moisten 0.2 g sample in
porcelain crucible with 1-2 drops H 2 S0 4 , add 2 mL MeOH,
stir well, and ignite. Green flame indicates presence of B.
Ref.: J AOAC 35, 285(1952).
CAS-7758-01-2 (potassium bromate)
CAS-7632-04-4 (sodium perborate)
923.10* Pyrogalloi in Hair Dyes
Final Action
Surplus 1970
A. Qualitative Test
See 35.058, 1 1th ed.
B. Colorimetric Method
See 35.059-35.061, 11th ed.
See 35.064, 11th ed.
952.19 Thioglycolate Solutions
in Cold Permanent Waves
Final Action
A. Qualitative Test
Dil. 2 mL sample to 10 mL with H 2 0, acidify with 10%
HO Ac, add 5 mL excess, and shake well. Add 2 mL 10%
Cd(OAc) 2 2H 2 soln, and shake. White gelatinous ppt forms
if thioglycolic acid is present. Add excess of NH 4 OH (2 + 3)
and shake. Ppt of Cd thioglycolate will dissolve.
B. Quantitative Method
(Applicable in absence of reducing substances other
than thioglycolates)
Pipet sample aliquot contg 250-300 mg thioglycolic acid
into wide-mouth 250 mL erlenmeyer. Dil. to 50 mL with H 2 0,
add 2-3 drops Me red (dissolve 0.1 g Me red in 50 mL al-
cohol, dil. to 100 mL with H 2 0, and filter if necessary), and
make slightly acid with HCI. Add 3-4 mL starch indicator
(mix ca 2 g finely powd. potato starch with cold H 2 to thin
paste; add ca 200 mL boiling H 2 0, stirring const., and im-
mediately discontinue heating; add ca 1 mL Hg, shake, and
let soln stand over the Hg), and titr. with AN 1 to purple end
point. 1 mL 0AN I = 0.009212 g thioglycolic acid.
Ref.: J AOAC 35, 285(1952).
CAS-68-11-1 (thioglycolic acid)
970.62 Dithiodiglycolic Acid
in Cold Permanent Waves
Titrimetric Method
First Action 1970
Final Action 1971
A. Principle
Soln contg mixt. of thioglycolic (TGA) and dithiodiglycolic
(DTDGA) acids is titrd with std O.l/V 1 soln, which selectively
titrs TGA. DTDGA is reduced to TGA in Jones reductor. Re-
sulting total TGA is titrd; increased TGA represents DTDGA.
B. Reagents
(a) Zinc metal.— 20— 30 mesh. MaJlinckrodt Chemicals
Analytical Reagent grade has been found suitable.
(b) Mercuric salt soln. — 2% aq. soln of Hg(N0 3 ) 2 or HgCl 2 .
AOAC Official Methods of Analysis (1990)
Suntan Preparations 367
C. Apparatus
(a) Jones reductor. — Glass tube, 50-65 cm long, 2 cm id,
with stopcock, preferably Teflon, and delivery tip extending
ca 8 cm below stopcock.
(b) Magnetic stirrer. — With glass- or Teflon- covered stir-
ring bar.
D. Preparation of Jones Reductor
Place 300 g Zn in 800 mL beaker; add 300 mL Hg soln and
2 mL HN0 3 . Stir 10 min with glass rod. Decant supernate and
repeat amalgamation with fresh portion Hg soln and 2 mL
HNO3. Wash amalgamated Zn 3 times, by decantation, with
FLO. (Zn should have silvery luster.) Maintain FLO layer over
Zn thruout.
Fill tube with H 2 0; then slowly add prepd Zn, draining ex-
cess H 2 0. Pass 500 mL H 2 thru column, maintaining H 2
layer over Zn.
Det. suitability of reductor as follows: Pass 20 mL H 2 thru
column, followed by 200 mL H 2 S0 4 (1 + 9), then 100 mL
FLO. Titr. combined washings as in 970.62E(c). If titrn is
>0.2 mL, wash column with addnl H 2 0.
Det. reductor efficiency by treating ca 350 mg DTDGA,
accurately weighed, in 5 mL FLO as in 970.62E(b). Recovery
must be ^97%. Lower recovery indicates unsuitable reductor.
Prepd reductor may be stored 3 months before use, provided
amalgamated Zn is always kept covered with H 2 0. Pre wash
column with 200 mL H 2 S0 4 (1+9) before use after overnight
storage.
E. Determination
(a) Titration J . — Pipet sample aliquot contg 350-400 mg
TGA and DTDGA into 500 mL Phillips flask contg mag. stir-
ring bar; dil. to 100 mL with FLO, add 2 drops Me red in-
dicator (dissolve 0.1 g Me red in 50 mL alcohol, dil. to 100
mL with FLO, and filter if necessary) and just acidify with
H 2 S0 4 (1 + 1). Add starch indicator soln (mix ca 2 g finely
powd. potato starch with cold FLO to thin paste; add ca 200
mL boiling H 2 0, stirring const., and immediately discontinue
heating; add ca 1 mL Hg, shake, and let soln stand over the
Hg) and titr. soln with 0AN I std soln, 939. 13A.
1 mL 0.1/V I - 9.212 mg TGA
(b) Reduction. — Arrange reductor column to deliver into 1
L suction flask contg mag. stirring bar. Connect flask to vac.
outlet which can be regulated to desired flow rate.
Dil. sample aliquot equal to that used in Titration 1 with
200 mL H2SO4 (1+9).
Add 50 mL H 2 S0 4 (1 + 19) to column, apply gentle suction,
and elute at ca 10 mL/min. When liq. reaches top of amal-
gam, immediately add dild sample soln, increase vac, and
elute at ca 17-20 mL/min. When liq. reaches top of amalgam,
rinse sample container with 100 mL FLS0 4 (1 + 19) in several
portions and add to column. Follow with three 100 mL por-
tions FLO at ca 50 mL/min. Release vac, rinsing column tip
into flask with H 2 0.
(c) Titration 2. — Place suction flask contg reduced sample
soln on mag. stirrer; add 5 drops Me red indicator and set
stirrer at medium speed. Add NH 4 OH to yellow indicator color
(ca 70 mL); then add H 2 S0 4 (1 + 1) dropwise just to indicator
red color. Stopper flask and place in ice bath. Cool sample
soln with min. swirling to <25°. Add 5 mL starch indicator,
place on mag. stirrer, and titr. with 0. IN I to purple end point.
Perform sep. blank detns for Titrations 1 and 2 and make
appropriate corrections.
mg DTDGA in sample aliquot - 9.111 (TV - M)
where M — mL 0. \N I from Titration I corrected for blank
and N = ml 0. 17V I from Titration 2 corrected for blank.
Ref.: JAOAC53, 78(1970).
gamma-BHC in Technical BHC, Pesticide Formula-
tions, and Lindane Shampoos and Lotions
See 984.05.
SUNTAN PREPARATIONS
970.63 Amyl p-Dimethylaminobenzoate
in Suntan Preparations
Spectrophotometric Method
First Action 1970
Final Action 1971
A. Apparatus
(a) Spectrophotometer. — Cary Model I 1 (replaced by Models
14 and 17, Varian Instrument Group) recording spectrophtr,
or equiv., with 1 cm quartz cells.
(b) Chromatographic equipment. — (/) Glass chromatgc tube,
55 cm x 22 mm id with glass wool plug in constricted tip.
(2) Brass tamper to fit chromatgc tube. (3) Source of variable
air pressure to regulate column elution rate at ca 2 mL/min.
B. Reagents
(a) Solvent. — (J) Immobile solvent. — n-Heptane-CCJ 4 (1 +
1). (2) Mobile solvents. — Acidified 50% and 60% alcohol, contg
2 mL HC1/500 mL.
(b) Silonized Celite.—See 974.28B(a).
(c) Amyl p-dimethylaminobenzoate . — 0.01 mg/mL. Dil. 10
mg std to 1 L with NH 4 OH-60% alcohol (1 -I- 99).
C. Preparation of Samples
In weighing bottle, weigh sample contg 10-20 mg amyl p-
dimethylaminobenzoate. Transfer quant, to 250 mL separator
with 50 mL H 2 0, slightly acidify with HC1, using test paper,
and ext with four 35 mL portions CHCI3. Combine exts, wash
with 10 mL H 2 0, and filter thru CHC1 3 - washed cotton plug in
powder funnel, into 250 mL beaker. Wash cotton with 10 mL
CHCI3. Evap. to ca 1 mL on steam bath under air jet. Remove
beaker from steam bath and continue evapn under air jet until
all CHCI.3 has evapd. Reserve prepd residue for chromatgy.
D. Chromatography
(a) Column preparation. — Prep, column in 2 layers as in
974. 28C, par. 1 and 2. (Proper prepn of columns may be
checked by eluting known amt of std from column, noting re-
covery.)
(b) Elution. — Elute column first with 100 mL acidified 50%
alcohol at 2 mL/min, then with 350 mL acidified 60% alcohol
at same flow rate. Collect eluates in consecutively numbered
50 mL vol. flasks. Add few drops NH 4 OH to each flask, mix
well, test for alky, and dil. to vol.
E. Determination
Obtain spectra at 220-360 nm for each eluate. For blanks,
use corresponding alcohol solns consisting of either NH 4 OH-
50% alcohol (1 + 99) or NH 4 OH-60% alcohol (1 + 99). Oil.
as necessary with NF^OH-alcohol solns of proper concn. For
quant, detn, obtain spectra of this compd in basic soln, not
acid soln. Calc. amt material in each eluate by comparing A
at 314 nm of sample with std amyl /?-dimethylaminobenzoate
368
Cosmetics
AOAC Official Methods of Analysis (1990)
soln in NH 4 OH-60% alcohol (1 + 99), (c), using straight line
background correction.
Ref.: J AOAC 53, 84(1970).
CAS- 14779-78-3 (amyl p-dimethylaminobenzoate)
942.22
Vanishing Cream
Final Action
A. Test for Type of Emulsion
Dust small amts of finely ground oil-sol. and H 2 0-sol. dyes
on sep. portions of sample. If color of oil-sol. dye spreads
rapidly, H 2 0-in-oil emulsion is indicated; if color of H 2 0-sol.
dye spreads, oil-in-H 2 emulsion is indicated.
B. Water
Transfer 5-20 g sample to erlenmeyer; add 50 mL toluene,
few glass beads, and ca 2 g lump rosin. Connect flask to Dean
and Stark distg tube receiver, and distil until no more H 2
collects in receiver. Cool, read vol. H 2 under the toluene at
room temp., and from this vol., calc. % H 2 0.
C. Ash
Place 2—1 g sample in flat-bottom Pt dish, and remove H 2
and volatile material by placing dish on steam bath or in 100°
oven. Ignite sample at low temp, and finally at 600° to const
wt.
D. Chloroform-Soluble Material
Place 2-10 g sample in separator, add 25-50 mL H 2 0, acid-
ify slightly with H 2 S0 4 (1 + 9), and ext with successive por-
tions CHCI 3 , collecting all exts in second separator. (Usually
4-5 portions CHCI3, each ca 35 mL, are enough to remove
all CHCI3-S0I. material.) Wash combined CHC1 3 exts with 10
mL H 2 0, filter thru cotton plug placed in separator stem, and
collect filtrate in weighed dish. Shake aq. washing with small
vol. of CHCI3, and filter this CHC1 3 into dish. Evap. CHC1 3
on steam bath and dry residue for 15 min intervals at 100° to
const wt.
Glycerol
-0.02 M. Dissolve 4.6 g
to ca 900 mL with H 2 0,
E. Reagents
(a) Potassium periodate soln.—
KIO4 in ca 500 mL hot H 2 0. Dil
cool to room temp., and dil. to I L.
(b) Sodium hydroxide std soln. — 0.027V. See 936.16.
(c) Bromocresol purple indicator. — Dissolve 0.1 g bromo-
cresol purple in 100 mL alcohol.
(d) Propylene glycol. — Bp 85-86°/ 10 mm.
(e) Arsenious oxide soln. — 0.02/V. Dil. 100 mL 0.17V As 2 3 ,
939. 12B, to 500 mL with H 2 0.
F. Isolation and Oxidation of Glycerol
(a) Isolation of glycerol. — Place 2—10 g sample in sepa-
rator, add 25-50*rnL H 2 0, acidify slightly with H 2 S0 4 (10 g/
100 mL), and ext with successive portions CHC1 3 . (Usually
4-5 portions, each ca 35 mL, remove all CHCJ 3 -sol. material.)
Wash combined CHC1 3 exts with 10 mL H 2 0. Filter aq. soln
and wash H 2 thru cotton plug to remove droplets of CHC1 3 ,
and collect filtrate in 250 mL vol. flask. Add 3 drops bromo-
cresol purple indicator to filtrate and neutze with C0 2 -free al-
kali (0. IN NaOH is satisfactory), making final adjustment with
0.027V NaOH. Dil, almost to vol. with H 2 0, and if necessary,
add more alkali to keep soln light but definite purple; then
complete diln to vol. and mix.
(b) Periodate oxidation. — Transfer aliquot neut. soln, pref-
erably contg 30-40 mg glycerol, to 100 mL vol. flask, and
add 50 mL KIO4 soln. Dil. to vol. with H 2 and let stand ca
1 hr. Test for excess periodate, which must be present in oxidn
mixt., by adding NaHC0 3 and KI to test portion. If excess is
present, I is liberated.
G. Determination
(a) By titration of formic acid. — (Applicable in absence of
substances yielding acid on periodate oxidn.) Transfer 50 mL
aliquot of oxidized mixt. to titrn flask, add 10 drops propylene
glycol (ca 0.5 mL), mix well, wash down sides of flask with
H 2 0, and let stand 10 min. Add 3 drops bromocresol purple
indicator and titr. with NaOH soln to light purple end point.
1 mL 0.027V NaOH = 1.842 mg glycerol.
(b) From periodate consumed. — Transfer 20 mL aliquot of
oxidized mixt., 942.22F(b), to titrn flask and dil. with ca 50
mL H 2 0. Add ca 1.0 g NaHCO.,, 0.5 g KI, and 5 mL starch
indicator (mix ca 1 g sol. starch with enough cold H 2 to make
thin paste, add 100 mL boiling H 2 0, and boil ca 1 min with
stirring). Titr. immediately with As 2 3 soln to disappearance
of blue. Stdze 10 mL KI0 4 similarly. Difference between the
2 titrns represents amt of periodate reduced in 20 mL aliquot
taken. To obtain amt of periodate reduced in original aliquot
obtained from 250 mL flask, multiply above difference by 5.
1 mL 0.027V As 2 3 = 0.4605 mg glycerol
Refs.: JAOAC 25, 903(1942); 26, 249(1943); 27, 462(1944);
30, 507, 651(1947); 31, 580(1948): 33, 362, 367(1950).
CAS-56-81-5 (glycerol)
SPECIAL REFERENCE
"Newburger's Manual of Cosmetic Analysis," 2nd ed., AOAC,
Arlington, VA 22201 (1977).
16. Extraneous Materials: Isolation
Jack L Boese and Roth Bandler, Associate Chapter Editors
Food and Drug Administration
GENERAL
945.75 Extraneous Materials
(Foreign Matter) in Products
isolation Techniques
A. Definition of Terms
Extraneous materials. — Any foreign matter in product as-
sociated with objectionable conditions or practices in produc-
tion, storage, or distribution; included are filth (see 970.66A(a)-
(d)), decomposed material (decayed tissues due to parasitic or
nonparasitic causes), and miscellaneous matter such as sand
and soil, glass, rust, or other foreign substances. Excluded are
bacterial counts.
B. Apparatus
(Avoid use of polyethylene beakers, funnels, containers, etc.,
as insect fragments and rodent hairs adhere to app. made from
this material.)
(a) Aerator, water, — For attachment to faucet to provide
smooth-flowing, aerated H 2 stream. Remove lower screen.
(Available from Faucet-Queens, Inc., 550 Palwaukee Dr,
Wheeling, IL 60090, No. 00200.)
(b) Autoclave. — (/) Slow exhaust type. — Set "slow exh" to
lower pressure from 15 to in 15-20 min. (2) Non-slow ex-
haust. — Let cool to psi before opening or venting.
(c) Blenders. — {]) High-speed. — Use 1 L, 4-lobe jar fitted
with 4-blade assembly, 2 blades tilted upward ca 30° with diam.
60 mm and 2 blades tilted downward ca 25° with diam. 55
mm. Operate at speed specified in method, using variable
transformer. Use tachometer to measure speed of blending jar
blades or blade shaft, after removing blades. (2) High-speed
overhead. — Alternative to high-speed blender: Mixer with 6
canted, sharp-edge stainless steel blades rotating on shaft of
suspended motor and speed control. Blades rotate at bottom
of stainless steel cup having 4 indentations, forming lobes.
Sorvall Omni-Mixer (DuPont Instrument Co., Sorva.1.1 Opera-
tions, Peck's Ln, Newtown, CT 06470), or equiv., meets these
requirements.
(d) Bolting cloth. — Silk cloth woven to std size opening and
thickness which is used in flour mills. Number of silk specifies
number of mesh/linear in. "X," "XX," or "XXX" after num-
ber refers to thickness of thread from which cloth is woven;
this also affects size of opening in cloth. Therefore, follow
designation exactly as to both number and "X" of bolting cloth.
(Available from Tetko, Inc., 420 Saw Mill Rd, Elmsford, NY
10523.)
Prep, disks by boiling large squares of silk before cutting
them into circles. Circles cut from unboiled silk shrink and
become misshapen. Make rulings ca 5-7 mm apart with India
ink or other permanent marking material, using fine pen, on
boiled and pressed cloth marked off in circles ca 85 mm diam.
When needed, dye ruled cloth by placing in hot (80-85°)
soln of 50 mg FD&C Blue No. I in 1 L H 2 contg 2.5 mL
HOAc, and holding at this temp, ca 15 min with frequent stir-
ring. Rinse well and store in dark.
(e) Butter stirrer. — See Fig. 945. 75 A.
(f) Centrifuge. — International type EXD centrf. (Interna-
tional Equipment Co.) with 8- place No. 240 head, No. 320
shield, No. 325 trunion ring, and No. 571 cushion, or other
centrf. giving equiv. max. relative centrifugal force. The fol-
lowing formula may be used to det. equiv. centrf.: N l "r 1 —
N 2 2 r 2 , where N { = 2200 rpm and r, = 19.6 cm (distance from
center of centrf. head to bottom of horizontal centrf. tube).
(g) Cyclone. — Laboratory cyclone or pulper consists of cy-
lindrical perforated metal screen in which revolves paddle which
forces soft material from food product out thru openings in
screen. Tough materials such as seeds, skins, and stems are
moved along and out opening in end of cylinder. Use as power
source } / 4 horsepower, 110 v, 1725 rpm elec. motor. Screen
is 22 gage material, 400 holes/sq in., each 0.027" diam. Screen
is 2.5" id and length of effective screen is 3". Paddle has 2
fins, each 25 /3 2 " wide, set alternately and extending l'Vie" from
center of shaft. Pulper is fed thru hopper which leads into ba-
sin 3.5" long and 2.5" id. Portion of paddle with fins inserted
at 30° angle forces material from basin into screening com-
partment. Cyclone is so constructed that waste opening may
be closed, as needed. Sieved material is caught in shield and
delivered thru spout to container. Machine may be readily dis-
assembled for washing. (Blueprints available from Div. of Mi-
crobiology (HFF-237), Food and Drug Administration, 200 C
St, SW, Washington, DC 20204.)
(h) Extraction vessels. — (I) Kilborn funnel . — 1 L, 3.5" od
by 9.5" high, 8 mm opening at tip. Rubber tubing 3 /s" id and
pinch clamp provides convenient cut-off.
(2) Percolator. — 2 L, Corning Glass Works No. 7040, or
equiv., conforming to following general size and shape: 115
mm id x 400 mm long, ca 90 mm id at 200 mm down from
top, with 8-9 mm bore tip, with cut-off as in (/). Use stirring
rod 370 x 10 mm diam, when specified, to prevent com-
pacting of sample in drain opening.
(3) Percolator with oversize bore tip. — Use std percolator
as in (2) but replace std bore tip (8-9 mm id) with 17-18 mm
id bore tip and appropriate size rubber tubing and pinch clamp.
Use stirring rod described in (2).
(4) Trap flask. — Wildman. — Consists of 1 or 2 L erlen-
meyer into which is inserted close-fitting rubber stopper or wafer
stopper (Entomological Supply Co., Inc., 2411 S Harbor City
Blvd. Melbourne, FL 32901) supported on stiff metal rod 5
mm ( 3 /]e") diam. and ca 10 cm longer than ht of flask. (Rod
of greater diam. is not desirable because of its greater dis-
placement of liq.) Rod is threaded (#10-32) at lower end and
furnished with nuts and washers to hold it in place on stopper.
Countersink lower nut and washer in the rubber to prevent
striking flask. See 970.668(b) and Fig. 945.75B.
(i) Filter paper. — Use smooth, high wet-strength, rapid-
acting filter paper ruled with oil-, alcohol-, and water-proof
lines 5 mm apart. S&S No. 8 is satisfactory.
(j) Filter paper defatting cup. — Center S&S 588 folded fil-
ter paper, or equiv., over bottom of smaller beaker specified
in method. Partially shape paper over bottom of beaker and
gently insert beaker and paper into larger specified beaker. Re-
move smaller beaker and transfer weighed sample into formed
paper cup.
369
370
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
■m
lO 1/2 "
DIAto
' 4 f/2 M DIAM.
3 /Q "DIAM.
FIG. 945.75A— Mechanical butter stirrer
(k) Funnels for filtration with suction. — Use funnels with
filter papers or bolting cloth cupped up on sides to eliminate
loss of solids. Use rapid filter paper for filtration thru Hirsch
funnel .
Use of wire screen or bolting cloth between perforated fun-
nel plate and filter paper accelerates filtration and gives more
uniform distribution of solids.
(I) Illuminators for widefield stereoscopic microscopes. —
Filth examination. — Illuminator for this purpose should have:
Metal Rod
Oily Layer
Nut
Stopper
Nut
Recess
Erlenmeyer
Flask
FIG. 945.75B— Wildman trap flask
compactness and flexibility; transformer or resistor to vary light
intensity; focusing adjustment to give uniformly lighted field
of view; blue- white color from cool low-voltage source.
(m) Howard mold-counting apparatus. — (/) Howard mold-
counting slide. — Glass slide of one-piece construction with flat
plane circle ca 19 mm diam. or rectangle 20 x 15 mm sur-
rounded by moat and flanked on each side by shoulders 0.1
mm higher than plane surface. Cover glass is supported on
shoulders and leaves depth of 0.1 mm between underside of
cover glass and plane surface. Central plane, shoulders, and
cover glass have optically worked surfaces. To facilitate cal-
ibration of microscope, newer slides are engraved with circle
1.382 mm diam. or with 2 fine parallel lines 1.382 mm apart.
(2) Reticle (accessory disk) for Huygenian eyepiece. — Glass
disk that fits into microscope eyepiece, ruled into squares each
side of which is equal to Ve of diam. of field. Since limiting
diaphragm is eyepiece field stop, rulings equal Vo of this dia-
phragm opening. Field viewed on slide with mold-counting
microscope has diam. of 1 .382 mm at magnification of 90-
125x. Reticles (accessory disks) available for widefield ocu-
lars may have field of view > 1.382 mm in diam. Center por-
tion of field of view is used for mold counting and is delin-
eated by inscribed circle having 1.382 mm specimen field diam.
Inscribed circle is ruled into squares each having side equal to
Ve diam. of circle.
(n) Magnetic stirring bar and stirrer -hot plate. — Teflon-
covered bars ca 47 mm long X 9 mm od; use with hot plate
having independent, continuously variable heat and speed con-
trols. See also 970.66B(c)
(o) Microscopes. — (/) Compound microscope. — For mold
counting and other filth and decomposition work, microscope
should have following min. specifications: binocular body with
inclined oculars; 4 parfocal achromatic objectives of ca 4, 10,
20, and 40X; revolving 4-piace nosepiece; Abbe condenser
with N.A. of 1.25; lOx Huygenian or widefield eyepieces;
fine adjustment; mech. stage.
(2) Widefield. stereoscopic microscope recommended for filth
examination. — Microscope should have following min. spec-
ifications: binocular body with inclined oculars; sliding or re-
volving nosepiece to accommodate 3 objectives; 3 parfocal ob-
jectives 1.x, 3x, and 6 or 7.5 X; paired 10 x and paired 15 x
widefield oculars; mounted on base and capable of illumina-
tion by transmitted or reflected light. 30 X is ordinarily used
for routine examination of filter papers. Verification at higher
magnification may be required.
(p) Pipet for tissue transfer. — Use 1 mL measuring pipet
with bore 3.0 ± 0.5 mm and tip cut off at 1.0 mL mark. In
pipetting, draw material slightly above 0.5 mL mark and let
it drop slowly to mark.
(q) Rot fragment counting plate and cover preparation. —
Glass plate; 55 x 100 mm, .1 .5-4.0 mm thick with cover 50
x 85 mm, ca 1.5 mm thick. Carefully paint on coat of resist
over the entire surface, avoiding pinholes. Asphaltum varnish
makes excellent resist; paraffin wax may also be used. Care-
fully scribe crosswise parallel lines, 4.5 mm apart with 15 mm
space at each end, thru resist. If asphaltum varnish is used,
lines may be scribed with new steel-wheel glass cutter.
Place coated scribed slides face down over HF in polyeth-
ylene container. Det. proper acid fume exposure by trial and
error. Following etching, remove resist by placing slide in H 2
contg detergent, if resist is not easily scrubbed off, use toluene
for cleanup.
Alternatively, use clear plastic plate; 55 X 100 mm, 4-6
mm thick with glass cover 50 x 85 mm, ca 2 mm thick. With
sharp needle, carefully scribe crosswise parallel lines, 4.5 mm
apart with 15 mm spaces at each end. Several slides can be
made at one time by using strip of plastic 100 mm wide and
AOAC Official Methods of Analysis (1 990)
General Techniques
371
any multiple of 55 mm long, allowing extra length to com-
pensate for each cut of 2-3 mm thickness.
Fasten V2 of square cover slip, ca 22 mm on side and ca
0.25 mm thick, at each end of counting plate to raise cover
plate above ruled plate. See Fig. 945. 75C. Glass slides are
available on special order from Ace Glass, Inc.
(r) Sieves. — See "Definitions of Terms and Explanatory
Notes. Sieves of No. 100 or finer should be "plain (not twill)
weave" of stainless steel. Plain weave is woven with one wire
alternately over and under next.
(s) Thin layer chromatographic apparatus. —
(1 ) Desaga/Brinkmann standard model applicator, or equiv.
(2) Desaga/Brinkmann standard mounting board, or equiv.
(3) Desaga/Brinkmann drying rack, or equiv. — Accom-
modates ten 8 x 8" plates.
(4 ) Desaga/Brinkmann model 51 stainless steel desiccating
cabinet, or equiv.
(5) Window glass. — 8 x 8", double strength window glass
plates of uniform width and thickness; smooth off corners and
edges with file or other tool.
(6) Chromatographic tank and accessories . — Metal instead
of glass troughs.
(7) Dipping tank and accessories. — Stainless steel, 8V2 X
8V2 x V4— 3 /ig" inside width with metal supports and close-
fitting U -shaped cover ca 9 X ] / 2 ". Capacity ca 300 mL.
(8 ) Spotting p ip ets . — 1 |ulL .
(9) Spray bottle.— % oz. (Thomas Scientific No. 2753-J10
or Lurex Scientific, Inc., No. 131-0514, 250 mL).
(10) Chromatography spray flask. — 250 mL (Microchem-
ical Specialties Co., 1825 Eastshore Hwy, Berkeley, CA 94710,
No. S-4530-D).
(77) Tank liner.— Cut 2 pieces, \2 l / 4 X 8 3 / 4 ", from desk
blotter, white or colored, and bend into L-shape to fit tank.
(72) Strong ultraviolet light source. — Such as germicidal
lamps (General Electric Co., Nela Park, Cleveland, OH 44112),
either (1) two 30 watt, 36" tubes, No. G30T8, mounted in std
30 watt reflector fixture ca 20 cm above papers; or (2) two 15
watt, 18" tubes, No. G15T8, mounted in std 15 watt desk lamp
fixture placed ca 10 cm above papers. Shield to protect eyes
and skin at all times.
C. Reagents
(a) Acid-alcohol soln.—UC\ and 60% alcohol (1 + 9) or
HC1 and 40% isopropanol (1 + 9).
(b) Alcohol. — 95% com. ethanol (not denatured) unless
otherwise specified. Make all dilns by vol.
(c) 60% Alcohol-calcium chloride so In. — To each 3 L 60%
alcohol (amt for 1. analysis), add 200 g anhyd. CaCl 2 . Stir until
salt dissolves. Cloudiness from traces of CaC0 3 will clear up
during analysis when soln is acidified.
(d) Allantoin std soln. — Prep. aq. soln contg 2 mg/mL. Pi-
pet 1 mL aliquot into 10 mL vol. flask and dil. to vol. with
acetone. Stable ca 3 months.
(e) Antifoam soln. — 1 g Dow Corning Antifoam A compd
dild with 20 mL EtOAc. Use supernate and keep tightly closed.
(f ) Carob bean soln. — Blend 0.75% carob bean gum in H 2 0.
Boil 2 min and cool to 20-25°. Add 2 mL HCHO/100 mL
and stir gently. Let settle and use clear supernate.
(g) Cellulose powder. — TLC grade, MN 300 (Brinkmann
Instruments, Inc., Cat. No. 66 00 100-8).
(h) Crystal violet soln. — Dissolve 10 g dye (Colour Index
42555) in 100 mL alcohol and filter.
(i) Detergent soln. — Prep. aq. Na lauryl sulfate soln as re-
quired.
(j) Emulsifiers. — Nonionic, H 2 0-sol. surfactants. (l)Non-
ylphenoxypoly(ethyleneoxy)ethanol. — Igepal CO-730 (GAF
Chemicals Corp., 1361 Alps Rd, Wayne, NJ 07470). (2) Di-
alkylphenoxypoly(ethyleneoxy)ethanol . — Igepal DM -7 1 (GAF
Corp.). (3) Nonylphenoxypoly(ethyleneoxy)ethanol. — Igepal
CO-630 (GAF Corp.).
(k) Flotation liquid. — Mineral oil, (p), and heptane, (1),
(85 + 15).
(1) Heptane. — Com. /i-heptane contg <8% toluene.
(m) Indoxyl sulfate {urinary indican) std soln. — Approx.
0.1 mg/mL. Available from Sigma Chemical Co. Stored in
light-resistant container in refrigerator, soln is stable ca 1 month.
(n) Isopropanol saturated with heptane. — To 600 mL iso-
propanol add 45 mL heptane and 430 mL H 2 0, mix, and let
stand overnight. Siphon from below interface.
(o) Kerosene, deodorized. — Sargent- Welch, Cat. No.
C12978, or equiv.
(p) Mineral oil. — Paraffin oil, white, light, 125/135 Say-
bolt Universal Viscosity (38°), sp gr 0.840-0.860 (24°). Re-
quest supplier to select lot meeting specifications and provide
certificate of analysis.
(q) Pancreatin soln. — Use USP or sol. pancreatin kept re-
frigerated at 10°. Use fresh soln. Mix at rate of 5 g/100 mL
H 2 at <40°. Use special soln for cheese, 10 g/100 mL. Stir
with malted milk unit or blender 10 min, or let stand 30 min
with frequent shaking. Centrf. at 1500 rpm and filter supernate
thru S&S No. 8 paper, or equiv. Alternatively, filter thru cot-
ton pads 10-13 cm thick and then thru rapid No. 8 paper in
Hirsch funnel with suction.
(r) p-Dimethylaminobenzaldehyde (pDMAB) spray. — Mix
30 mL MeOH and 1.0 mL HC1 and let cool. Dissolve with
stirring 0.5 g pDMAB in 25 mL cooled mixt. Stable ca 1 month.
(Caution: Spray reagent is toxic and corrosive. See safety notes
on spraying chromatograms.)
(s) Sodium acetate spray. — Satd aq. soln.
(t) Sodium oleate. — Tech. grade.
COVER PLATE
COVER SLIP
FIG. 945.75C— Rot fragment counting slide
372
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
(u) Sodium phosphate soln. — Tech. grade Na 3 P0 4 . Prep.
5% soln.
(v) Stabilizer solns. — 0.5% Na carboxymethylcellulose
preferred (Hercules Inc., Cellulose and Protein Products Dept,
1313 Market St, Wilmington, DE 19899). Place 500 mL boil-
ing H 2 in high-speed blender. With blender running, add 2,5
g Cellulose Gum and 10 mL ca 37% HCHO soln w/w, and
blend ca 1 min. Alternatives: 3-5% pectin or 1% algin. Add
required amt of stabilizer directly to H 2 while agitating in
high-speed blender. Treat soln with vac. or heat to remove air
bubbles. Add 2 mL HCHO soln/ 100 mL soln as preservative.
(If blender is not available, mix dry stabilizer with alcohol to
facilitate incorporation with H 2 0.) Adjust to pH 7.0-7.5. Fil-
ter soln thru 8 |xm membrane filter (Millipore No. SCWP-047-
00, or equiv.) using suitable vac. filtration app. (Millipore No.
XX15-047-00, or equiv.).
(w) Tween 80-60% alcohol soln. — To 40 mL polysorbate
80 (ICI Americas, Inc.), add 210 mL 60% alcohol, mix, and
filter. (Proportionate vols may be prepd.)
(x) Tween 80-40% isopropanol soln. — To 40 mL polysor-
bate 80 (ICI Americas, Inc.), add 210 mL 40% isopropanol,
mix, and filter. (Proportionate vols may be prepared.)
(y) Tetrasodium EDTA— alcohol soln. — Dissolve 5 g
Na 4 EDTA in 100 mL H 2 0, add 150 mL alcohol, mix, and
filter. (Proportionate vols may be prepd.)
(z) Tetrasodium EDTA-40% isopropanol soln. — Dissolve
5 g Na 4 EDTA in 150 mL H 2 0, add 100 mL isopropanol, mix,
and filter. (Proportionate vols may be prepd.)
(aa) Urea std soln. — 20 mg/mL H 2 0. Stable ca 3 months.
(bb) Wetting agents.- — (/) Tergitol Anionic 7. — Na hep-
tadecyl sulfate (Sigma Chemical Co.). (2) Triton X-IJ4.—AI-
kylaryl polyether alcohol (Rohm & Haas Co.).
970.66
Light and Heavy Filth
General
A. Definition of Terms
(a) Filth. — Any objectionable matter contributed by animal
contamination of product such as rodent, insect, or bird matter;
or any other objectionable matter contributed by insanitary
conditions.
(b) Heavy filth. — Heavier filth material sepd from product
by sedimentation based on different densities of filth, food par-
ticles, and immersion liqs such as CHC1 3 , CCI 4 , etc. Examples
of such filth are insect and rodent excreta pellets and pellet
fragments, sand, and soil.
(c) Light filth.— Lighter filth particles that are oleophilic and
are sepd from product by floating them in an oil-aq. liq. mixt.
Examples are insect fragments, whole insects, rodent hairs,
and feather barbules.
(d) Sieved filth. — Filth particles of specific size ranges sepd
quant, from product by use of selected sieve mesh sizes.
B. Special Technics
(a) Wet sieving technique. — Use clean sieve of correct diam.
(8" min.), mesh type (plain not twill weave) and mesh number
(100, 140, 230, etc). Hold sieve under aerator, 945.75B(a),
spray of specified temp. H 2 at approx. 30° angle. Pour well-
mixed sample, portionwise (not so much that clogging or ex-
cessive foaming results), onto sieve so that moderate pressure
spray of H 2 contacts material on sieve. Increase H 2 pressure
to achieve max. spray action on sieve, but not so violent that
sample froths over lip of sieve. Keep sample material washed
to lower inside edge of sieve (while held at 30° angle) and
direct H 2 spray onto sample material until majority of de-
tergent foaming subsides and thru H 2 is essentially clear. Re-
peat portionwise addn of sample and wash sample container
thoroly on final addn. Continue washing material on sieve un-
til all detergent foaming subsides and thru H 2 is clear. Quant,
transfer sieve retainings as specified in method. Clean sieve
inside walls using rubber policeman and direct H 2 spray on
screen, held at angle, to collect all sample residues at lower
edge of sieve. Repeat side wall and screen washing, as nec-
essary, to ensure quant, transfer of sieve retainings.
(b) Operation of Wildman trap flask. — Unless otherwise
directed in specific method, cool mixt. in flask to room temp.
Bring vol. of liq. to ca 900 mL in 2 L flask and to ca 600 mL
in 1 L flask. Add vol. of flotation liq. as stated in method by
pouring down stirring rod. Stir mag., 970.66B(c). Add enough
liq. to bring flotation liq. well into neck of flask. (Note: Deaer-
ate all flotation liqs before use.)
Unless otherwise stated, let mixt. stand 30 min, intermit-
tently stirring bottom layer every 3-6 min during first 20 min
of standing. Spin stopper (wafer) to remove sediment and trap
off by raising stopper (wafer) as far as possible into neck of
flask, being sure that oil layer and >1 cm of liq. below in-
terface are above stopper (wafer). Hold stopper (wafer) in place
and pour off liq. into beaker. Rinse out material on rod and
in neck of flask with liq. extn medium in which floating was
performed and add to beaker.
Do not wash out neck of flask with 95% alcohol or other
liq. which may interfere with surface relationships of the 2
phases; this will cause loss in recovery in subsequent trap-
pings.
Filter trapped material and rinsings with suction thru rapid
paper in Hirsch funnel. Add flotation liq. as specified to trap
flask and stir vigorously. Add enough liq. extn medium to bring
flotation liq. into neck of flask. Trap off again, rinse, and filter
as above.
(c) Operation of magnetic stirrer. — To disperse flotation
liq. thru sample, dil. liq. extn medium to vol. specified in
method and bring to proper temp. Add mag, stirring bar,
945.75B(n), and proper vol. of flotation liq. Slowly bring unit
to max. speed that does not produce visible or audible splash-
ing (central portion of stirring bar is usually just visible at bot-
tom of vortex) and stir for time stated in method. Time stirring
interval after achieving proper speed and vortex.
(d) Filtration technic. — (Treatment of trapped-off mate-
rial.) If material trapped off in beaker contains appreciable
starchy debris, add enough HC1 to make soln 1-2% of HCI
(1 + 99 - 49), bring to boil, and filter while hot. If fats or
colloidal material retard filtration, hasten by playing stream of
hot H 2 over paper during filtration.
(e) Clearing of plant materials. — With sedimentation or
flotation procedures, some food material may be trapped off
with filth particles. By proper clearing, filth may be made to
stand out in contrast with white background of filter paper by
one of following technics:
(J) For heavy filth, moisten paper with H 2 or 50% alco-
hol. (This method does not clear material completely, but it
leaves rodent pellets and other filth soft and pliable.)
(2) For light filth examination, wet paper with glycerol -al-
cohol (1 + 1) immediately after filtering. Place enough liq.
on paper to fill fibers but not enough to cause flowing of extd
materials. This clearing agent does not harden filth material
on paper, as do many oils which might be used as clearing
agents.
(3) Clove oil can be used for clearing plant materials. This
oil has high refractive index and clears more completely than
does alcohol-glycerol soln.
(f ) Illumination for the widefield stereoscopic micro-
scope. — (/) By direct light. — Focus and adjust light to strike
AOAC Official Methods of Analysis (1990)
Beverages
373
paper from above at ca 70° angle from horizontal. Light may
come from right or left.
(g) Microscopic examination of filter papers. — Make ex-
amination at30x (unless otherwise specified), using widefield
stereoscopic microscope, on properly cleared paper on opaque
white background. Continually tease and probe particles while
observing thru microscope. Turn over all large pieces of ma-
terial, such as bran, which might obscure filth elements. Ex-
amine all doubtful pieces of material at 60-75 X . At least twice
magnification used in original examination is necessary to show
new details not observable at lower power. If doubt still re-
mains, mount piece, clear thoroly, and examine under compd
microscope. Thoro knowledge of appearance of authentic ma-
terials is assumed.
(h) Counting insect and other animal filth. — Diagnostic
characteristics of insect fragments: Count as of insect origin
any fragment showing one or more of the following characters:
(/) characteristic shape of whole or portion of specific appen-
dage or body part; (2) articulation point (various types of joints);
(3) one or more body hairs or setae; (4) one or more setal
scars; (5) surface pattern (sculpturing) characteristic of a spe-
cific insect; (6) one or more sutures present (various types sep-
arating body plates or sclerites). Diagnostic characteristics of
animal hairs: See Vazquez, A. W., Structure and Identifica-
tion of Common Food-Contaminating Hairs, J AOAC 44,
754(1961).
(i) Format for reporting filth. — Container: Describe size,
type, and closure(s) of immediate container and note condition
if not intact. Product: Common name, if identity is known,
or simple description. Code(s): Manufacturer's or distributor's
name and identification marks. Method(s): Cite AOAC para-
graph number(s) and note any modifications made. Amount
examined: Number subsamples analyzed and amount per sub.
If amount is variable, report for each sub under Findings.
Findings — Report findings on analyst's worksheet by subsam-
ple number. Use only categories that apply and report any filth
element that is found under no more than one category. Within
categories, group filth elements by identity, when known, and
then by size or other appropriate descriptive feature. If amt
filth present makes exact count impractical, report either ap-
proximate or minimal figure rather than term "too numerous
to count." Summarize sample results by category totals and
averages. Note whether or not sample was fumigated before
shipment or on receipt at laboratory if there are whole insects,
mites, or other arthropods.
(1) No. whole insects or equivs (i.e., sep. heads or body
portions with head attached). Distinguish whole insects and
equivalents in subtotals. Give identity, stage of life cycle, and
size (mm). State whether whole insects are alive or dead.
(2) No. insect cast skins. Give identity (if known), size (mm),
and state whether nymphal, larval, or pupal. Distinguish whole
cast skins (with head portion) and cast skin fragments.
(3) No. insect eggs. Give identity, if known.
(4) No. insect fragments, other than sep. setae. Give iden-
tity (if known), dimensions or size range (mm), and name of
part. State whether identified fragments are from adult or im-
mature insects.
(5) No. setae (if fly, state).
(6) Insect excreta. Report wt (mg) and/or count of excreta
pellets with dimensions or size range (mm). Give identity, if
known.
(7) Insect penetration of container. Report number, size
(mm), and direction. Note container integrity and complete-
ness of closures and seams.
(8) No. mites. Give identity, if known. State whether alive
or dead. Report mite fragments here as subcategory.
(9) No. arthropods other than insects and mites. State whether
alive or dead. Give identity (e.g., spiders, pseudoscorpions).
Report fragments here as subcategory.
(10) No. rat or mouse fecal pellets (state which or give length
and diameter in mm). Give wt (mg) if from condimental seeds,
spices, cocoa beans, coffee, or grains.
(11) No. rat or mouse fecal pellet fragments. Give basis for
identification. Give dimensions or size range (mm). Give wt
(mg) if from condimental seeds, spices, cocoa beans, coffee,
or grains.
(12) Other mammalian feces. Report size (mm) and wt (mg).
Give identity (e.g., cat, cow), if known, and basis for iden-
tification.
(13) No. rat or mouse hairs or hair fragments. Report length
(mm) of each hair and hair fragment or, if numerous, group
into categories of limited size range.
(14) No. other hairs and hair fragments. Report length (mm),
grouping as in (13). If unidentified, state whether striated or
nonstriated.
(75) No. feathers, feather fragments, and barbules. Give di-
mensions or size range (mm) of feathers and fragments.
(76) Urine on container or food (state which). Report odor
of urine, if detected. Give number and dimensions or size range
(in.) of stains and note if penetration is to product. Report
component(s) detected by AOAC test.
(17) Bird excreta on container or food beneath (state which).
Report amount as wt (mg) or no. and dimensions of droppings,
as appropriate.
(18) Other extraneous materials (describe and report each
type by appropriate quantitative figure).
BEVERAGES AND BEVERAGE MATERIALS
965.38 Filth in Cocoa, Chocolate,
and Press Cake
Flotation Method
First Action 1965
Final Action 1988
A. Apparatus and Reagent
(a) Hirsch porcelain funnel with plug. — Size 0, fitted with
fixed perforated filtering plate. Diam. at top, 94 mm; diam.
of plate, 56 mm. Fit stem end of funnel with rubber tubing ca
10 cm (4") long which can be plugged with plastic or cork
stopper.
(b) Sodium hypochlorite soln. — Approx. 0.25%. Oil. 5 mL
com. NaOCl soln, 5.25% by wt, with 95 mL H 2 0. Prep, fresh
daily.
B. Determination
(a) Cocoa.— Mix 50 g cocoa into 500 mL hot (55-70°) 2%
detergent soln, 945.75C(i), or 500 mL hot 2% IgepaJ CO-630,
945.75C(j)(J). Pour portionwise onto No. 230 sieve,
945.75B(r), and wash with forcible stream of 55-70° tap H 2 0,
using aerator, 945.75B(a). Remove fat by tilting sieve ca 20°
and play H 2 thru liq. which collects at side. When fat and
fine material have washed thru and foam is gone, transfer res-
idue to 2 L trap flask, 945.75B(h)(4), with H 2 0. Add ca 500
mL H 2 and boil 10 min. Cool to room temp, and add H 2
to total vol. of 1 L. Pour 50 mL heptane, 945.75C(1), down
stirring rod. Lower mag. stirring bar, 945.75B(n), into flask
on stirring rod stopper. Raise rod above liq. and secure with
clamp. Stir mag., 970.66B(c), 5 min. After stirring, fill flask
with H 2 0. Let stand 30 min, gently stirring bottom layer every
4-5 min with stirring bar for first 20 min. Trap off heptane.
Add 35 mL more heptane, stir by hand gently 1 min, let stand
15 min, and again trap off. Filter combined trappings, using
374
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
Hirsch funnel. Remove paper and examine microscopically. If
debris on filter paper is excessive, proceed as in (d) after ex-
amining paper for hairs only.
(b) Chocolate. — Use 100 g finely shaven chocolate and
proceed as in (a).
(c) Press cake. — (/) Method}. — Heat sample (usually very
hard lumps) 2-3 hr at 60-70° and break into <0.5" (1 cm)
pieces. Mix 50 g into 500 mL hot 2% detergent soln,
945.75C®, or 500 mL hot 2% igepal CO-630, 945.75C(j)(3),
in 800 mL beaker. Stir with butter stirrer, 945.75B(e), or mag.
stirrer, at low speed 2-3 hr until completely dispersed, or let
soak overnight. Stir thoroly, pour portionwise onto No. 230
sieve, 945.75B(r), and proceed as in (a).
Refs.: JAOAC 48, 543(1965); 50, 496(1967); 57, 957(1974).
(2) Method If. — Break sample into ^1 cm (0.5") pieces,
using hammer or similar implement. Weigh 50 g into 1 L beaker
and add 100 mL peanut oil. Add mag. stirring bar, and heat
with gentle stirring to 150°. Transfer to cool mag. stirring unit
and stir 10 min at speed where no splashing occurs. Add 500
mL aq. 5% Triton X-l 14 soln, 945.75C(bb)(2), and stir 5 min.
Proceed as in (a), beginning "Pour portionwise onto No. 230
sieve, . . ."If oil drops are visible after completion of siev-
ing, wash material on sieve with 25-50 mL 2% aq. Triton X-
114 soln by spraying from wash bottle. Repeat as necessary.
Continue sieving until surfactant is removed and proceed as in
(a).
Ref.: JAOAC 54, 567(1971).
(d) Bleaching technic. — Return paper to Hirsch funnel in
suction flask. Wash thoroly with H 2 0. (If paper contains al-
cohol and glycerol from examination for hairs, wash first with
alcohol and then with H 2 0.) Apply vac. until paper appears
dry, turn off vac, and plug rubber tubing with stopper. Cover
paper with ca 3 mm (5-7 mL) NaOCl soln and let stand until
bleaching of cocoa tissue is complete, but <30 min. Maintain
Jevel of soln entire period and do not let soln flow over rim
of paper. Turn on vac, which will remove stopper. Wash pa-
per with H 2 0. Examine microscopically for insect fragments
and other extraneous materials other than hairs.
Refs.: JAOAC 58, 1302(1975); 61, 400(1978).
988.16 Filth in Ground Coffee
and Coffee Substitutes
Sedimentation and Flotation Method
Final Action 1988
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
(a) Heavy filth, sand, and soil. — Weigh 100 g sample in
600 mL beaker, add 350 mL CHC1 3 , and boil 15 min, stirring
occasionally. Wash down sides of beaker with CHC1 3 . Let mixt.
cool and settle 15 min with occasional stirring of top layer.
Carefully decant CHC1 3 and floating tissue onto smooth ca 15
cm filter paper in buchner without disturbing heavy residue on
bottom of beaker. Repeat decanting with small amts of CHC1 3
until practically no plant tissue remains with residue on bottom
of beaker. (Sp gr of CHC1 3 may be increased by addn of CC1 4 ,
if necessary to float plant tissue. Do not add CC1 4 beyond 1
part CC1 4 to 1 part CHC1 3 .) Transfer residue from beaker to
ashless filter paper and examine for filth. If residue is appre-
ciable, ignite filter and det. wt sand, soil, etc.
(b) Light filth (ground coffee) — Make filter paper cup from
24 cm paper and 150 and 250 mL beakers, 945.75B( j). Weigh
25 g sample into this cup. Add 100 mL CHC1 3 by pouring
most of liq. outside paper cup into the 250 mL beaker. Press
cup down into CHC1 3 and place on steam bath. Boil gently 5
min, avoiding excessive loss of CHC1 3 . Lift paper, clamp with
clothespin, and let drain to slow drip. Discard solv. and repeat
extn with two 100 mL portions CHC1 3 . Position paper on Hirsch
funnel and aspirate to apparent dryness. Complete by drying
(/) overnight in hood, (2) 1 hr in 80° convection oven, or (3)
30 min in 80° vac. oven at >5" (13 cm, 16.9 kPa) vac.
Wash sample into 2 L trap flask with total of 400 mL H 2 0.
Boil gently 15 min, remove from hot plate, and set aside. Dil.
to 600 mL with H 2 0, add 400 mL undild isopropanol while
stirring mag., heat to bp, and boil 2-3 min. Add 40 mL min-
eral oil, 945.75C(p), and heat to vigorous boil. Transfer to
cool stirrer and stir mag., 970.666(c) , 5 min. Slowly fill flask
with 40% isopropanol by letting liq. flow down rod while top
of stopper is held just above contents. Stir with rotary swirl
to re suspend solids, and trap off after 2 min. Filter onto ruled
paper. Add 30 mL mineral oil, stir by hand 30 sec, and trap
off after 5 min. Filter onto another ruled paper and examine
papers microscopically.
If filter paper debris is excessive, examine for rodent hairs
and then bleach as in 965.38B(d).
Ref.: JAOAC 55, 57(1972).
(c) Light filth (other substitutes except chicory). — Air dry
decanted material on paper overnight or for 1 hr in oven at ca
80°, transfer dried material to 2 L trap flask, and add 400 mL
hot H 2 0. Boil 15 min and, if necessary, add small amts cold
H 2 intermittently to prevent foaming. Cool mixt. to <20°,
Trap off twice, using 35 and 25 mL portions heptane,
945.75C(1), resp. In first trapping, after stirring heptane, let
stand 5 min before filling flask. Filter and examine micro-
scopically.
(d) Light filth (ground chicory). —Add 50 g sample to 1 L
beaker contg soln of 5 g Na lauryl sulfate and 10 g NaHC0 3
in 500 mL H 2 0. Stir and place in steam bath. Heat 20 min,
stirring twice at 5 min intervals, and wash down sides with
few mL H 2 after each stirring. Transfer to No. 230 sieve and
wash until foam is gone. Rinse sieve retainings with ca 100
mL 40% isopropanol. Transfer (use of teaspoon suggested) to
2 L trap flask with 40% isopropanol. Dil. to 800-900 mL with
40% isopropanol. Boil 4 min with gentle stirring. Add 50 mL
mineral oil and heat until boiling starts again. Move to cool
mag. stirrer and stir 5 min. Fill trap flask with 40% isopro-
panol added down rod with stopper held just above liq. Stir
immediately to resuspend settlings. Stir 2 more times at 3 min
intervals. Raise rod; wash with few mL 40% isopropanol and
clamp with stopper ca 75 mm below interface. Let stand 4
min. Trap off, rinse neck, and filter onto ruled paper. Add
25-30 mL mineral oil, stir by hand 45 sec at moderate speed,
and add 15-20 mL H 2 0. Let stand 20 min, trap off, rinse flask
neck with undild isopropanol, and filter onto sep. ruled papers.
Examine papers at 20-30 x.
Ref.: JAOAC 54, 571(1971).
967.23 Aphids in Hops
Flotation Method
First Action 1967
Final Action 1988
AOAC-ASBC Method
A. Reagents and Apparatus
(a) Flotation soln. — Satd Na 2 B 4 07 soln, 100 g borax/L H 2 0.
(b) Iodine stain. — Dissolve 0.5 g 1 and 1.5 g Kl in 25 mL
H 2 0.
AOAC Official Methods of Analysis (1 990)
Dairy Products 375
(c) Blender. — 'Tntensifier" Twin Shell Blender, Patterson-
Kelley Co., Div. of Harsco, 100 Burson St, PO Box 458, East
Stroudsburg, PA 18301-0458, or equiv.
B. Preparation of Sample
Place sample in blender, using 4 qt (3.8 L) size shell for
small samples or 8 qt (7.6 L) size shell for large samples.
Activate blender and "intensifier" for 1 min intervals until
blending and breakage of sprigs are complete. Draw off 10 g
samples from bottom plate.
C. Determination
Mix 10 g representative sample in 100 mL satd borax soln
in 2 L Wildman trap flask. Bring to slow boil. Keep mixt.
from boiling onto sides of flask by keeping boiling to min.
and by washing down sides with H 2 0. Boil 1.5 hr, and cool
to room temp. Fill flask to 1600 mL with H 2 and 35 mL
heptane, 945.75C(1), and stir vigorously 10 sec. Fill flask with
H 2 0, let stand 30 min, and trap off. Perform second trapping,
using 25 mL heptane, stirring 10 sec, and letting flask stand
15 min. Wash neck of flask with isopropanol. Pour trappings
onto ruled paper(s), add 10—12 drops I stain, and examine mic-
roscopically. If excess plant tissue is present in trappings, pour
trappings thru 5" No. 10 sieve held over paper. Wash plant
tissue on sieve with alcohol onto filter paper to remove any
adhering insects.
Count as aphid any whole aphid or part contg head. Count
individually aphid cast skins and other insects.
Ref.: JAOAC 50, 499, 520(1967).
970.67
See 960.51.
Filth in Tea
Direct Sieving Method
First Action
Heavy Filth in Tea
Sedimentation Method
First Action
970.68
See 975.48.
981.18 Light Filth in Tea
Flotation Method
First Action 1981
Final Action 1988
A. Reagents
Sequestering agent.— Mix 50 mL Tween 80-40% isopro-
panol, 945.75C(x), 50 mL Na 4 EDTA-40% isopropanol,
945.75C(z), and 200 mL 40% isopropanol.
B. Determination
Weigh 10 g sample into 800 mL beaker, add ca 300 mL
boiling H 2 0, heat to boiling, and simmer 6 min with mag.
stirring using Teflon-coated bar. Wash material from beaker
to No. 230 sieve with forcible stream of aerated H 2 0, but do
not let mixt. splash out of sieve. When rinse H 2 is clear,
drain residue and quant, transfer with 40% isopropanol to 2 L
Wildman trap flask. Rinse sides of flask with 40% isopropa-
nol, and bring total vol. in flask to 900 mL with 40% isopro-
panol. Pour 50 mL flotation oil, 945.75C(k), down stirring rod,
with top of disk or rubber stirring stopper held just below sur-
face of liq. Clamp rod above liq. Mag. stir 6 min. Let stand
2-3 min. Pour 300 mL sequestering agent down stirring rod,
with top of disk or rubber stirring stopper held just below sur-
face of liq. Mix 1 min by gently swirling with stirring rod
beneath surface of liq. Pour 40% isopropanol slowly down
stirring rod to fill flask. Rinse rod with 40% isopropanol and
clamp so that stopper is held above layer of settled material.
Let stand 5 min. Rotate stopper to free settled material. Let
stand 25 min and trap off, rinsing neck with isopropanol until
all residue is removed. Rinse neck with 40% isopropanol and
add rinse to trappings. Add 30 mL flotation oil by pouring
down stirring rod into trap flask. Hand-stir 1 min to bring flo-
tation oil down. Add 40% isopropanol to refill flask. Let stand
30 min, and trap off into same beaker of first trappings. Rinse
neck with isopropanol until all residue is removed. Transfer
combined trappings onto one or more ruled filter paper(s). Ex-
amine paper(s) microscopically at 30 x for insect fragments
and rodent hairs.
Ref.: JAOAC 64, 287(1981).
952.21
DAIRY PRODUCTS
Sediment in Milk
Sediment Test Method
Final Action 1965
A. Apparatus and Materials
(a) Tester. — Simply constructed, easily cleaned, and ad-
justable between samplings to permit sanitary removal of used
disk and replacement with clean disk. Before using, check tester
for reproducibility as in 952. 21B. Milk or sediment must not
bypass disk. Select type according to method of sampling:
(J) For mixed sample method. — Pressure, gravity, or vac.
type: (a) For 1 gal. sample use any suitable device that will
filter sample thru disk with exposed area V/s" diam. (b) For;
1 pt sample, equip single-unit, off-bottom tester with No. 125
BNC 0.40" safety head (available from Sediment Testing Sup-
ply Co., 7366 N Greenview Ave, Chicago, IL 60626) having
filtering area 0.40" diam., or use any suitable device having
filtering area 0.40" diam.
(2) For off-bottom method. — Single-unit type for intake of
1 pt on upstroke of plunger and discharge thru disk on down
stroke, or 2-unit type, contg 1 unit for removal of 1 pt milk
from bottom of can and another for filtering sample. Use sam-
pling device long enough to permit reaching bottom of milk
can, with filtering area lVs" diam.
(b) Cotton sediment disks. — Std lintine cotton disks or pads,
\ X W diam., for use over flat wire screen in tester to expose
filtration area 1 Vs" diam. Disk must not contain phenolic resins
or other chem that may contaminate milk.
Test sediment disks as follows: Filter 12 mg std sediment
mixt. (60 mL aliquot (d)) thru pad, using clean flask to catch
filtrate. Transfer filtrate to beaker, rinse flask 3 times with
H 2 0, and add rinsings to beaker. Filter fitrate thru 7 or 9 cm
S&S White Ribbon paper (or equiv.) that has been washed
with ca 200 mL H 2 0, dried to constant wt at 100°, and cooled
in covered dish in desiccator before weighing. Thoroly rinse
beaker and paper with H 2 and dry to const wt as above. Test
2^3 disks; av. wt sediment passing thru each disk should be
<2.8 mg. in addn, std disk prepd from fine mixt. should not
appear to have sediment buried beneath surface.
(c) Sediment filtering apparatus. — (/) For Z'/s" diameter
stds. — App. must hold I 1 // sediment disk and have effective
filtering area lVs" diam. This iVs" area must be unobstructed
except for wire screen or wire screen and perforated plate sup-
376
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
port for filter disk. App. should be supported in filter flask so
vac. can be used for rapid filtration or flask air outlet can be
closed to stop filtration. App. should have ca 80° funnel with
capacity of 5:80 mL but <450 mL. Test app. by filtering H 2
suspension of C thru std disk. Disk should have clean, sharply
defined border. When sediment suspension is filtered, sedi-
ment should be evenly distributed over disk with no pattern
formation. Figs 952. 21A and B show suitable app.
(2) For 0.40" diameter stds. — Vac. type that holds I 1 //
sediment disk and uses only 0.40" diam. filtering area. Test
app. as in (I).
(d) Preparation of coarse std sediment disks. — Prep, uni-
form mixt. of oven-dried (100°) materials which meet follow-
ing screening specifications. Grind all materials by hand with
mortar and pestle.
Cow manure, thru No. 40
Cow manure, thru No. 20, retained on No. 40
Garden soil, thru No. 40
Charcoal, thru No. 40
Charcoal, thru No. 20, retained on No. 40
53%
2%
27%
14%
4%
Place 2.00 g above mixt. in 100 mL vol. flask, thoroly wet
with 4-6 mL 0.1% Aerosol OF soln (prep. 1-2% soln in ace-
tone and dil. with H 2 0) or other suitable wetting agent, add
46 mL 0.75% carob bean gum soln prepd as in 945.75C(f),
and bring level of liq. just into neck of flask by adding 50%
(by wt) sucrose soln. Let stand >30 min, add few drops al-
cohol, and dil. to vol. with the sucrose soln. Mix thoroly, pour
into 250 mL beaker or other suitable container, and stir with
mech. or mag. stirrer at speed (ca 200-300 rpm) such that
mixt. is thoroly agitated but very little air is whipped into sus-
pension. Observe with bright reflected light to see that sus-
pension is uniformly stirred.
Transfer, while stirring, 10 mL portion (200 mg std sedi-
ment) with large-tip, graduated pipet to 1 L vol. flask, and dil.
to vol. with the 50% sucrose soln. When thoroly mixed, each
FIG. 952.21 A— Sediment filtering apparatus, unassembled
FIG. 952.21 B — Sediment filtering apparatus, assembled
mL contains 0.2 mg sediment. Mix, pour into 1.5 L beaker,
and stir as above. If particles accumulate on side of beaker,
wash down with portions of sediment suspension or push un-
der with tip of pipet. While stirring, pipet definite vols of sed-
iment mixt. and add to 3 / 4 pt filtered sweet skim milk. Mix
thoroly and pass mixt. thru std sediment disk in filtering app. ,
(c)(7). Gently pour milk down side of filtering app. and filter
with very little or no suction. Wash container promptly with
l U pt filtered skim milk. Let last portion of milk flow thru pad
with no suction applied. If sediment does not appear to be
evenly distributed over pad, add 15 or 20 mL skim milk and
let it filter thru without suction. Repeat addn until sediment
appears evenly distributed. Suck air thru disk ca 1 min to re-
move excess skim milk.
For permanent record, mount and spray disks with 40%
HCHO soln or with ale. soln contg 2.5 g each of menthol and
thymol in 100 mL. Alternatively, if most of milk is removed
by thoro aspiration, no preservative is needed. Dried pads may
be coated with colorless plastic cement dild with 1-3 vols ace-
tone so that mixt. is thin enough to pour easily. If acetone
dissolves pigment from paper and stains pads, place pads on
flat glass plate for treating with dild cement. Move pads while
drying to prevent sticking to glass. When pads are almost dry,
place light wt (e.g., petri dish) directly on them to prevent
curling. Pads may be mounted with plastic cement. (Std disks
made from manure contg large amt of chlorophyll cannot be
coated with plastic cement, as sol v. exts chlorophyll and stains
pad green. Use this method of preserving pads only if there is
no leaching of pigment from sediment on addn of dild plastic
cement.)
Following above method, prep, series of disks contg sedi-
ment remaining from 0.0, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0,
6.0, 7.0, 8.0, 9.0, 10.0, 12.0, and 14.0 mg std mixt. Mark
disks to show mg of sediment used to prep, each pad. Do not
use as std any pad on which sediment is not evenly distributed.
For comparison with tests on samples, entire series of disks
may be used, but usually it is more convenient to select few
AOAC Official Methods of Analysis (1990)
Dairy Products 377
disks denoting variations in grade that are applicable to par-
ticular investigations being made. If grading charts are prepd
and reports made, indicate on chart and report whether mixed
or off-bottom sample was used. If stds are to be handled or
used for appreciable length of time, place them under glass,
transparent plastic sheets, or other suitable materials. In using
stds, use either of following methods; (7) Grade sediment disk
to nearest std disk regardless of whether actual amt of sediment
is above or below std; or (2) grade sediment disk of sample
as "more than mg" or "less than mg." Choose stds to
fit method of grading. When grading disks, disregard gross
pieces of material (whole flies, hairs, large chunks of dirt or
manure, etc.) but if such matter is present, list each sep. on
report.
(e) Preparation of fine std sediment disks. — (7) l x /$" di-
ameter stds. — Grind oven-dried (100°) cow manure, garden
soil, and wood charcoal (not powd) in impact mill, Wiley mill,
or other suitable type, using fine screen in mill. Pass cow man-
ure thru Wiley mill or similar type 2 or 3 times. Sift materials
sep. in max. batches of 50 g as follows: Dry 25-50 g at 100°
for 3-4 hr. While still warm, place in 8" No. 140 sieve nested
over No. 230. Add cover and receiver. Shake nested sieves
by hand 5 min at ca 120 strokes /min. Sep. sieves and brush
off material adhering to underside of No. 230 and discard be-
fore emptying sieve. Dry material retained on No. 230 ca 2
hr (max. batch 20 g) and resift 5 min as above. Sep. sieves
and brush off material adhering to underside of No. 230 before
emptying. Use "on 230" fractions from second siftings and
mix uniformly in following proportions: cow manure 66%,
garden soil 28%, and charcoal 6%.
Combine above 2 "on 140" fractions of each of the 3 ma-
terials and resift as above, except use No. 120 sieve nested
over No. 140. Resift new "on 140" fraction, retaining "on
140" fraction from second sifting. (Dry before each sifting and
brush material from underside of No. 140 sieve before emp-
tying.) Mix manure, soil, and charcoal in above proportions.
Place 1.80 g mixt. from "on 230" fractions and 0.20 g mixt.
from "on 140" fractions in 100 mL vol. flask. Proceed as in
(d), beginning "... thoroly wet with 4-6 mL 0.1% Aerosol
OT soln ..." except use H 2 instead of 50% sucrose soln
for dilg 10 mL aliquot to 1 L.
Where (d) states "While stirring, pipet definite vols ..."
proceed as follows: Det. approx. funnel capacity of filtering
app., (c)(7), by pouring H 2 into assembled app. with filter
flask air outlet closed. Include H 2 that filters thru as part of
funnel capacity. While stirring, pipet aliquots of sediment sus-
pension into beakers. Add H^O to make total vol. 20-50 mL
less than funnel capacity, using total vol. of ^60 mL but <400
mL.
With filter flask air outlet closed to prevent filtration, mix
dild aliquot and pour into app. , (c)(7), fitted with wet std disk,
(b). (Use alcohol or wetting agent if necessary to wet disk.)
Add 20-50 mL H 2 to beaker and rinse by swirling. Pour into
funnel, keeping lip of beaker touching surface of H 2 if pos-
sible. (Rinse H 2 should nearly fill funnel if capacity is ^450
mL.) Open flask air outlet. After H 2 has filtered thru pad,
apply vac. and aspirate disk for ca 1 min. Remove pad and
let dry in covered dish. If sediment is not evenly distributed,
discard pad. After some practice, ca 75% of pads prepd should
be acceptable. No preservative is required. Pads may be coated
with dild plastic cement and used as in (d).
(2) 0.40' diameter stds. — While stirring, pipet 100 mL above
dild fine suspension into suitable container and dil. to 800 mL
with H 2 0. Each mL contains 0.025 mg sediment and when
filtered thru 0.40" diam, area is equiv. to 0.2 mg filtered thru
lVs" diam. area. Prep, series of stds by filtering suitable ali-
quots thru disks in app., (c)(2). Dil. each aliquot to 50-60 mL
and filter with min. suction. Rinse beaker with small vol. H 2
and add to funnel. Carefully rinse side of funnel with small
vol. H 2 0. Use min. suction necessary to remove excess H 2
from disk. Designate 0.40" diam. stds as " mg equiv." and
use in grading 1 pt mixed sample test disks in same manner
as V/s diam. stds are used.
(f ) Photographic stds. — Photographic stds (obtainable from
Photography Div., Office of Governmental and Public Affairs,
US Dept of Agriculture, Washington, DC 20250) may be used
as guide in grading sediment pads, but it is preferable to use
actual disks prepd as in (d) or (e). Stds that more nearly re-
semble disk being graded should be used in each case. Do not
use photographs that have become faded, stained, soiled, or
otherwise damaged.
B. Checking Sediment Testers
To check sediment testing devices, proceed as follows:
Measure actual vol, of milk delivered to assure that 1 pt is
withdrawn and passes thru disk. Transfer 10 mL 2% sediment
suspension in sucrose soln, 952.21 A(d), using large-tip, grad-
uated pipet, to 10 gal. clean filtered H 2 in clean milk can.
After thoroly agitating mixt., remove 1 pt with clean pt mea-
sure and filter thru lVs" diam. area of sediment disk,
952.21 A(b), mounted on suitable funnel of correct size, e.g.,
952.21A(c)(7). After thoro agitation of contents of milk can,
again remove pt sample with the sediment testing device and
pass thru sediment disk in exactly same manner as for testing
milk. Repeat this operation with the tester several times to det.
whether all disks so obtained give same sediment as disk ob-
tained by filtering thru funnel, 952.21A(c)(/).
C. Collection of Sample
(a) Mixed sample method. — For retail containers, 5 to 10
gal. cans, and storage tanks, use 1 pt or 1 gal. samples. Before
mixing milk, transfer with small strainer any floating extra-
neous matter, such as flies, hairs, large chunks of debris, etc.,
to mounted disk, 952.21D(a), or mount on sep. disk, properly
identified. Thoroly mix milk in container before removing test
portion. Avoid contamination of sample with foreign matter
on stirrers or by any other means. For retail containers take I
pt from mixed container or composite sufficient number to make
1 gal. Proceed as in 952.21D(a).
(b) Off-bottom method. — For 5 to 10 gal. cans take pt sam-
ple with either type of off-bottom tester from unstirred can of
milk. Before withdrawing sample, remove with small strainer
any floating extraneous matter as in (a). Take sample < l W off
bottom of unstirred can of milk by inserting sampler and, dur-
ing upstroke of plunger, drawing head of instrument once across
diam. of can bottom or around circumference if can has high
center. Expel milk with gun in can and then with short stroke
remove excess fluid from pad. Proceed as in 952.21D(b).
D. Determination
(a) Mixed samples . — Pass sample thru properly adjusted disk,
952.21 A(b), held in correct position in tester. Warm 1 pt sam-
ple to 90-100°F and filter thru restricted area 0.40" diam.,
952.21A(a)(7). If single-unit off-bottom tester with special head
is used, warm sample larger than 1 pt to 90-100°F and with-
draw 1 pt with tester while stirring, or draw 1 pt into tester
and warm milk by holding tester under running hot H 2 before
discharging milk thru disk.
Warm 1 gal. sample to 80-90°F or filter cold thru lVs" diam.
area of disk, 952.21A(b). If milk is filtered at temp. <90°F,
rinse disk by filtering ca V2 pt sediment-free warm (90~100°F)
H 2 thru disk before removing from tester. If milk is to be
salvaged, do not dil. with H 2 0. (Milk varies in its rate of flow
thru disks; pasteurized milk may be more difficult to filter than
raw milk. Other factors influencing rate of flow are temp., fat
378
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
content, degree of clumping of fat globules, stage of lactation,
presence of mastitic milk, and amt of sediment in sample.)
Remove disk from tester and mount on special sized paper
or store in individual transparent waxed envelope. (If disk is
placed on paper or in envelope while still moist, drying milk
acts as adhesive.) Grade by comparison with std disks,
952.21 A(d) or (e), and indicate on report whether pad was
graded wet or dry. (Character of sediment may be detd by
microscopic examination.)
To prevent decomposition on storage, disk may be sprayed
with HCHO soln or ale. menthol-thymol soln as in 952.21A(d).
Do not use glue to affix disk to paper; if disk becomes de-
tached, moisten with few drops H 2 and remount. Protect from
contamination.
(b) Off-bottom samples. — Remove disk from tester,
952.21C(b), and proceed as in (a), third par., beginning "...
mount on special sized paper ..."
Refs.: JAOAC 35, 340(1952); 36, 310(1953); 37, 117(1954);
48, 559(1965). Am. J. Public Health 37, 728(1947).
948.27 Sediment in Dairy Products
Sediment Test Method
Final Action 1988
(a) Rapid method for sweet cream and cream in which curd
is easy to disperse and in absence of mold.— Place 1 pt (500
mL) sample in beaker or pan of convenient size, ca 2 L, and
add ca 500 mL hot H 2 (70-90°). More or less H 2 may be
added so that mixt. when ready for filtration is at 45-60°. Re-
move whole flies or other large filth particles which float to
surface and which would be broken up by stirrer. Place these
on sediment pad when completed. Place pan under malted milk
stirrer, and add, while stirring, 25 mL 40% Na hexameta-
phosphate soln, if necessary, add more Na hexametaphosphate
soln to make mixt. alk. to litmus. Stir 30-60 sec or until curd
is broken up. Filter with vac. thru std sediment disk,
952.21A(b). If pad clogs, filter remaining portion thru fresh
disk. Rinse pan and funnel with hot H 2 onto sediment disk.
(b) Other dairy products. — Proceed as in 960. 49C or D,
and filter thru std sediment disk, 952.21A(b). Violent mech.
agitation, such as is provided by malted milk stirrer, may be
used to facilitate dispersion of product.
Compare with std sediment disks, 952.21 A(d), (e)(7), or
(f).
Ref.: JAOAC 31, 93(1948).
960.49 Filth in Dairy Products
Filtration Methods
First Action 1960
Use following methods independently or in various combi-
nations. Weigh 225 g, except in 960. 49F, into suitable con-
tainer and use S&S ruled No. 8 paper for filtration. Cut hard
cheese into small pieces.
A. Evaporated Milk, Condensed Milk, Sweet Cream, Spray-
Dried Whole or Skim Milk
Reconstitute dried or coned products. Dil. reconstituted
product with equal vol. hot FLO, hot 3% Na 2 C 2 4 soln, or hot
2% Na 2 C0 3 soln, and filter with suction. During filtration,
continually wash paper with stream of near boiling H 2 to
prevent accumulation of layer of particles which clogs paper.
Examine paper microscopically.
B. Butter
Place container in H 2 bath or oven at ca 80°. When fat
seps, filter directly thru paper with suction, retaining most of
curd and H 2 in container. After fat passes thru, filter re-
maining material. To facilitate filtration of curd, wash paper
with near boiling H 2 during filtration. (For butter not filter-
able by this process, use 960. 49C.) Examine paper micro-
scopically.
C. Soft and Semi-Soft Cheese and Sour Cream; Some Dried
Whole and Skim Milks; and Butter That Cannot Be
Filtered by 960.49B
Cut 225 g cheese into 6 mm cubes and add to 800-1000
mL boiling H 3 P0 4 (1 + 40) in 1.5-2 L beaker, stirring con-
tinuously with slow speed mech. stirrer, 945.75B(e), or on
mag. stirrer-hot plate, 945.75B(n), with stirring bar ca 75 x
12 mm, until sample is dispersed (usually >20 min). Filter,
without letting mixt. accumulate on paper, and continually wash
filter with stream of near boiling H 2 to prevent clogging.
When filtration is impeded, add H 2 0, dil. (1-5%) alkali, H 3 P0 4
(1 + 40), or hot alcohol, until paper clears; then resume addn
of sample suspension and H 2 0, Examine paper microscopi-
cally.
D. Hard Cheeses, Hard Skim, Part Skim Milk Cheeses
(Romano, Ricotta, Feta, Pecorino, Sardo, Goats' Milk
Cheeses, Sbrinz, Goya, Whey Cheeses, etc.)
(Not applicable to cheese contg herbs, spices, or molds
thruout)
Prep, cheese for sampling by trimming and discarding thin
layers to remove all "old" cut surfaces and to keep paraffin
coating and mold out of sample. Cut and break up 225 g trimmed
cheese into 4 L beaker. Add ca 700 mL ca 55° filtered H 2 0.
Set beaker under mech. stirrer, 945.75B(e), and stir 15 min,
maintaining mixt. at 55°. Add 100 mL 20% aq. Na 4 EDTA
soln, stir, and adjust mixt. to pH 8 with NH 4 OH or dil. HCI
(1 + 2). Rinse sides of beaker free of adhering cheese particles
with ca 60° H 2 0. Maintain pH 8 by addn of NH 4 OH and keep
adding ca 60° H 2 to dil. cheese mixt. to ca 3 L. if foaming
occurs, place wet vegetable parchment paper, 27 lb wt, for-
merly Patapar paper (available from James River Corp., KVP
Group, 100 Island Ave, Parchment, Ml 49004), or Parafilm
M (Cat. No. 13-374, Fisher Scientific Co.), split to accom-
modate stirrer blades, over top of beaker to break foam. Con-
tinue stirring until cheese becomes finely dispersed.
Cool dispersion to 40° and adjust to pH 8 with NH 4 OH or
HCI (1 + 2). Add 300 mL pancreatin soln, 945.75C(q), (ex-
cept use 600 mL pancreatin soln for ricotta). Let mixt. digest
at <40° with continued stirring ca 1.5 hr. Maintain pH 8 by
addn of NH 4 OH.
After digestion, place beaker on hot plate and heat to 65-
68°, continuing mech. stirring. Adjust to pH 6.0 ± 0.2 with
HCI (1 +2). Carefully adjust stirrer blades close to bottom
of beaker to pick up any cheese particles which settle. Con-
tinue stirring 15 min or until cheese appears completely sol-
ubilized. Rinse inside of beaker, stirrer blades, etc., with ca
65° H 2 and filter thru ruled paper, using ca 65° H 2 and then
alcohol to rinse beaker. If filtration becomes slow (e.g., cream
cheese), let paper clear, wash with alcohol, and use addnl pa-
per. Mixt. will filter more easily if No. 60 screen (ca 5 cm
diam.) is placed under paper and small amt of mixt. is allowed
to suck dry before filtering is continued. Examine paper mi-
croscopically.
Ref.: JAOAC 50, 501(1967).
AOAC Official Methods of Analysis (1990)
Nut Products
379
E. Cheese Containing Mold, Plant Tissues, and Spices
Disperse cheese by 960. 49C or first par. of 960. 49D. Pour
thru No. 140 sieve, 945.75B(r), washing thoroly with forcible
stream of H 2 0. Transfer material retained on sieve to beaker.
Add 200 mL H^P0 4 (1 + 49), boil until lumpy residue dis-
solves, and pour again thru No. 140 sieve, washing thoroly
with forcible stream of hot H 2 0. Transfer material on sieve
with ca 200 mL 60% alcohol to trap flask and cool. Trap off,
using heptane, 945.75C(B), and H 2 0, filter, and examine mic-
roscopically.
F. Casein
Weigh 50 g sample into 1 L beaker. Slowly stir in 170 mL
20% Na 4 EDTA soln until well mixed with sample. With con-
stant stirring, bring vol. to 1 L with hot tap H 2 (55-70°).
Wet sieve on No. 230 sieve, 945.75B(r), with forcible spray
of hot tap H 2 until foam subsides. Wash sieve retainings into
beaker and filter thru ruled filter paper. Examine papers mic-
roscopically.
Ref.: JAOAC 53, 552(1970).
spatula and finally rinse paper clean with 60% alcohol -CaCl 2
soln, 945.75C(c). Bring vol. to 1 L with 60% alcohol-CaCU
soln and add 50 mL HC1. Add mag. stirring bar, 945.75B(n),
to flask, place flask on mag. stirring hot plate, and heat to full
boil with gentle stirring. Immediately transfer flask to cool
stirring unit and add 40 mL mineral oil, 945.75C(p), by pour-
ing down stirring rod. Stir mag., 970.666(c), 2 min.
Fill with 60% alcohol-CaCL soln and gently stir 5-10 sec
with stirring rod. Let stand 2 min and trap off. Add 25 mL
mineral oil, hand stir gently 30 sec, and let stand 10 min.
Repeat trapping. Wash flask neck thoroly with isopropanol,
and transfer washings to beaker with trappings. Filter onto ruled
paper and examine microscopically.
Ref.: J AOAC 53, 553(1970).
968.34
Filth in Pecans
Final Action 1988
Light Filth
—First Action 1968
NUTS AND NUT PRODUCTS
968.33 Filth in Shelled Nuts
Final Action 1988
(Not applicable to pecans)
A. Heavy Filth by Sedimentation
—First Action 1968
(Caution: See safety notes on distillation, flammable solvents,
and petroleum ether.)
Weigh 100 g sample into 600 mL beaker. Add ca 350 mL
pet ether and boil gently 30 min, adding pet ether to maintain
original vol. Decant solv., taking care not to lose any coarse
nut tissue, and discard. Add ca 300 mL CHCl 3 to beaker and
let settle 10—15 min. Pour off floating nutmeats and ca % of
the CHCI3, and discard. Repeat sepn with smaller vols of mixt.
of CHCI3 and CCI4 (1 + 1) until residue in beaker is relatively
free of nutmeat particles. Transfer residue in beaker to ashless
paper and examine for heavy filth. If appreciable amt of sand
and soil is present, ignite paper in weighed crucible at ca 500°
and weigh.
Ref.: JAOAC 51, 527(1968).
B.
Light Filth by Flotation
—First Action 1970
(Caution: See safety notes on flammable solvents, toxic sol-
vents, ethanol, and chloroform.)
Nutmeats, all sizes, except pecans. — Weigh 100 g sample
into 1.5 L beaker. Add 600 mL CHC1 3 ; boil 15 min. Prep.
>24 cm paper for 100 mm plate diam. buchner by moistening
with H 2 and forming around base of 1 L beaker. Place 7 cm
disk of bolting cloth, 945.75B(d), (mesh size not critical) in
buchner, insert paper, apply vac, and press moistened paper
until good seal is obtained. Rinse paper with isopropanol. Quant.
transfer nutmeats and CHC1 3 onto previously prepd paper.
Maintain suction on nutmeats in buchner 5 min after visible
dripping ceases. Release vac, add isopropanol until nutmeats
are covered, let stand few min, and reapply vac until dripping
ceases. Repeat isopropanol wash step and aspirate 5 min after
visible dripping ceases. Quant, transfer nutmeats on paper to
2 L trap flask, 945.75B(h)(4). Scrape all fines from paper with
(Caution: See safety notes on toxic solvents and chloroform.)
Form cup, using 32 cm paper with 1-1.5 L beaker,
945.75B(j). Weigh 100 g sample into filter paper cup and place
in 1.5 L beaker. Add 400 mL CHCJ 3 and boil 5 min. After
cooling few min, lift paper and drain. Repeat 5 min boil and
drain with two addnl 400 mL portions CHCJ 3 . Proceed as in
968. 33B, beginning "Place 7 cm disk of bolting cloth, ..."
Ref.: JAOAC 51, 527(1968).
B. Curculio Larvae in Pecan Pieces
—First Action 1970
Weigh 115 g (ca l / 4 lb) sample into 1.5 L beaker and add
mag. stirring bar, 945.75B(n). Add 300 mL undild isopro-
panol and stir on mag. stirrer 5™ 10 sec. Add FLO (200 mL
for midget pieces and 300 mL for small, small medium, me-
dium, and mixed pieces) and stir 5-10 sec on stirrer. After
few sec, gently agitate settled nutmeats with stirring rod to
release any entrapped curculio. Remove all floating material
and examine for curculio larvae. Reclaim flotation soln by
pouring thru No. 12 sieve and use for one addnl sample.
Ref.: JAOAC 53, 550(1970).
978.19 Filth in Coconut (Shredded)
First Action 1978
Final Action 1988
(a) Heavy filth. — Proceed as in 941. 16 A, using 100 g sam-
ple in 800 mL beaker.
(b) Light filth. — Weigh 100 g sample into 1.5 or 2.0 L
beaker. Add 1 L detergent soln, 968. 35E. Heat in steam bath
10 min, stirring immediately and after ca 5 min.
Pour entire sample onto 8" No. 230 sieve, rinse beaker with
hot H 2 0, and add rinse to sieve. Wash sieve with forcible stream
of hot H 2 until all foam is gone; then rinse well with 40%
isopropanol and let drain. Place wide stem funnel in 2 L trap
flask, and transfer bulk of sample to flask with spoon. Rinse
remaining material to edge of sieve with aerator spray, and
transfer quant, to trap flask with 40% isopropanol. Dil. to 1
L with 40% isopropanol, add mag. stirring bar, 945.75B(n),
and place on mag. stirrer-hot plate, 945.75B(n). Add 40 mL
mineral oil, 945.75C(p), and stir vigorously 1 min. Turn stir-
rer to slow rate, add 50 mL HCI, and heat to vigorous boil.
380
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1 990)
(Caution: Soln may froth violently upon reaching bp with high
heat input.)
Place sample flask on cool stirrer and stir mag. , 970.66B(c),
3 min. Let stand 2 min; then slowly fill flask with 40% iso-
propanol added down stirring rod to bring oil interface 1 cm
above fully raised stopper or wafer. Lower stopper to midpoint
of flask, clamp, and let stand undisturbed 2 min. Trap off into
beaker, rinsing neck of trap flask with 40% isopropanol. Add
25 mL flotation .liq. 945.75C(k), and stir vigorously by hand
1. min. Adjust oil level as above, and let stand undisturbed 10
min. Trap off into second beaker, rinsing neck of flask well
with isopropanol.
Filter each trapped off layer onto sep. identified papers, rinsing
beakers with isopropanol, and examine papers microscopically
at 30x.
Ref.: JAOAC 61, 898(1978).
968.35 Fifth and Extraneous Material
in Peanut Butter
Sedimentation/Flotation Methods
Final Action 1988
4. Preparation of Sample*
—Surplus 1970
.See 40.031, 11th ed.
B. Water-Insoluble Inorganic Residue ("WIIR") and Excreta*
—Surplus 1970
See 40.032, llth ed.
C. Rocks and Decomposed Peanuts*
—Surplus 1970
See 40.035, llth ed.
D. Glass*
—Procedure 1960
—Surplus 1970
See 40.036, llth ed.
Light Filth
First Action 1968
E. Reagent
Detergent .yo/w— Dissolve sep. 20 g USP Na lauryl sulfate
and 10 g tech. Na 2 B 4 7 10H 2 O in H 2 0, combine, and dil. to
1 L.
F. Determination
Weigh 100 g sample into 1.5 L beaker and heat on steam
bath until softened. Add 1 L filtered hot detergent soln, and
stir well. Heat 10 min in steam bath. Stir well, pour portion-
wise onto No. 230 sieve, 945.75B(r), and wash with forcible
stream of 55-70° tap H 2 0, using aerator, 945.75B(a). When
foam is gone, transfer material on sieve to 2 L trap flask,
945.75B(h)(4), with 55% alcohol (or 40% isopropanol) and
bring vol. to 1 L. Add 50 mL HC1. Lower mag. stirring bar
into flask on stirring rod stopper. Heat to bp and boil 10 min
while slowly stirring on mag. stirring hot plate 945.75B(n).
Transfer flask to unheated stirring unit and immediately add
40 mL mineral oil, 945.75C(p), by pouring down stirring rod.
Stir mag. 2 min. Fill with deaerated 55% alcohol (or 40% iso-
propanol) and gently stir 5-10 sec with stoppered rod. Let
stand 5 min. Trap off. Add 25 mL mineral oil, stir by hand
gently 30 sec, and let stand 5 min. Repeat trapping. Wash
flask neck thoroly with isopropanol. Filter onto ruled paper
and examine microscopically.
Ref.: JAOAC 51, 531(1968).
GRAINS AND THEIR PRODUCTS
950.86 Light Filth (External) in Grains
and Seeds
Rotation Method
Procedure
Transfer 225 g sample to 2 L trap flask, 945.758(h)(4).
Add 600 mL 40% alcohol and boil gently, with frequent stir-
ring, 5 min. Cool, trap off, using heptane, 945.75C(S), and
40% alcohol, filter, and examine microscopically.
982,31 Insect Infestation
(Internal) of Wheat
Cracking Flotation Method
First Action 1982
Final Action 1988
A, Preparation of Sample
Mix grain by passing 6 times thru Jones sampler, recom-
bining sepns before each pass. Sep. slightly >50 g and weigh
50 g. Transfer weighed sample, small amt at a time, to 5 or
8 in. No. 12 sieve, and with stiff bristle brush, work insects
thru sieve as completely as possible.
Grind screened sample in cutting-type mill set at 0.061 in.
(see 985. 36B). Dry damp or tempered grain in forced-draft
oven 1 h at 70-80° or 2 h in oven without draft.
B. Isolation
Transfer cracked grain, including any residue in mill, to 2
L glass beaker contg mag. stirring bar. 945.75B(n), and mixt.
of 600 mL H 2 + 50 mL HC1. Stir gently while boiling 15
min on hot plate.
Transfer sample to No. 100 sieve, 945.75B(r), with gentle
stream of hot tap H 2 0. Wash material on sieve with very gentle
stream of hot (55-70°) tap H 2 until washings show no acidity
when tested with blue litmus paper.
Add mag. stirring bar, 945.75B(n), to 2 L trap flask,
945.75B(h)(4). Place wide-stem funnel in flask opening and
quant, transfer residue on sieve to flask with 40% isopropanol.
Add 40% isopropanol to total vol. of 800 mL.
Clamp stirring rod so stopper or wafer is above liq. in flask.
(Trap flask may stand overnight at this point.) Stir gently while
boiling 7 min ± 10 s on mag. stirring hot plate. Remove flask
from hot plate and wash down sides with min. of 40% iso-
propanol and immediately add 100 mL 1 + 1 mixt. Tween-
80 and 40% isopropanol soln, 945.75C(x), and Na 4 EDTA and
40% isopropanol soln, 945.75C(z), slowly down rod. Hand-
stir gently 1 min and let stand 3 min.
Add 50 mL mineral oil, 945.75C(p), down stirring rod. Stir
mag. 970.66B(c), 5 min on cool mag. stirrer, and let stand 3
min.
Fill flask with 40% isopropanol, added slowly down stirring
rod to avoid mixing or agitation of flask contents, and let stand
20 min undisturbed. Trap off, rinsing neck of flask with 40%
isopropanol, and add rinse to trappings in beaker.
Add 35 mL mineral oil to flask and hand- stir 1 min. Clamp
stirring rod so stopper or wafer is at midpoint of flask. Let
stand 5 min, spin stirring rod to free settlings from stopper or
wafer, and adjust oil level with 40% isopropanol to ca 1 cm
above fully raised stopper. Let stand undisturbed 15 min. Trap
off, and combine trappings in beaker. Rinse neck of flask well
with isopropanol, adding rinsings to beaker. Filter trappings
on ruled filter paper, rinsing beaker well with isopropanol. Ex-
amine papers at 15x, counting only whole or equiv. insects
and cast skins.
Ref.: JAOAC 64, 1408(1981).
AOAC Official Methods of Analysis (1990)
Grains 381
985.36 Insect Infestation
(Interna!) of Oats
Cracking Flotation Method
First Action 1985
A. Reagent
Tween 80~Na 4 EDTA [(ethylenedinitrilo)-tetraacetic acid
tetrasodium salt] premix soln. — Measure 420 mL 40% iso-
propanol in 500 mL graduate. Add 80 mL Tween 80 (poly-
sorbate 80) to 100 mL g-s graduate. Invert 100 mL graduate
over 2 L glass beaker and drain briefly. Rinse 100 mL grad-
uate with several portions of the 420 mL 40% isopropanol,
pouring each rinse into beaker. Add rest of 40% isopropanol
to beaker, add mag. stirring bar, 945.75B(n), and start mag.
stirring. Add 10 g Na 4 EDTA to beaker while stirring rapidly.
Add 500 mL 40% isopropanol and stir until uniform. Mixed
reagent stored in g-s flask is stable 1 week.
B. Preparation of Sample
Mix grain by passing 6 times thru Jones sampler, recom-
bining sepns before each pass. Sep. slightly >25 g and weigh
25 g. Transfer weighed sample, small amt at a time, to 5 or
8 in. No. 12 sieve, and with stiff bristle brush, work insects
thru sieve as completely as possible.
Grind screened sample in cutting-type mill set at 0.061 in.
(Elec. coffee grinder can be used.) Particle size of cracked
oats, ca 0.061 in., can be checked by passing the 25 g cracked
oats thru No. 14 sieve, 945.75B(r). Amt of cracked oats re-
tained on sieve should range from 75 to 80% by wt and amt
passing thru sieve should range from 20 to 25% by wt. When
using Labconco mill, rotate blade adjusting wheel counter-
clockwise until cutting blades are touching. Then rotate ad-
justing wheel clockwise 3 / 4 revolution. This setting should give
cracked oats within particle size range mentioned above. Dry
damp or tempered grain in forced-draft oven 1 h at 70-80° or
2 h in oven without draft.
C. Isolation
Transfer cracked grain, including any residue in mill, to 2
L glass beaker contg mag. stirring bar, 945.75B(n), and add
600 mL isopropanol . Cover beaker and stir gently while boil-
ing 5 rnin on hot plate.
Transfer sample to No. 100 sieve, 945.75B(r), with gentle
stream of hot tap H 2 . Wash material on sieve with very gentle
stream of hot (55-70°) tap H 2 to remove isopropanol. Quant.
transfer material on sieve to original 2 L beaker with H 2 0.
Add mag. stirring bar, 945.75B(n), and mixt. of 600 mL H 2
+ 50 mL HO. Cover beaker and mag. stir contents gently
while boiling 15 min on hot plate. Transfer material to No.
100 sieve, 945.75B(r), with gentle stream of hot tap H 2 0.
Wash material on sieve with very gentle stream of hot (55—
70°) tap H 2 until washings show no acidity when tested with
blue litmus paper.
Add mag. stirring bar, 945.75B(n), to 2 L trap flask,
945.75B(h)(4). Place wide-stem funnel in flask opening and
quant, transfer material on sieve to flask with 40% isopropa-
nol. Add 40% isopropanol to total vol. of 800 mL.
Clamp stirring rod so stopper or wafer is above liq. in flask.
(Trap flask may stand overnight at this point.) Stir gently while
boiling 7 min ±10 s on mag. stirring hot plate. Remove flask
from hot plate and wash down sides with min. of 40% iso-
propanol and immediately add 100 mL Tween 80-Na 4 EDTA
soln slowly down rod. Hand-stir gently 1 min and let stand 3
min.
Add 50 mL mineral oil, 945.75C(p), down stirring rod. Stir
mag., 970.66B(c), 5 min on cool mag. stirrer, and let stand
3 min.
Fill flask with 40% isopropanol, added slowly down stirring
rod to avoid mixing or agitation of flask contents, and let stand
20 min undisturbed. Trap off into beaker, rinsing neck of flask
with 40% isopropanol, and add rinse to trapping in beaker.
Transfer trapping to ruled filter paper, rinsing beaker well
with isopropanol. Add 35 mL mineral oil, 945.75C(p), to flask
and hand-stir 1 min. Clamp stirring rod so stopper or wafer is
at midpoint of flask. Let stand 5 min, spin stirring rod to free
settlings from stopper or wafer, and adjust oil level with 40%
isopropanol to ca 1 cm above fully raised stopper. Let stand
undisturbed 15 min. Trap off into beaker, rinsing neck of flask
well with isopropanol and adding rinsings to trapping in beaker.
Transfer trapping to ruled filter paper, rinsing beaker well with
isopropanol. Examine papers at 15 x, counting only whole or
equiv. insects and cast skins.
Ref.: JAOAC 68, 699(1985).
955.42 Insect Infestation (Interna!)
of Grains and Seeds (Except Wheat and Oats)
Cracking Flotation Method
First Action
Mix grain by passing 6 times thru Jones sampler, recom-
bining sepns before each pass. Sep. slightly >100 g and weigh
100 g. Transfer weighed sample, small amt at time, to 5" or
8" No. 12 sieve, and with stiff bristle brush, work insects thru
sieve as completely as possible.
Grind screened sample in cutting-type mill set at 0.061".
(Dry damp or tempered grain in forced-draft oven 1 hr at 70-
80° or 2 hr in oven without draft.) Transfer cracked grain,
including any residue in mill, to 2 L trap flask, 945.75B(h)(4),
trap as in 970.66B(b), using 60% isopropanol satd with hep-
tane, 945.75C(n), and heptane, 945.75C(I), as solvs, and fil-
ter on 10XX bolting cloth, 945.75B(d). If considerable starchy
material is in ext, hydrolyze with HCl as in 970.66B(d). Ex-
amine as in 970.66B(g) except use 1 5 x as lower limit of mag-
nification. Count only whole and equivalent insects and cast
skins.
955.43 Foreign Matter in Brewer's Grits
A. Rodent Excreta — Procedure
See 941.16A.
B. Light Filth— First Action
See 941. 16B.
971.31 Light Filth
in Cracked Wheat and Flours
Flotation Methods
First Action 1971
Final Action 1973
(Applicable to rye, pumpernickel, and buckwheat flours)
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Weigh 25 g sample into 94 x 33 mm Soxhlet thimble and
cover with pad of glass wool. Add ca 300 mL CHC1 3 and 3-
4 glass beads to 500 mL Soxhlet extn flask. Ext in Soxhlet
extractor at medium rate ca 90 min (counting time from first
overflow and siphoning ca every 5 min). Place extn thimble
in 250 mL beaker and dry with air on steam bath until no
CHC1 3 odor remains.
Quant, transfer contents of thimble and any material adher-
382
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
ing to glass wool, with spatula and acid-alcohol rinses,
945.75C(a), to 1 L trap flask for trap flask method, (a), or to
1-1.5 L beaker for percolator method, (b).
Place trap flask or beaker on mag. stirrer, add stirring bar,
and slowly add acid-alcohol soln, stirring constantly to form
smooth slurry. Dil. to ca 600 mL with acid-alcohol soln and
add ca 40 mL mineral oil, 945.75C(p). Mag. stir mixt. 10
min, 970.66B(c). Continue as in (a) or (b).
(a) Trap flask method. — At end of stirring period, fill flask
with acid-alcohol soln and let stand 30 min; stir gently every
3-4 min for first 25 min. Trap off, rinsing neck of flask with
acid-alcohol soln. Add ca 30 mL mineral oil and stir mag. ca
5 min. Let stand 30 min, stirring and trapping as above. Com-
bine trappings in beaker and transfer quant, to Kilborn funnel,
945.75B(h)(/) or percolator (2), contg ca 125 mL H 2 0. Retain
beaker. Add tap H 2 to ca 1 cm from top of funnel . Let oil
layer sep. 5-10 min and drain until interface is ca 5 cm above
constriction, discarding lower aq. layer. Repeat H 2 washes
until lower phase is clear. Drain interface-oil layer into re-
tained beaker, washing sides of funnel with ca 50-100 mL
H 2 0. If product residue is present in retained beaker contents,
add ca 10 mL HC1. Boil ca 5 min on hot plate and filter thru
ruled paper. After mineral oil layer has passed thru paper, rinse
all glassware used (except trap flask) with alcohol followed by
H 2 0, then 5% detergent soln, 945.75C(i), and finally with H 2 0.
Filter rinses thru original filter paper. Examine microscopi-
cally.
(b) Corning percolator method. — Place stirring rod in drain
opening inside percolator, 945.75B(h)(2), Add ca 250 mL H 2
to percolator. Quant, transfer beaker contents to percolator.
Add acid-alcohol soln to ca 6 cm from top. Let stand ca 30
min; gently stir contents with stirring rod every 3-4 min dur-
ing first 25 min in such manner as to prevent product dropping
into drain opening. After 30 min, raise stirring rod and drain,
discarding lower layer but leaving ca 250 mL in percolator.
Rinse stirring rod with acid-alcohol soln and add rinsings to
percolator contents. Retain stirring rod for further rinsings.
Add acid-alcohol soln to percolator to ca 6 cm from top and
let stand ca 30 min, stirring as before. Drain and discard lower
layer as before to 250 mL. Add H 2 to ca 6 cm from top and
proceed as in (a), beginning "Let oil layer sep. 5-10 min ..."
Refs.: JAOAC 53, 723(1970); 54, 903(1971).
981.19 Light Filth in Corn Meal
(White and Yellow)
Brine Flotation Method
First Action 1981
Weigh 50 g sample into 600 mL beaker, add stirring bar
[945.75B(n)], and 300-400 mL alcohol. Boil 2-3 min with
const stirring on mag. stirrer plate [945.75B(n)]. Transfer to
230 mesh sieve [945.75B(r)], wash with ca 100 mL alcohol,
then with hot tap H 2 0, <80°, and then with brine [prepd by
dissolving ca 360 g rock salt, H 2 0-softener salt, ice cream salt,
or equiv. per L tap H 2 and filtering]. Transfer to 2 L trap
flask [945.75B(h)(f)] and dil. to 800 mL with brine. Add 30
mL olive oil and mag. stir [970.66B(c)] 10 min. Remove from
stirrer, insert trap rod, wash insides of flask with brine, and
wait 3-5 min. Fill flask very slowly, ca 50 mL/min, with tap
H 2 down trap rod. Percolator method given below is most
efficient way to do this:
Set trap flask on base of ring stand and suspend percolator
[945.75(h)(2)] in ring directly above so that tip is ca 20 in.
above base. Attach 6 in. piece of l U in. id rubber tubing to
tip and close with pinch cock capable of adjusting flow. Insert
rod of trap flask into tubing and, after filling percolator with
H 2 0, open pinch cock only slightly and let H 2 flow down
trap rod into flask. By inserting 6 in. piece of J -/s in. rod or
wire (spent refill of ballpoint pen works well) into tubing along
with trap rod, tendency for stream of H 2 to spin off trap rod
is eliminated.
After filling flask, stir bottom brine layer with rotary motion
to release any trapped oil. Do not stir entire contents of flask,
because it is desirable at this point to maintain integrity of the
2 layers, brine /H 2 0. Let flask remain still 30 min before trap-
ping. Trap oil layer and rinse neck of flask with H 2 0. Add 35
mL olive oil and stir in. Repeat trapping but, at this time,
hand-stir entire flask contents with plunger with rotary motion
to release any oil adhering to flask sides. Wait 30 min, trap
into beaker contg first trapping, and rinse neck of flask, rod,
and plunger with alcohol, then. CHC1 3 to remove all adhering
oil. Filter on ruled paper, rinsing beaker as above, and ex-
amine microscopically.
Ref.: JAOAC 64, 191(1981).
986.28 Mammalian Feces in Corn Meal
Alkaline Phosphatase Detection Method
First Action 1986
A. Principle
Intestinal tract of most mammals contains alk. phosphatase
enzyme. Enzyme at test pH and temp, splits phosphate radicals
from substrate /pH indicator phthln diphosphate to produce light
pink to red-purple color from free phthln.
B. Apparatus
(a) Hot water bath. — Maintained at 42 ± 1°.
(b) Hot plate stirrer and 41 mm ovoid stirring bar. — Fisher
1451 158A or equiv.
(c) Petri dishes. — Plastic disposable, 150 x 20 mm or 150
x 15 mm (Falcon 1058, available from BBL Microbiology
Systems, or plastic/glass equiv.).
(d) Weighing boats.— -8.1 X 8.1 x 1.9 cm, 100 mL ca-
pacity (Fisher Scientific Co., Cat. No. 02210B, or approx.
size equiv.).
C. Reagents
(a) Magnesium chloride soln. — Dissolve 0.203 g
MgCl 2 .6H 2 and dil. to 500 mL with FLO. Indefinite shelf
life.
(b) Stock test reagent.— To prep, soln, dissolve 19.0 g borax
(NaB 4 7 10H 2 O) and 6.28 g anhyd. Na 2 C0 3 in I L H 2 with
stirring. Add 0.94 g phthln diphosphate and stir while adding
2 mL MgCl 2 soln. Prepn is stable ca 4 months at room temp.
Soln should be colorless and ca pH 9.5. Discard if not col-
orless. Degraded phthln diphosphate produces pink color in
reagent. Store phthln diphosphate in desiccator below 0°. (Phthln
diphosphate, Sigma Chemical Co., No. P 9875.)
(c) Liquid test agar. — Prep, fresh before using, 150 mL per
10 g sample to be analyzed. Measure equal vol. of stock test
reagent, (b) (half of total test agar vol. needed), and H 2 into
sep. appropriate size beakers. Beaker for H 2 must be large
enough to accommodate 2 times vol. of H 2 0. Reserve stock
test reagent. Place beaker of H 2 on hot plate stirrer, add stir-
ring bar (ovoid 41 mm), and, with rapid stirring, add sufficient
agar to H 2 to yield 2% agar soln (1.5 g agar/75 mL H 2 0).
Continue stirring, and heat to boil (watch for foam-over). Cover
beaker with cover glass to prevent heat loss. When agar begins
to foam, add reserved stock test reagent, pouring reagent down
side of beaker to prevent agar from coming out of soln. Stir
rapidly with heat ca 1 min.
AOAC Official Methods of Analysis (1990)
Grains 383
D. Determination
Weigh 10 g corn meal into weighing boat from each well
mixed subsample. Prep, appropriate amt of liq. test agar, (c).
Cool boiled test agar by placing beaker of test agar into larger
beaker of cold H 2 0. Continually stir test agar and maintain
temp, check until soln is 55°. Pour test agar into petri dish,
ca 150 mL per dish. Immediately distribute monolayer of corn
meal onto surface of test agar. This is accomplished by gently
tapping weighing boat held so that corn meal flows over one
side, not from corner, while tilting and moving boat above
agar surface as corn meal flows. Let corn meal become wet
with test agar and sink before adding another layer. Continue
in this manner until entire 10 g sample has been added. Dis-
tribution time should be ca 1 min per 10 g corn meal sample.
Best sepn of corn meal and excreta occurs while test agar is
hot. Multiple samples can be added to resp. dishes, one at a
time or a little of each sample to its resp. dish sequentially,
until all of each sample has been distributed.
Let test agar gel (requires ^20 min). Agar is gelled when
no agar flows when dish is slightly tipped. (Note: Take care
not to disturb dispersed material in liq. test agar. If particles
are moved, color concn around particles will be diffused and
pos. spots will be missed.) When gelling is complete, check
for pink spots, viewing plate against white background. Mark
spots on lid of dish, using grease pencil. Mark lid and bottom
of dish, using H 2 0-proof marker, so that lid can always be
placed in same position.
Incubate petri dish at 42° in H 2 bath 10 min. Submerge
plate in H 2 bath just enough to cover agar level in dish. When
incubating several dishes at one time, place plates in H 2 in
pairs, staggering times so that reading delays are avoided, and
small, rapidly diffusing pink spots are not missed. Remove
plate from H 2 bath after 10 min. Wipe inside lid to remove
fog and hold lid so that bottom edge of lid is 2-3 mm above
top edge of petri dish base while reading plate. Replace lid
and repeat 10-min incubation 2 times, marking addnl pink spots
on the petri dish lid after each period. Tally and record number
of spots as fecal particles/10 g sample. Spots which appear
and then are not seen on subsequent checks and spots which
are seen on bottom of petri dish with corn meal are to be counted
in tally.
E. Positive Controi for Feces and Test Agar Medium
Scatter some ground known rodent feces on petri dish of
liq. test agar in place of corn meal sample and continue with
method. One control plate is needed for each batch of test agar
prepd.
F. Response
Amt, intensity, and range of color (light pink to red-purple)
observed will vary depending on size of fecal particles, species
source, and diet of animal. Particles as small as 250 |xm can
be identified.
ReL: J AOAC 69, 496(1986).
941.16
Filth in Grain Products
First Action
(Applicable to corn grits, rye and wheat meal, whole wheat,
flour, farina, semolina)
A. Rodent Excreta
(Caution: See safety notes on toxic solvents, carbon tetra-
chloride and chloroform.)
Weigh 50 g sample in 250 mL hooked-lip beaker. Add CHC1 3
to within ca 1 cm of top, mix thoroly, and let settle >30 min,
stirring surface layer occasionally. Carefully decant CHC1 3 and
float tissue onto buchner, without disturbing heavy residue in
bottom of beaker. Before decanting, take care that floating layer
has not become so compact as to render this operation diffi-
cult. Add amt of CC1 4 equal to amt of CHC1 3 and tissue left
in beaker, let settle again, and decant as before. Repeat this
process with mixt. of equal parts CHCl 3 and CC1 4 until very
little tissue remains in beaker. Do not decant any rodent ex-
creta fragments that may be present. Wash residue in beaker
onto 7 cm ruled paper with stream of CHC1 3 or CC1 4 and ex-
amine microscopically. Retain decanted floating tissues for light
filth analysis.
Ref.: Cereal Chem. 18, 655(1941).
B. Light Filth
(Not applicable to whole and degerminated corn meal)
Draw air thai material in buchner, 941. 16 A, until liq. evaps.
Air dry overnight, or dry in oven at ca 80°. (Caution: In oven
drying, phosgene is liberated and adequate ventilation must be
provided.) Transfer residue to I L trap flask, 945.75B(h)(4).
Add 100 mL 60% isopropanol satd with heptane, 945.75C(n),
and mix thoroly. Wash down sides of flask with isopropanol-
heptane soln until ca 400 mL is added, and soak 30 min. Trap
off twice with 20-30 mL heptane, 945.75C(l), for each trap-
ping and 60% isopropanol satd with heptane as liq. extn me-
dium. In first trapping, let stand 5 min after stirring in heptane
before filling flask. Filter, and examine both trappings micro-
scopically.
970.69 Light Filth in Wheat Germ
Flotation Method
First Action 1970
Final Action 1973
(Applicable to raw or processed wheat germ)
(Caution: See safety notes on toxic solvents and chloroform.)
Form paper cup, using 32 cm paper and 250 and 400 mL
beakers, 945.75B(j). Tare 400 mL beaker and paper, weigh
50 g sample into filter paper in beaker, and add ca 150 mL
CHC1 3 . Boil on steam bath 5 min, occasionally rinsing down
sides of filter paper with CHC1 3 to maintain original level. Re-
move from heat. Carefully lift paper contg sample from beaker
so as to prevent any loss of sample. Let most of CHC1 3 drain
into beaker; then discard drainings. Repeat operation 2 addnl
times beginning "... add ca 150 mL CHC1 3 . w After last CHC1 3
defatting, place paper contg sample in buchner. Apply vac.
until draining slows to drip. Rinse sides of paper and sample
with undild isopropanol and apply vac. until draining has ceased.
Turn off vac. Add ca 50-60 mL undild isopropanol to sample.
Let stand 2 min; then apply vac. until dripping ceases and sam-
ple appears dry.
Transfer sample from paper to 1 L beaker with hot tap H 2
(55-70°). Fold filter paper in half and rub together; then wash
with hot tap H 2 into beaker. Repeat several times until paper
appears clean. Discard paper and bring vol. of hot H 2 to 600
mL. Add 30 mL HC1 and 1 mL antifoam, 945.75C(e). Boil
on stirrer-hot plate, 945.75B(n), 10 min with const stirring;
then remove from heat. Pour contents of beaker onto No. 230
sieve, 945.75B(r), and wash with forcible hot H 2 spray (55-
70°), 945.75B(a), until all starchy material has passed thru and
only bran remains (color of sample will change from light tan
384
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1 990)
to dark brown). Transfer material from sieve to 2 L trap flask
with 55% alcohol or 40% isopropanol, diL to ca 1 L, and add
50 mL HCL Heat to 60-70° on hot plate (do not boil), remove
flask from heat, and add 50 mL mineral oil, 945.75C(p). Stir
mag., 970.66B(c), 3 min. Fill flask with same diL alcohol
used previously, stir gently by hand 1 min, let flask stand 10
min, and trap off, rinsing neck of flask with same dil. alcohol
used previously. Perform second extn, using 25 mL mineral
oil. Stir gently by hand 1 min, let stand 15 min, and trap off.
Rinse neck of flask with undild isopropanol or alcohol. Filter
trappings thru ruled paper and examine microscopically.
Ref.: JAOAC 53, 560(1970).
972.32 Light Filth (Pre- and Post-Milling)
in Flour (White)
Flotation Method
First Action 1972
Final Action 1988
Digest 50 g flour in 2-2.5 L beaker with ca 600 mL HC1
(3 + 97) by autoclaving 5 min at 121°. Immediately transfer
digest to 1 L beaker, using HCI (3 + 97) at room temp, to
asist in transfer. Add 50 mL mineral oil, 945.75C(p), and stir
mag. 970.66B(c), 5 min. Quant, transfer to Kilborn funnel,
945.75B(h)(/), or percolator, (2), retaining beaker. Let stand
30 min, stirring gently with long glass rod several times during
first 10 min. Drain lower layer to ca 3 cm of interface, wash
sides with cold tap H 2 0, and let layers sep. ca 2-3 min. Re-
peat drain and H 2 wash until lower phase is clear. After final
wash, drain oil layer into retained beaker, rinsing sides of fun-
nel with H 2 and alcohol. Add HCI to ca 3% (v/v) and boil
3-4 min on hot plate. Filter hot soln thru ruled paper, and
thoroly rinse beaker and funnel with H 2 0, alcohol, and 5%
detergent soln, 945.75C(i). Filter each rinse sep. thru same
paper. Examine microscopically.
Ref.: JAOAC 55, 514(1972).
940.34
Insect Eggs in Flour
Sieving Method
First Action
Transfer 50 g flour to No. 100 sieve (if >ca 0.1 g residue
is obtained, No. 60 or No. 80 sieve should be used to prevent
slow filtration after digestion) and sift gently until no more
flour passes thru. Transfer residue on sieve to 250 mL beaker
and wet with 2-3 mL alcohol. Add 30 mL H 2 S0 4 (1 + 19),
cover beaker, and heat on steam bath 10 min. Filter thru paper
on suction funnel, using min. suction necessary to filter. Keep
beaker partially inverted over funnel and rinse with H 2 0. Turn
off suction. Add 15-20 mL ca 0. IN 1 to paper in funnel. Al-
low 10-15 sec for I to stain contents. Apply gentle suction.
After I passes thru filter, wash paper with 25-30 mL 1% H 2 S0 4 ,
followed by several small H 2 washes. Transfer paper to petri
dish and examine at once under 20 x magnification.
Ref.: Food Ind. 12, 36(1940).
943.06
Insect Excreta in Flour
Final Action 1988
(a) Optional for 1-4 samples. — Weigh 0.20 g flour on
weighed flat glass disk 7-7.5 cm diam. Add clove oil and
spread mixt. into thin uniform layer. (Enough oil should be
present to clear flour and present smooth surface of oil, but
not so much that mixt. flows off disk.) Place wire grid over
disk and examine microscopically with dark background and
intense reflected light. Depending upon size of plate, larger
amts of flour and ruled glass plate can be used and oil-flour
mount covered with glass, e.g., use 0.5 g flour on tomato rot
count plate, 945.75B(q). Weigh flour in counterbalanced scoop
or directly on plate. Thoroly sat. flour on counting plate, cover
with glass, and count insect excreta. To move or turn sus-
pected particles, gently apply pressure or move cover slightly
while observing thru microscope.
(b) Optional in multiple-sample schedule. — Tare 2—8 small
numbered vials on each balance pan and weigh by shifting
weights from one side to other. (If desired, larger portion may
be weighed in beaker and some of flour floated off in CHC1 3 -
ether or CHCl 3 -toluene mixt., sp gr 1.40, before transferring
to filter paper.) Rinse contents of each vial onto smooth-sur-
face, ruled paper in Hirsch funnel with CHC1 3 or CC1 4 . Trans-
fer paper to petri dish, flood with clove oil, and examine with
dark background and intense reflected light.
Ref.: JAOAC 26, 257(1943).
982.32 Light Filth in Rice Flours
(Powders), Extruded Rice Products,
and Rice Paper
Flotation Method
First Action 1982
Final Action 1988
A. Sample Preparation
(a) Rice flours (powders). — Preheat hot plate to max. heat.
Add mag. stirring bar to 2 L beaker, and tare. Add 100 g
sample. With forceful stream, add ca 100 mL hot tap H 2 0.
Add 75 mL HCI and fill to 800 mL mark with hot tap H 2 0.
Place hot mixt. on hot plate and, with mag. stirring, bring
mixt. to vigorous boil. Boil 5 min. In small increments, trans-
fer hot mixt. to No. 230 sieve. Reserve 2 L beaker. Wash
residue with forceful stream of hot tap H 2 until foaming has
subsided and H 2 is clear. Transfer residue to reserved 2 L
beaker with 40% isopropanol. Add mag. stirring bar. Fill with
40% isopropanol to 800 mL mark. With mag. stirring, bring
to boil on hot plate. Add 95 mL mineral oil, 945.75C(p), and
boil and stir 3 min.
(b) Extruded rice products and rice paper. — Preheat hot plate
to max. heat. Add mag. stirring bar to a 2 L beaker, and tare.
Break up 225 g extruded product or paper while weighing into
tared beaker. Add ca 1450 mL hot tap H 2 mixed with 150
mL HCI. Hand-stir with glass rod while heating to vigorous
boil. When product is fluid enough, use mag. stirring. Boil 10
min. In small increments, transfer hot mixt. to No. 230 sieve.
Reserve 2 L beaker. Wash residue with forceful stream of hot
tap H 2 until foaming has subsided and H 2 is clear. Transfer
residue to reserved 2 L beaker with 40% isopropanol. Add
mag. stirring bar. Fill with 40% isopropanol to 800 mL mark.
With mag. stirring, bring to boil on hot plate. Add 95 mL
mineral oil, 945.75C(p), and boil and stir 3 min.
B. Filth Analysis
(a) Rice flours (powders). — Clamp off rubber hose on per-
colator, 945.75B(h)(2). Add 300 mL 40% isopropanol. Trans-
fer hot sample mixt. from above to percolator. Rinse 2 L beaker
with 40% isopropanol and pour rinse into percolator. With same
beaker, add enough 40% isopropanol (ca 1 L) to fill percolator
within 3 cm of top. Let stand 5 min and drain contents to 5
cm from bottom of oil layer. Repeat fill and drain steps at 2
min intervals with hot tap H 2 until aq. phase is clear. Drain
as above. Drain oil layer into 1 L beaker. Rinse percolator
sides with several alternate washes of H 2 0, 40% isopropanol,
AOAC Official Methods of Analysis (1990)
Baked Goods
385
and isopropanol, collecting rinsings in same I L beaker. A 1%
sodium lauryl sulfate soln may also be used if needed for final
rinse. Filter onto ruled filter paper and examine microscopi-
cally at ca 30 x.
(b) Extruded rice products and rice paper. — Proceed as in
(a), except for second cycling. Refill percolator with 20% iso-
propanol. Let stand addnl 5 min. Drain oil layer into 1 L beaker
and rinse percolator as above.
Ref.: J AOAC 65, 1086(1982).
965.39 Light Filth in Flour (Corn)
Final Action 1989
A. Pancreatin Digestion Method
Light filth. — Weigh 50 g flour into 600 mL beaker; stir into
smooth slurry with 50 mL pancreatin soln, 945.75C(q), dild
with 100 mL H 2 0. Dil. with H 2 to total vol. of ca 400 mL,
and adjust to pH 8 with Na 3 P0 4 soln, 945.75C(u). Readjust
pH after ca 15 min and again in ca 45 min. Add, with stirring,
3 drops HCHO soln and digest 16-18 hr at room temp, or
<40°. Transfer to 2 L trap flask and ext as in 970.66B(b),
using 30 and 20 mL deodorized kerosene, 945.75C(o), and
H 2 as sol vs. Combine trappings and rinsings in beaker, trans-
fer to 2 L trap flask, and trap off as above. If considerable
starchy material is in ext, hydrolyze with HC1 as in 970.666(d) .
Examine papers microscopically.
B. Acid Hydrolysis Method
Light filth. — Disperse 50 g flour in 1 L beaker with ca 400
mL HCJ (5 + 95) and 20 mL mineral oil, 945.75C(p). Place
on hot plate, bring to rolling boil with stirring, and boil 10
min. Remove from heat and transfer quant, to extn vessel,
945.75B(h)(7) or (2). Rinse beaker and rod with ^50 mL hot
H 2 0, transfer rinsings to extn vessel, and retain beaker and
rod. Fill extn vessel with cold H 2 to ca 3 cm from top. Let
settle 30 min, and drain carefully without forming vortex, until
upper layer is ca 5 cm from bottom. Add 25 mL kerosene,
945.75C(o), to extn vessel and drain oil layer into retained
beaker. If excessive starchy material has sepd with oil layer,
hydrolyze with 100-200 mL HC1 (5 + 95) before continuing.
Wash sides of extn vessel with 5% detergent soln, 945.75C(i),
in wash bottle, and collect washings in retained beaker. Filter
entire contents of beaker thru ruled paper, 945.75B(i), in Hirsch
funnel. Rinse beaker with 5% detergent soln, and filter. Ex-
amine microscopically at 30 x.
C. Rodent Excreta
Proceed as in 941.16A.
Ref.: JAOAC 48, 554(1965).
972.33 Light Filth in Flour (Soy)
Flotation Method
First Action 1972
Final Action 1988
Weigh 50 g sample into 500 mL beaker and add ca 50 mL
isopropanol, with stirring. Add mag. stirring bar and, with
stirring, slowly add 300 mL satd NaCl soln at room temp. Stir
to thin slurry and transfer in small increments to No. 230 sieve,
945.75B(r). Wash residue with forceful stream of cold H 2 0,
using aerator, 945.75B(a), until effluent is clear and foaming
has subsided. Let residue drain in sieve. Wash residue with
isopropanol and let drain. Transfer residue to 1.5 L beaker
with 40% isopropanol and dil. to 800 mL with 40% isopro-
panol. Heat to bp with mag. stirring, and add 50 mL mineral
oil, 945.75C(p). Stir mag., 970.66B(c), 3 min while con-
tinuing to boil.
Transfer quant, to 2 L percolator, 945. 75B (h)(2). Retain 1.5
L beaker as vessel to fill percolator with H 2 during refill cycles.
Fill percolator with 40% isopropanol to within 3 cm of top.
Let stand 5 min and drain contents to within 3 cm of bottom
of oil layer. Repeat drain and refill steps at 3 min intervals
with hot tap H 2 (55-70°) until aq. phase is clear. Drain most
of aq. phase and discard. Drain oil layer into 400 mL beaker.
Wash percolator with alternate washes of H 2 and isopropa-
nol, and collect washings in 400 mL beaker. Filter onto ruled
paper and examine microscopically.
Ref.: J AOAC 55, 60(1972).
972.34 Light Filth in Wheat Gluten
Flotation Method
First Action 1972
Final Action 1988
Add 900 mL 40% isopropanol, 100 mL HO, and mag. stir-
ring bar to 2 L trap flask, 945.75B(h)(4). Using mag. stirrer,
without heat, stir at max. speed where no splashing occurs,
and slowly add 50 g sample thru long-stem funnel with wide
diam. bore to avoid getting sample on stoppered rod or sides
of trap flask. Stir 3 min, and then boil 15 min with gentle
stirring on mag. stirrer-hot plate. Immediately transfer flask to
cool stirring unit and add 40 mL light mineral oil, 945.75C(p).
Stir mag., 970.66B(c), 3 min. Slowly fill flask with 40% iso-
propanol by letting liq. flow down stoppered rod while top of
stopper is maintained just above flask contents. After filling
flask, gently stir settled material 5-10 sec with stoppered rod.
Let stand undisturbed 5 min and immediately trap off. Add 25
mL light mineral oil, stir gently by hand 30 sec, and let stand
10 min. Repeat trapping. Wash flask neck thoroly with iso-
propanol and transfer washings to beaker contg trappings. Fil-
ter onto ruled paper and examine microscopically.
Ref.: JAOAC 55, 64(1972).
972.35 Light Filth in Starch
Sieving Method
First Action 1972
Final Action 1988
Weigh 225 g sample into 2 L beaker, add 1.2 L cold H 2 0,
and stir well to disperse lumps. Pour onto No. 230 sieve,
945.75B(r), and wash with forcible stream of cold tap H 2 0,
using aerator, 945.75B(a). (If excessive residue remains on
sieve, wash into beaker with H 2 0, add HC1 to make to (1 +
9), bring to bp, and repeat sieving.) Quant, transfer sieve re-
tainings to beaker, filter onto ruled paper, and examine mic-
roscopically.
Ref.: JAOAC 55, 62(1972).
BAKED GOODS
972.36 Light Filth in High
Bran Content Breads
Flotation Method
First Action
Post-milling contamination. — Add 225 g sample to 2 L beaker
contg ca 1 L H 2 and 50 mL HC1. Stir well. Add 1 mL an-
tifoam soln, 945.75C(e). Autoclave ca 20 min as in
386
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
945.75B(b)(7) or ca 15 min as in (2). Transfer digest in small
portions onto No. 140 sieve, 945.75B(r), with hot tap H 2
(55-70°) until amt of residue remains const. Place sieve in
pan, cover residue to depth of ca 2 cm with alcohol or iso-
propanol, let stand 5 min, and drain. Repeat this step 3 times
with CHC1 3 , then twice more with alcohol or isopropanol, and
drain completely. Promptly transfer sieve retain ings quant, to
1 L beaker with acid-alcohol, 945.75C(a), dilg contents to ca
600 mL with acid-alcohol. Add 50 mL mineral oil, 945.75C(p),
and stir mag., 970.66B(c), 5 min.
Completely transfer beaker contents to percolator,
945.75B(h)(2), or Kilborn funnel, (7), retaining beaker. Let
stand 30 min, stirring gently ca every 5 min with long glass
stirring rod for first 20 min; drain contents to ca 250 mL. Add
acid-alcohol to ca 3 cm of top and let stand 30 min, stirring
as before. Again drain to ca 250 mL. Fill funnel with cold tap
H 2 0, let settle ca 1.5 min, and drain to ca 250 mL. Continue
drain and refill cycles until lower aq. phase is clear and free
of suspended material.
After last wash, drain oil-H 2 interface into retained beaker.
Promptly wash sides of percolator with 50-100 mL portions
hot tap H 2 0, isopropanol or alcohol, and 5% detergent soln,
945.75C(i), if necessary. Filter thru ruled paper, washing sides
of beaker as above, using rubber policeman, if necessary. Ex-
amine microscopically.
Ref.: JAOAC 55, 516(1972).
972.37 Light Filth in Baked Goods
with Fruit and Nut Tissues
First Action 1972
A. Pancreatin Digestion Method
Post-milling contamination. — Weigh 225 g sample into 2 L
beaker, add enough hot H 2 to soften and sat. material, and
proceed as in (a). If lumps persist or if H 2 is not immediately
absorbed uniformly thru entire mass, proceed as in (b).
(a) Adjust mixt. to pH 7-8 with ca 5% Na 3 P0 4 soln. Stir
and break up material as much as possible. Cool to 40° and
add 100 mL pancreatin soln, 945.75C(q). Stir thoroly and
readjust to pH 7-8. Let stand 30 min, stir, and readjust pH.
(b) Est. vol. of mixt. and add HC1 to ca 1 + 49 concn. Boil
until solids become finely divided and so digested that mixt.
will not froth over when covered during boiling. Neutze to ca
pH 6 with NaOH soln; then add Na 3 P0 4 soln to pH 8 and
continue as in (a).
For white flour products, add 0.2 mL or 4 drops HCHO and
digest overnight. For products made from whole wheat and
rye flours and from similar materials of high bran content, di-
gest only 2-3 hr.
Pour digested material thru 5" or 8" No. 140 sieve,
945.75B(r). While pouring, play forcible stream of hot H 2
from tap on this material. Wash well with large stream of hot
H 2 0. After complete washing (no starchy material visible un-
attached to bran), wash twice alternately with alcohol and CHC1 3
in that order, and then rinse thoroly with alcohol and finally
with H 2 0.
Transfer material to filter paper if little residue remains or
to 1 or 2 L trap flask, 945.75B(h)(4), if large amt remains.
Transfer bulk of material with spoon. Rinse residue from screen
with 60% alcohol from wash bottle. Wash screen with forcible
stream of hot H 2 0, collecting final residue at one edge of screen
and transferring to trap flask with stream of 60% alcohol as
above. Add 400 or 900 mL 60% alcohol, depending on size
of trap flask.
Boil 20 min. Cool to <20° and add 20 or 40 mL heptane,
945.75C(1); fill flask with 60% alcohol, and trap off twice.
Use care in stirring and adding alcohol to prevent emulsions
or inclusion of air. If residue in flask tends to rise, stir material
down 2 or 3 times. Filter trapped-off material and examine
microscopically.
B. Acid Hydrolysis Method
(Rapid method; also applicable to flours)
Post-milling contamination. — Add 225 g sample to 2 L beaker
contg ca 1 L H 2 and 30 mL HC1. Wet product completely
and, for flour, stir until slurry is practically lump-free. Add
antifoam soln, 945.75C(e), cover with watch glass, and heat
15-20 min in autoclave at 121°. Let pressure fall to before
opening vent valve. Transfer digest in small portions to 5" or
8" No. 140 sieve, 945.75B(r), washing thoroly between addns
with needle spray from aerator. After all sample has been
transferred, continue washing until there is no further reduc-
tion in amt of residue. Proceed as in 972. 37 A, par. 5, begin-
ning "After complete washing ..."
Ref.: JAOAC 45, 660(1962).
950.94* Light Filth (Pre- and Post-MiBiing)
in Baked Goods with Fruit and Nut Tissues
Pancreatin Digestion Rotation Method
First Action
Surplus 1975
See 44.056, 12th ed.
970.70 Light Fiith in White Breads
and High-Fat Products
Flotation Method
First Action 1970
Final Action 1988
Add 1 L hot (55-70°) tap H 2 to 2 L beaker. Add 20 mL
emulsifier, 945.75C(j)(2) ? and" 5 mL (j)(7), and mix well.
Add 225 g sample, breaking any crust to <1 sq in. Stir well.
Proceed with either autoclave, (a), or steam bath, (b), diges-
tion.
(a) Autoclave. — Add 30 mL HC1 with stirring. Add 1 mL
antifoam soln, 945.75C(e). Autoclave as in 945.75B (b)(7) or
(b)(2) for 30 min at 121°.
(b) Steam bath. — Add 90 mL HO with stirring. Heat in
steam bath 10 min. Add 1 mL antifoam soln. Boil 15 min on
mag. stirrer-hot plate, 945.75B(n), keeping beaker covered with
watch glass.
Wet sieve on No. 230 plain weave sieve, 945.75B(r), with
hot H 2 (55-70°). Sieve until effluent is clear and foam is
gone. Transfer sieve retainings to original beaker. (Caution:
Do not allow sample in beaker or sieve to cool.) Add 30 mL
HC1 and dil. to 1 L with H 2 0. Stir on stirrer-hot plate, and
bring to bp. Boil 6 min, add 50 mL mineral oil, 945.75C(p),
and continue heating until boiling resumes. Transfer beaker to
cool mag. stirrer and stir mag., 970.66B(c), 3 min.
Promptly transfer beaker contents to percolator,
945.75C(h)(2), contg ca 250 mL H 2 0. Rinse beaker into per-
colator and bring vol. to 1700 mL mark with H 2 0. After 1
min, stir percolator contents with glass rod. Place rod in beaker
AOAC Official Methods of Analysis (1990)
Cereals
387
and set aside to receive final oil drain. Let stand 2 min. Drain
oil to 250 mL mark and discard drainings. Refill percolator
with H 2 0. Continue drain and refill cycles until lower aq. phase
is almost clear. Drain oil to 250 mL mark. Drain oil into orig-
inal beaker. Wash percolator sides with min. of 50 mL H 2
and alcohol or isopropanol. If sides do not appear clean, fol-
low with H 2 and 5% detergent wash, 945.75C(i). Filter onto
ruled paper and examine microscopically.
Ref.: J AOAC 53, 562(1970).
975.46 Light Filth in Breading
of Frozen Food Products
Flotation Method
First Action 1975
Final Action 1988
Using clip tongs, place each portion individually in H 2 G bath
maintained at 17-49°C (63-l20°F). Let remain until breading
becomes soft (5—1 10 sec for portions held in storage at — 18°C
(0°F) and can easily be removed with round tip, 10 cm (4")
blade spatula or table knife. Limit dip time in >100°F H 2 to
15 sec max.
(Note: Several preliminary trials may be necessary to det.
dip time required for debreading sample units. For these trials
only, prep, satd soln of CuS0 4 .5H 2 (450 g (1 lb)/2 L tap
H 2 0). Correct dip time is min. time of immersion in CuS0 4
soln required before breading can be easily scraped off, pro-
vided that debreaded portions are still solidly frozen, and only
slight trace of blue color is visible on surface of debreaded
portions. As guide, use lower temps with raw and higher temps
with precooked products.)
After immersion, remove portion and blot lightly with dou-
ble thickness paper towel. Complete this step in <7 sec. Scrape
and remove breading and batter with spatula, removing ma-
terial from narrow sides and ends in initial movements fol-
lowed by removal from wide flat surfaces. If breading is dif-
ficult to remove, redip partially debreaded portion in H 2 at
room temp. (17-30° C; 63-86° F) ca 2 sec. Blot with towel
and remove residual batter and breading material.
Weigh 50 g sepd breading in 1 L beaker. Add ca 300 mL
hot tap H 2 (55-70°), 5 mL emulsifier CO-730, 945.75C(j)(/),
20 mL emulsifier DM-710, 945.75C(j)(2), and 60 mL HC1.
Fill beaker to 600 mL with hot tap H 2 0, stir well, and heat
beaker in steam bath 20 min, stirring at 5 min intervals. Add
1 mL antifoam soln, 945.75C(e). Boil 15 min on preheated
hot plate, keeping beaker covered with watch glass. Wet-sieve
on No. 230 plain weave sieve, 945.75B(r), with hot H 2 0, un-
til effluent is clear and foam has dispersed. Transfer sieve re-
tainings to original beaker with HCl-60% alcohol soln (1 +
9), and bring vol. to ca 600 mL. Add 50 mL mineral oil,
945.75C(p), and stir mag., 970.66B(c), 10 min. Place stirring
rod in drain opening of percolator, 945. 75B (h)(2), and add ca
250 mL acid-alcohol soln. Quant, transfer beaker contents to
percolator. Add acid-alcohol soln to ca 6 cm from top, let stand
5 min, resuspend solids with stirring rod, and let stand addnl
5 min. Drain to ca 250 mL mark, refill percolator to original
vol. with acid-alcohol soln, and repeat previous steps until lower
aq. phase is almost clear. Drain oil to 250 mL mark and trans-
fer to original beaker. Wash percolator sides with 50 mL H 2 0,
and alcohol or isopropanol. If sides do not appear clean, fol-
low with H 2 and 5% detergent wash, 945.75C(i). Filter onto
ruled paper and examine microscopically.
Ref.: J AOAC 58, 441(1975).
969.41 Light Filth
in Alimentary Pastes
Flotation Method
First Action 1969
Final Action 1988
Weigh 225 g sample into 1.5-2.0 L beaker. Add 1 L HC1
(30 + 970) and 0.3 mL antifoam soln, 945.75C(e). (For spa-
ghetti, break into lengths that will not lie flat on bottom of
beaker.) Autoclave 30 min at 121° as in either 945.75B(b)(/)
or (2).
Wet sieve on No. 230 sieve, 945.75B(r), with hot tap H 2
(50-70°) to remove all original liq. and major portion of fine
material.
Return sieve retainings to original beaker with hot H 2 (60-
100°), dilg to ca 1 L. Add 30 mL HCI, mag. stirring bar, and
50 mL mineral oil, 945.75C(p).
Stir mag., 970.666(c), 6 min. Promptly transfer to perco-
lator, 945.75B(h)(2), contg ca 250 mL H 2 0. Rinse beaker into
percolator with hot tap H 2 to bring to 1700 mL. After 3 min,
drain oil interface to 250 mL. Discard drainings and refill by
pouring hot tap H 2 down percolator sides to loosen adhering
material and refill to 1700 mL mark. After 2-3 min, drain and
refill for 2 more cycles. (Lower layer should be almost free
of suspended material after last refill; if not, continue thru >1
recycles.) Finally, drain oil-H 2 interface to 250 mL mark,
change to original beaker, and drain. Promptly wash down sides
successively with >50 mL portions hot tap H 2 0, isopropanol
or alcohol, and hot tap H 2 0. Use 5% detergent soln, 945.75C(i),
if necessary.
Transfer beaker contents to ruled filter paper with min. of
50 mL washes of hot H 2 0, alcohol or isopropanol, and H 2
or detergent, using rubber policeman if necessary to clean sides
of beaker. Examine microscopically.
Ref.: JAOAC 52, 463(1969).
BREAKFAST CEREALS
970.71 Light Filth in Cereals
(Corn and Rice) and Corn Chip Products
Flotation Method
First Action 1970
Final Action 1988
(Caution: See safety notes on flammable solvents.)
(a) Cereals and food products containing no fats or oils. —
(Check ingredient label.) To 1-1.5 L beaker (depending on
bulk of product), add 50 g sample, 500 mL hot (55-70°) tap
H 2 0, and 40 mL HCI. Bring mixt. to full boil on mag. stirrer-
hot plate, 945.75B(n), using slow stirring speed. Boil 20 min
and wet sieve immediately on No. 230 sieve, 945.75B(r), with
forceful hot (55-70°) H 2 spray until residue no longer passes
thru sieve and H 2 is clear. Wash sieve retainings either into
2 L Wildman trap flask, 945.75B(h)(4), or back into original
beaker if Kilborn, (h)(7), or percolator, (h)(2), is to be used,
using 40% isopropanol.
(!) Trap flask-— Bring vol. to 800 mL with 40% isopro-
panol and add 30 mL HCI. Raise stirring rod plunger and
secure above liq. with clamp. Add mag. stirring bar,
945.75B(n), and stir at slow speed while bringing mixt. to bp.
Boil 5 min. Add 50 mL mineral oil, 945.75C(p), and stir mag.,
970.66B(c), 3 min.
Remove from heat and fill with 40% isopropanol. Let stand
388
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
10 min and trap off, rinsing neck of flask and rod with iso-
propanol or alcohol. Filter trappings thru ruled paper. Examine
microscopically.
(2) Kilborn or percolator . — Bring vol. in original beaker
to 600 mL with 40% isopropanol and add 25 mL HC1. Bring
to bp with slow stirring, boil 5 min, add 50 mL mineral oil,
and stir mag. 3 min, 970.66B(c).
Transfer from beaker to separator, rinsing beaker into sep-
arator with 40% isopropanol. If residue in separator is heavy,
resuspend with glass rod. Rinse rod into separator.
Let stand 3 min and drain contents to within 3 cm of bottom
of oil layer. Refill with hot (55-70°) tap H 2 0. Repeat drain
and refill steps with 3 min intervals, until H 2 phase is free
of plant material. Discard drainings. Drain oil layer into orig-
inal beaker, rinsing sides of separator alternately with isopro-
panol or hot H 2 and alcohol, using rubber policeman to clean
sides. Filter contents of beaker thru ruled paper. Examine mic-
roscopically.
(b) Cereals and food products containing natural and syn-
thetic fats or vegetable oils. — Proceed as in (a), beginning
"... 500 mL hot (55-70°) tap H 2 0, ..." but also add to
this mixt. 20 mL emulsifier, 945.75C(j)(2); then proceed as
in (a) with no further changes.
Ref.: J AOAC 53, 558(1970).
971.32 Light Filth in Cereals
(Whole Wheat)
Flotation Method
First Action 1971
Final Action 1988
Weigh 50 g sample into 1 L beaker. Add 500 mL H 2 and
40 mL HC1. Boil 20 min with mag. stirring to keep solids from
scorching. Immediately after boiling, wet-sieve on No. 230
sieve, 945.75B(r), with hot (55-70°) tap H 2 until effluent is
clear. Transfer sieve retain ings with isopropanol to preshaped
filter paper cup, using >24 cm paper and 150 and 250 mL
beakers, 945.75B(j). Transfer paper and retainings to Hirsch
funnel. Apply vac. to apparent dryness. Turn vac. off, add
150 mL isopropanol, and vac. dry. Repeat isopropanol wash-
ing and vac. drying 2 more times.
Transfer filter paper retainings with 40% isopropanol to 2
L trap flask contg 25 mL oleate soln (10 g Na oleate,
945.75C(t), dild to 100 mL with 40% isopropanol and stirred
in at room temp.). Bring vol. of flask to 800 mL with 40%
isopropanol. Boil 5 min with mag. stirring. Cool contents of
flask to 23 ± 2° with either air or H 2 cooling. Add 50 mL
flotation liq., 945.75(k), and stir mag. 3 min, 970.66B(c). Fill
flask with 40% isopropanol by slowly running isopropanol down
rod onto top of stopper, held ca 3 mm above liq. Let stand 3
min and' trap off. Add 50 mL flotation liq. and stir ca 15 sec.
Add 40% isopropanol as needed to fill flask. Let stand 20 min
and perform second trapping. Combine trappings. Filter thru
ruled filter paper and examine microscopically.
Ref.: JAOAC54, 573(1971).
980.27 Light Filth in Barley,
Oatmeal, and Mixed Dry Infant Cereal
Flotation Method
First Action 1980
Final Action 1988
Place 50 g sample in 2 L beaker and, with forceful stream
hot (55-70°) H 2 directed at side of beaker, add 1 L H 2 0.
Add 80 mL HCL If product ingredient list includes vegetable
oil, add 20 mL DIVL710, 945.75C(j)(2). Place mag. stirring
bar, 945.75B(n), in beaker and put beaker on preheated mag.
stirring hot plate set at max. heat. Initially stir vigorously to
prevent scorching, then slowly as mixt. thins while heating 20
min. If product darkens, reduce heat to next lower step. Wet-
sieve with forceful stream hot H 2 on No. 230 sieve until H 2
becomes clear. Wash residue to one side of sieve. Drench wet
residue with isopropanol and let drain. Form paper cup by
wrapping No. 588 (S&S) 32 cm filter paper, or fluted equiv.,
around 600 mL beaker and forcing it into 1 L beaker. Remove
600 mL beaker from paper cup. Leave paper cup in 1 L beaker.
Using 6" (15 cm) powder funnel to aid transfer, wash residue
from sieve into paper cup with isopropanol. Add enough iso-
propanol to bring to 400 mL mark. Boil gently on preheated
hot plate 10 min in hood. Drain isopropanol from paper cup
by gravity or vac. (Do not let residue dry out.) Discard iso-
propanol. Moisten sides of 2 L trap flask with 40% isopro-
panol. Place powder funnel in neck of 2 L trap flask. Wash
residue from filter paper cup into trap flask with 40% isopro-
panol .
Fill to 800 mL mark with 40% isopropanol. Add 50 mL
mineral oil, 945.75C(p), and mag. stir 3 min. After stirring,
add 50 mL 1 + 1 mixt. Tween-80 and 40% isopropanol soln,
945.75C(x) and Na 4 EDTA plus 40% isopropanol soln,
945.75C(z), slowly down stirring rod. Do not mix. Let stand
5 min. Fill trap flask with 40% isopropanol by pouring slowly
down stirring rod. Let stand 20 min and trap off. Add 35 mL
mineral oil, stir gently by hand 30 sec, and let stand 10 min.
Repeat trapping. Filter onto ruled paper and examine micro-
scopically at 30 x.
Ref.: J AOAC 63, 187(1980).
EGGS AND EGG PRODUCTS
(Eggs may be contaminated with chicken excrement, dirt, sand,
metal fragments, hairs, and feathers, depending upon condi-
tion of the eggs, method of manufacture, and storage condi-
tions. Method of isolation of contaminants depends upon na-
ture of product (whole, whites, or yolks) and physical state
(fresh, frozen, or dried).)
960.50 Filth in Eggs and Egg Products
First Action 1960
Final Action 1989
A. Reagents
(a) Anionic surfactant. — Na N-methyL/V-tall oil acid taur-
ate, Igepon TK-32 (GAF Chemicals Corp, 1361 Alps Rd,
Wayne, Nj 07470), or equiv.
(b) Phenolphthalein soln. — Prep. 5% soln in alcohol, dil.
with equal vol. H 2 0, and filter.
(c) Disodium phosphate soln. — Filtered satd soln (ca 100 g
anhyd. salt/L), and filtered 6% (anhyd. basis) aq. soln.
(d) Trisodium phosphate soln. — Filtered satd soln.
(e) Tetrasodiwn EDTA soln. — 10% filtered aq. soln of
Na 4 EDTA.
B. Light and Heavy Filth and Other Extraneous Materials
(a) Whole eggs or yolks. — Thaw frozen sample at room temp,
or in cold running H 2 0. Weigh 100 g thawed sample into 250
mL centrf. bottle. Add 30 mL 6% Na 2 HP0 4 and stir. Shake
vigorously 1.5-2.0 min, add addnl 30 mL 6% Na 2 HP04, and
shake ca 2 min. Dil. with 6% reagent to fill bottle and centrf.
AOAC Official Methods of Analysis (1990)
Meat and Poultry
389
whole eggs 5 min at 1 500 rpm and yolks 5 min at 800 rpm.
Decant ca 2 /s liq. into 1.5 or 2.0 L beaker and isolate light
filth as in (b). Add ca equal vol. 6% Na 2 HP0 4 soln to residue
in bottle, shake well, and recentrf. Decant closely. To sedi-
ment in bottle add ca l / 2 vol. H 3 P0 4 and warm on steam bath.
Transfer to 250 mL beaker, boil 3-5 min, and filter while
boiling. Examine at 30 X for metal and glass fragments, and
chicken excrement. Check amorphous white material for uric
acid as in 962.20.
(b) Egg whites. — Use decanted whole egg or yolk material
from (a) or weigh 100 g thawed whites into 1.5 or 2.0 L beaker
and add ca 300 mL 6% Na 2 HP0 4 soln in small portions with
thoro stirring. Add 16 mL Na 4 EDTA soln, (e), then 12 mL
phthln, (b). Let stand 10 min; then adjust to pH 7.6-8.0, using
H 3 P0 4 (1 +9) or Na 3 P0 4 soln as needed. Add 2 mL surfac-
tant, (a), and readjust to pH 7.6-8.0, using short range pH
paper. Add 200 mL pancreatin soln, 945.75C(q), and readjust
to 7.6-8.0.
Place in 37-38° H 2 bath to ca depth of digestion mixt.;
stir, and adjust to pH 7.6-8.0 at ca 15 min intervals for ca 2
hr. Add 2 mL surfactant and dil. to 1.0-1 .2 L with H 2 0. Ad-
just to pH 8.0 and place in incubator at 37° overnight. Readjust
to pH 8.0 and let stand 15-20 min without stirring. Decant in
small portions onto ruled paper, using full suction, while
washing paper with hot tap H 2 0. Examine paper microscopi-
cally.
(c) Dried egg yo/fcs.— Defat egg yolks as follows: Weigh
25 g sample into 150 mL tall-form beaker, add 50 mL pet
ether, and stir thoroly (until smooth). While stirring, add sol v.
to almost fill beaker. Stir top again after 1 min, let stand 1
min, and decant soJv. into larger beaker. Repeat defatting step
twice more with pet ether. Filter combined washes thru smooth
textured paper, air dry paper thoroly, and hold for pancreatin
digestion. Discard sol v. Place 150 mL beaker on steam bath
and remove solv. completely from residue with continuous
stirring to prevent bumping. Transfer dried residue in beaker
and dried residue on paper, using spatula, to 600 mL beaker
and proceed as in (d).
(d) Dried whole eggs. — Weigh 25 g sample into 600 mL
beaker, or continue with dried yolk residue from (c). Add mixt.
of 90 mL satd soln of Na 2 HP0 4 and 10 mL alcohol in small
portions with stirring. (Suspension must be smooth and finely
divided at this point.) With stirring, add 12 mL Na 4 EDTA soln,
(e), then 5 mL phthln, (b). If intense red develops, discharge
with H3PO4 (1 + 9). Adjust to pH 7.6-8.0 with satd Na 3 P0 4
soln, using short range indicator paper. Add 200 mL pancre-
atin soln, 945.75C(q), to suspension. Continue as in (b), be-
ginning "Place in 37-38° H 2 bath . . ."
(e) Dried whites. — Weigh 25 g sample into 250 or 400 mL
beaker. Dil. 4.5 mL surfactant, (a), to 35 mL with H 2 and
add to beaker in portions of 5 mL, rotating and shaking beaker
until sample absorbs each portion. Let soak 10-15 min; then
add 20 mL H 2 in 4-5 portions with thoro stirring after each
addn. Stir to smooth slurry. (Material must be finely dispersed
before proceeding.) Add 7 mL Na 4 EDTA soln, (e), then 3 mL
phthln, (b). Transfer to 1.5 or 2 L beaker and dil. with H 2
to 700-800 mL. Adjust to pH 7.2-7.6 and add 200 mL pan-
creatin soln, 945.75C(q). Continue as in (b), beginning "Place
in 37-38° H 2 bath ..."
C. Sedimentation Method for Chicken Excrement and Heavy
Fiith
(a) Frozen whole eggs or yolks. — Examine by 960.50B(a).
(b) Dried egg yolk. — Add 25 g sample in small portions
with continuous stirring to mixt. of 75 mL H 3 P0 4 (1+9) and
5 mL surfactant, 960.50A(a), in 150 mL tall-form beaker. Stir
to smooth paste and add H 3 P0 4 , few mL at time, to fill beaker
while stirring. Stir top layer 1 min and Jet stand 5 min. Decant
ca 2 / 3 vol. into 250 mL beaker and add H 3 P0 4 (1 +9) to both
beakers equal to vol. present. Stir contents of both beakers 1
min and let stand 5 min. Again stir top layers 1 min and slowly
add H 3 P0 4 with stirring to fill both beakers. Let stand 5 min
and repeat stirring and standing. Decant both beakers closely
into 1 L beaker.
Dil. material in 1 L beaker with H 2 0, stirring continuously,
until full. Stir top layer 1 min and let stand 5 min, and repeat
stirring and standing. Decant closely, discarding supernates.
Composite all residues in 250 mL beaker by transferring with
H 3 P0 4 (1 + 9) from wash bottle. Decant acid and floating egg
material and transfer residue to ruled paper with H 2 0, using
min. suction. Wash residue with two 30 mL portions H 2 0,
using min. suction. Examine microscopically, keeping paper
moist. Check amorphous white material for uric acid as in
962.20 or 986.29.
Refs.: JAOAC 43, 565(1960); 48, 545(1965).
POULTRY, MEAT, AND FISH AND OTHER
MARINE PRODUCTS
950.87* Filth and Sand
in Chicken Giblet Paste
Procedure
Surplus 1970
See 40.063, 11th ed.
950.88* Glass in Meat Scraps
Procedure
Surplus 1970
fe 40.065, 11th ed.
968.36 Shell in Crabmeat (Canned)
Digestion Method
First Action 1968
Weigh 57 g (2 oz) representative sample into 400 mL beaker.
Add 150 mL 1.5% NaOH soln and stir to break up lumps.
Add 10 drops 1% aq. Alizarin Red S indicator. Heat until meat
has been digested (10 min at ca 80°), stirring 3 or 4 times.
Pour on No. 12 sieve nested in No. 60 sieve, 945.75B(r), and
wash with H 2 0. Wash shell from both sieves onto weighed
paper, dry at 100°, and cool to room temp. Weigh and count
shell. Report shell as number of pieces and wt/lb.
Ref.: JAOAC 51, 521(1968).
976.27 Light Filth
in Crabmeat (Canned)
Flotation Method
First Action 1976
Final Action 1988
Transfer entire contents of <7 oz (200 g) can (or 7 oz por-
tion of larger sample) to 2 L trap flask, 945.75B(h)(4). Tho-
roly wash can (and parchment, if present) with tap H 2 and
add washings to flask. Add ca 800 mL hot (55-70°) tap H 2 0.
390
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
With mag. stirring, 970.66B(c), heat to bp. Add 50 mL min-
eral oil, 945.75C(p), and stir mag. 3 min while continuing to
boil. Remove flask from heat, fill, with hot tap H 2 0, and let
stand 30 min, stirring gently by hand at 10 and 20 min. Trap
off into 400 mL beaker. Add 30 mL mineral oil to trap flask.
With stirring bar spinning at max. speed, disperse oil thruout
aq. (lower) phase, stirring by hand. Stir mag. 5 min at max.
speed. Fill flask with hot tap H 2 and let stand 20 min, stirring
gently by hand at 10 min. Trap off into same beaker. Wash
mouth of trap flask with isopropanol and decant washings into
beaker.
Transfer to 2 L percolator, 945.75B(h)(2), contg ca 250 mL
H 2 0. Rinse beaker into percolator and bring vol. to ca 1700
mL with H 2 at room temp., ca 20°. Let stand 3 min. Drain
oil to 250 mL mark and discard drainings. Repeat fill and drain
cycle 5:2 more times. Drain remaining oil and H 2 into orig-
inal 400 mL beaker. Wash percolator sides with 1 % detergent
soln, 945.75C(i), and isopropanol, and collect washings in
beaker. Filter onto ruled paper and examine microscopically.
If filtering action slows, use new filter paper.
Ref.: J AOAC 59, 825(1976).
968.37 Shell in Clams
and Oysters (Canned)
Digestion Method
First Action 1968
Weigh 57 g (2 oz) representative sample into 600 mL beaker.
Continue as in 968.36, except digest by boiling ca 15 min with
mag stirring.
Ref.: JAOAC51, 521(1968).
972.38 Light Filth in Fish (Canned)
and Fish Products
Flotation Method
First Action 1972
Final Action 1988
For <8 oz (225 g) samples, transfer entire contents of can
(or 8 oz portion of larger sample) to 1.5 L beaker and break
up lumps with spatula. Wash can thoroly with small amt of
isopropanol and add washings to beaker. Add 50 mL HC1 and
H 2 to make 800 mL. With mag. stirring, heat to bp and boil
20 min. (If product foams, add H 2 occasionally.) Add 50 mL
mineral oil, 945.75C(p), and stir mag., 970.66B(c), 5 min
while continuing to boil.
Transfer to 2 L percolator, 945.75B(h)(2), contg ca 250 mL
H 2 0. Retain the 1.5 L beaker to fill percolator with H 2 dur-
ing refill cycles. Fill percolator with hot tap H 2 (55—70°) to
within 3 cm of top. Let stand 3 min and drain contents to ca
3 cm of bottom of oil layer. (If large amt of suspended solids
is present, let stand longer to permit sepn of oil.) Repeat drain
and refill steps at 3 min intervals until aq. phase appears clear.
Finally, slowly drain percolator to min. vol. of aq. phase with-
out loss of oil phase. Drain oil layer into 600 mL beaker. Wash
percolator with warm H 2 0, 5% detergent soln, 945.75C(i),
H 2 0, and isopropanol in sequence, using ca 50 mL per wash,
and collect washings in 600 mL beaker. Filter onto ruled paper
and examine microscopically.
Ref.: J AOAC 55, 69(1972).
974.32 Light Filth in Shrimp (Canned)
Flotation Method
First Action 1974
Final Action 1988
For shrimp <2.5 cm long, place entire contents of can into
2 L beaker contg mag. stirring bar. For larger shrimp, skewer
on probe, and wash each shrimp with hot (55-70°) H 2 from
plastic squeeze bottle over 2 L beaker contg stirring bar. Dis-
card shrimp. Wash can thoroly, pouring washings into beaker.
Bring H 2 level in beaker to ca 925 mL with hot tap FLO.
Add 25 mL HC1 and 50 mL light mineral oil, 945.75C(p).
Boil and stir mag., 970.666(c), 3 min. Transfer promptly to
percolator. 945.75B(h)(2), which has its rubber hose fitting
clamped shut as close to tubulation opening as possible and
contg ca 200 mL hot tap H 2 0. Reserve beaker. Add ca 800
mL addn I hot tap H 2 0. Let stand 10 min. Drain oil layer to
ca 7.5 cm from bottom, using rod to force shrimp tissue thru
tabulator, if necessary. Remove rod, and wash with hot H 2
into reserved beaker. Reserve rod for further washings. Drain
percolator to 300 mL mark, let stand 1 min and slowly drain
and discard remaining aq. phase until min. vol. remains. Do
not let vortex form, as it may cause loss of oil. Drain oil layer
into reserved beaker. Filter thru ruled paper. Wash paper with
55-70° hot tap H 2 0. Alternately wash percolator and rod with
5% detergent soln, 945.75C(i), and hot tap H 2 0. Filter onto
the ruled filter paper and examine microscopically.
Ref.: JAOAC 57, 691(1974).
973.60 Light Filth in Pork Sausage
(Uncooked) and Ground Beef or Hamburger
Enzyme Digestion Method
First Action 1973
Final Action 1988
A. Determination
Grind sausage, using meat grinder with end plate having
3 / i6 " holes. Weigh 225 g sample into 1.5-2 L beaker. Add 980
mL warm H 2 and 20 mL emulsifier, 945.75C(j)(/), and stir
5 min. Add 20 mL HC1 and stir 1 min. Proceed with overnight
digestion, (a), or rapid digestion, (b).
With ground beef, use only overnight digestion with 5.0 g
1:10,000 or 10 g NF pepsin. In flotation, (c), omit addn of 50
mL HC1 and subsequent boiling. After addn of light mineral
oil, let stand 20 min, instead of 10 min.
(a) Overnight digestion. — Add 0.5 g 1:10,000 pepsin (No.
0151, Difco Laboratories, or equiv.) or 2.0 g NF pepsin and
stir 1 min. Digest in 50° FLO bath or incubator 18 hr. Add 5
mL Triton X-114, 945.75C(bb)(2), and stir 1 min. Keep all
samples at digestion temp, in bath until ready to sieve. Sieve
portionwise on No. 230 sieve with hot H 2 spray. Transfer to
ruled filter paper if small amt residue remains on sieve or pro-
ceed with flotation, (c).
(b) Rapid digestion. — Add 2.0 g 1:10,000 pepsin or 10 g
NF pepsin and stir 1 min. Digest in 62° FLO bath 2 hr. Add
5 mL Triton X-114, 945.75C(bb)(2), and stir 1 min. Keep all
samples at digestion temp, until ready to sieve. Sieve portion-
wise on No. 230 sieve. Proceed with flotation, (c).
(c) Flotation. — Wet residue on sieve with 40% isopropanol
and immediately transfer quant, to 2 L trap flask, using 40%
isopropanol. Bring vol. to 1 L with 40% isopropanol and add
50 mL HC1. Add mag. stirring bar, 945.75B(n), and, with
gentle stirring, boil 10 min on mag. stirrer-hot plate. Cool to
AOAC Official Methods of Analysis (1990)
Fruits 391
room temp, in cold H 2 bath and add 40 mL flotation liq.,
945.75C(k). Stir mag.~3 min, 970.66B(c). Let oil phase sep.
1 min, and slowly fill flask with 40% isopropanol by letting
liq. flow down stoppered rod while top of stopper is main-
tained just above flask contents. After filling flask, gently stir
settled plant material with stoppered rod 5-10 sec. Let stand
undisturbed 5 min and immediately trap off. Add 25 mL light
mineral oil, 945.75C(p), stir gently by hand 30 sec, and let
stand 10 min. Repeat trapping. Wash flask neck thoroly with
isopropanol and transfer washings to beaker contg trappings.
Filter onto ruled paper and examine microscopically.
Refs.: JAOAC 56, 631(1973); 59, 51(1976).
B. Alternative Method for Sausages
{Caution: See safety notes on chloroform.)
(a) Bulk or link sausages that are easily teased apart. —
Weigh 225 g sample into 1.5-2 L beaker. Add 1 L 10% Ter-
gitol soln, 945.75C(bb)(7), and 75 x 12 mm stirring bar; stir
mag., 5 min, or until thoroly dispersed. Sieve portion wise on
No. 230 sieve, 945.75B(r). Form filter paper around 1 L beaker,
945.75B( j), moistening with H 2 to make paper pliable. In-
sert paper into buchner, 91 mm id plate, wash with isopro-
panol, and aspirate to near dryness.
Wet residue on sieve with isopropanol and quant, transfer
to filter paper cup with isopropanol. Add enough isopropanol
to cover residue and, after 1 min, apply vac. until dripping
ceases. Transfer paper cup to 1 L beaker and add 300 mL
CHC1 3 . Boil on steam bath 5 min. Let soln cool few min, lift
paper, drain, and transfer to 300 mL fresh CHC1 3 . Repeat 5
min boil and drain. Return paper cup to buchner and apply
vac. until dripping ceases. Cover residue with isopropanol for
1 min, reapply vac, and continue to aspirate 5 min. Quant,
transfer residue to 2 L trap flask, using 40% isopropanol, and
proceed as in 973.60A(c), second sentence.
(b) Link sausages compressed into casings so that product
is not easily teased apart. — Remove casing and weigh 225 g
sample into 2 L beaker. Add 1 L 10% Tergitol soln,
945.75C(bb)(i), and stir with meeh. stirrer, 945.75B(e), at
max. speed at which no splashing occurs, 15 min or until tho-
roly dispersed. Proceed as in (a), beginning, "Sieve portion-
wise on No. 230 sieve, ..."
Ref.: JAOAC 55, 66(1972).
FRUITS AND FRUIT PRODUCTS
945.76 Filth in Apple Butter
Flotation Method
Final Action 1988
Weigh 100 g well mixed sample into 400 mL beaker, add
enough cold H 2 to obtain uniform dispersion, and transfer to
2 L trap flask, 945.75B(h)(4). Add 35 mL heptane, 945.75C(i),
and stir. Add cold H 2 to bring heptane into neck of flask.
Let stand 30 min with occasional stirring, and trap off. Trans-
fer trapping to second flask contg ca 1 L H 2 0, stir, fill flask
with H 2 0, and let stand 15 min with occasional stirring. Trap
off, filter onto ruled paper, and examine paper microscopi-
cally.
Ref.: JAOAC 56, 522 (1973).
945.77* Filth in Apple Chops (Dried)
First Action
Surplus 1969
A. Heavy Filth
See 40.069, 11th ed.
B. Insects and Light Filth
See 40.070, 11th ed.
981.20 Thrips and Other Insects
in Frozen Blackberries and Frozen Raspberries
Flotation Method
First Action 1981
Final Action 1988
(Caution: See safety notes on chloroform and carcinogens.)
A. Sample Preparation
Place entire frozen contents of retail package(s) in 2-3 L
beaker and add 500 mL hot tap H 2 (55-70°). Gently sep.
frozen mass during thawing. Nest 8 in. SS funnel in 3 L beaker.
Place 8 in. No. 8 sieve over funnel. After berries are com-
pletely thawed, pour entire contents of beaker onto sieve and
distribute berries evenly over sieve. Rinse both beaker used
for thawing and retail package with 100 mL H 2 each and
pour rinses thru sieve contg berries.
Do not wash berries once on the sieve. Let berries drain 10
min (±30 s). Remove berries to 1 L tared beaker and record
10 min drained wt. Retain berries in tared beaker for analysis.
Retain drainings and beaker washings for detn of amt filth
present in proportionate liq.
B. Determination
Add 100 g drained berries to 1 L beaker contg mag. stirring
bar (945.75B(n)). Mash berries with fork. Add 300 mL tap
H 2 (55-70°) and 70 mL HC1. Fill beaker to 800 mL mark
with H 2 (55-70°). Place beaker on preheated hot plate ad-
justed to maintain berry- HC1 mixture at 55-70°. Mag. stir 5
min while maintaining temp. Wet-sieve portionwise on 8 in.
No. 25 sieve nested in No. 80 sieve, using 55-70° H 2 until
only seeds remain and drainings are clear. Discard seeds on
No. 25 sieve. Gently rinse residue to edge of No. 80 sieve and
wet thoroly with isopropanol. Let drain. Form paper cup
(945.75B(j)) by forming 32 cm No. 558 filter paper (S&S)
around 600 mL beaker and forcing it into 1 L beaker. Nest
wide-mouth glass powder funnel in paper cup. Wash residue
from sieve into paper cup using isopropanol initially and min-
imal amt of 40% isopropanol for final rinse. Add enough iso-
propanol to bring to 400 mL mark. Boil 1 min on preheated
hot plate. Transfer paper cup into wide-mouth funnel and drain
into original beaker. Discard drainings. Place paper cup in
beaker, repeat boil in 400 mL fresh isopropanol, and save
drainings. Place filter paper cup contg sample in clean I L
beaker and add 400 mL CHC1 3 . Cover beaker with watch glass
contg ice cube to minimize loss of CHCJ 3 during boiling. Boil
5 min on steam bath, and drain CHCI 3 from cup as in isopro-
panol drain above. Place filter paper cup contg sample in iso-
propanol saved from last isopropanol boil. Let cup soak 5 min
with occasional up and down movement of cup. Wash residue
from filter paper onto No. 230 sieve with hot H 2 0, and then
wet residue with 40% isopropanol and let drain. Wash residue
from sieve with 40% isopropanol into 2 L trap, using powder
funnel nested in neck of flask. Fill flask with 40% isopropanol
to 800 mL mark. Add mag. stirring bar. Place trap flask on
392
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
preheated mag. stirring hot plate and, with gentle stirring, let
come to gentle boil. Turn off mag. stirrer. Boil 5 min. Turn
off hot plate. Add 50 mL light mineral oil (945.75C(p)) and
mag. stir 2 min. Remove trap flask from mag. stirrer/hot plate.
Add 50 mL 1 + 1 mixt. Tween-80 and 40% isopropanol soln,
945.75C(x) and Na 4 EDTA plus 40% isopropanol soln,
945.75C(z) gently down stirring rod so that soln is just under
oil layer. Stir in slowly 1 min. Let trap flask stand 4 min. Fill
trap flask with 40% isopropanol gently down stirring rod. Let
stand 10 min. Trap off into 250 mL beaker. Add 35 mL light
mineral oil to trap flask. Stir in 1 min. Let stand 6 min. Trap
off into second 250 mL beaker. Filter onto ruled filter papers
(945.75B(i)) and examine papers under 10-30X.
Det. vol. of drained liq. to be filtered onto ruled paper, us-
ing following formula:
mL drained liq. to be filtered
= (100 g/10 min drained wt(g))
x total mL drained liq.
Mix drained liq. to res u spend all solids and immediately
measure vol. calcd above.
Pour drained liq. (vol. calcd above) thru No. 25 sieve nested
in No. 80 sieve, and then wash No. 80 sieve residue onto ruled
filter paper and examine at 10-30X as required.
Ref.: J AOAC 64, 194(1981).
945.78 Maggots in Blueberries
and Cherries
Sedimentation Method
Procedure
Weigh 567 g (20 oz) fresh fruit or use No. 2 can of pro-
cessed fruit. Add 100 mL H 2 to fresh or frozen fruit and boil
5 min, with frequent stirring. (Omit this step with canned fruit.)
Transfer 1 cm layer of fruit to No. 6 sieve immersed in pan
of H 2 0. Shake loose maggots and debris thru sieve. Carefully
mash fruit under H 2 to rub any remaining maggots thru sieve.
Rinse and discard any pulp and seeds. Repeat process with
another portion of fruit.
After all fruit is screened, transfer mixt. to black-bottom
pan. (With cherries, transfer first to No. 6 sieve resting in ca
3 cm H 2 0, shake sieve until maggots drop thru, and discard
pulp on sieve.) Slowly decant H 2 and pulp from pan. Add
more H 2 and repeat decantation. Pick out and count maggots
by examination of contents of pan. Transfer contents of this
pan to white-bottom pan and count maggots in pan.
964.23 Filth in Fig and Fruit Paste
First Action 1964
A. Determination
(a) Light filth. — Weigh 100 g paste into l L beaker. Add
400 mL boiling H 2 and mag. stirring bar. Boil on mag. stir-
rer-hot plate, 945.75B(n), until all lumps are disintegrated. Wet
sieve mixt. on 8" No. 1 40 sieve, 945.75B(r), with hot tap H 2
to remove fine and sol. material. Transfer residue from sieve
to 2 L trap flask, 945.758(h)(4), with H 2 0. Add H 2 to bring
vol. to ca 900 mL, and ext twice with 35 and 25 mL kerosene,
945.75C(o), as in 970.66B(b). Pour onto ruled filter paper.
Examine papers microscopically as in 970.66B(f)(7) and (g).
(b) Heavy filth. — Empty remaining trap flask contents and
rinsings onto 8" No. 140 sieve. Wet sieve with hot tap H 2
to remove kerosene as completely as possible. Transfer ma-
terial from sieve to 1 L beaker and add hot H?0 to ca 400 mL.
Boil vigorously 15 min, frequently adding 10% Na 4 EDTA soln
to keep pH ca 8. (Check with pH paper.) Transfer hot mixt.
to 8" No. 140 sieve. Wet sieve until seeds are completely sepd
from fig tissue. Return residue on sieve to the 1 L beaker. Add
H 2 to ca 300 mL, swirl, and quickly decant suspended fig
tissue and filth elements onto ruled paper in Hirsch funnel,
retaining seeds in beaker. Add H 2 and repeat decanting,
changing paper as necessary. Examine papers for heavy filth
elements at ca 30 x.
Ref.: JAOAC 47, 897(1964).
B. Alternative Method for Light Filth
Weigh 100 g paste into 1 L beaker. Break paste into small
lumps. Add 400 mL boiling H 2 and mag. stirring bar. Boil
and stir on mag. stirrer-hot plate, 945.75B(n), until all lumps
are disintegrated. Wet-sieve mixt. on 8" No. 140 sieve,
945.75B(r), with hot tap H 2 to remove fine and sol. material.
Transfer residue from sieve to 2 L trap flask, 945.75B(h)(4),
with H 2 0. Add H 2 to bring vol. to ca 900 mL, add 35 mL
kerosene, 945.75C(o), and ext as in 970.66B(b). Make second
extn with 25 mL kerosene.
If trapped material is relatively free of plant tissue, filter on
ruled paper. Examine microscopically for whole and equiv.
insects only. Det. insect head count for fig paste from number
of whole or equiv. forms of lepidoptera and coleoptera.
If trapped material contains excessive plant tissue, transfer
with ca 150 mL H 2 to percolator, 945.75B(h)(2), contg ca
250 mL H 2 0. Fill percolator to within 5 cm of top, let kero-
sene layer sep., and drain slowly to 250 mL mark. Repeat
refill and drain cycle if necessary. Drain remaining liq. into
original beaker, rinsing percolator alternately with H 2 and
acetone; filter and examine as above.
Ref.: JAOAC 58, 443(1975).
950.89
Filth in Jam and Jelly
Final Action 1988
(a) Jam. — Empty contents of jar into dish and mix thoroly.
Weigh 100 g into beaker, add 200 mL H 2 (ca 50°), transfer
to 1 L trap flask, 945.758(h)(4), add 10 mL HC1, and boil
ca 5 min. Cool to room temp., add 25 mL heptane, 945.75C(1),
and stir thoroly. Trap off, filter, and examine microscopically.
(b) Jelly. — Empty contents of jar into dish and mix thoroly.
Weigh 100 g into beaker and add*300-400 mL hot H 2 0; warm
beaker, with stirring, until jelly dissolves, filter, and examine
microscopically.
When so-called "jellies" contg small amts of fruit tissue will
not filter thru paper, proceed as in (a).
970.72 Filth in Citrus
and Pineapple Juices (Canned)
Final Action 1988
A. Fly Eggs and Maggots
Filter 250 mL thoroly mixed sample thru buchner fitted with
10XX bolting cloth, 945.75B(d) (wire mesh screen under bolt-
ing cloth facilitates filtration). Pour juice slowly to avoid ac-
cumulation of excess pulp on cloth (2 or 3 cloths may be nec-
essary). Examine filters microscopically.
B. Light Filth
To 250 mL juice in 2 L trap flask add 15 mL castor oil,
USP, and fill with enough hot H 2 (ca 70°), stirring vigor-
AOAC Official Methods of Analysis (1990)
Sugars
393
ously, to bring oil layer into neck of flask. Let stand 30 min.
Trap off, filter, and examine.
969.42 Light Filth in Raisins
Microscopic Examination Method
First Action 1969
Final Action 1988
(Caution: See safety notes on toxic solvents and chloroform.)
Add 500 mL CHC1 3 to 225 g sample in 1 L beaker and boil
on steam bath 10 min, keeping CHC1 3 vol. at ca 500 mL. De-
cant CHC1 3 , holding back raisins with glass rod, onto 7.5 cm
ruled filter paper in Hirsch funnel. Retain paper. Repeat 10
min CHCI3 boil and decant. Using H 2 0, wash filter retainings
from paper back into beaker contg raisin tissue. Bring vol. in
beaker to 700 mL with hot H 2 (55-70°) and rehydrate in
steam bath 30 min. Sieve portion wise onto 8" No. 8 sieve nested
in 8" No. 140 sieve, 945.75B(r). Thoroly wash each portion
with stream of hot H 2 while gently rubbing raisins over sieve
with fingers. Microscopically examine any decomposed raisins
for fly eggs and maggots.
Wet retainings on No. 140 sieve with 25% isopropanol,
transfer to 2 L trap flask, 945.75B(h)(4), with 25% isopro-
panol, and bring vol. to 1 L. Add 70 mL HO and mag. stirring
bar to flask, heat to boiling, and continue for 10 min, slowly
stirring on mag. stirrer- hot plate, 945.75B(n). Cool to <25°
in cold H 2 bath. Add 40 mL flotation oil (mix kerosene,
945.75C(o), and mineral oil, 945.75C(p), (1 + 2)) and stir
mag., 970.66B(c), 5 min. Let stand 1 min after gentle 10-15
sec stir with stoppered rod (see 970.66B(b)). Fill with deaer-
ated 25% isopropanol by slowly running alcohol down rod onto
top of stopper maintained ca 3 mm above liq. Let stand 15
min, gently stirring mixt. 2-3 times during first 10 min. Trap;
filter first and second extns sep. Add 25 mL flotation oil and
gently hand stir 1 min. Let stand 1 min; gently disturb oil-
alcohol interface with several up-and-down strokes of stop-
pered rod to cause fine plant material to settle. Let stand 10
min. Perform second trapping. Thoroly wash flask neck with
isopropanol. Pour trappings onto ruled filter paper and ex-
amine at 30 X. If second extn is difficult to filter, pour onto
No. 230 sieve, 945.75B(r), and wash twice alternately with
undild isopropanol and hot H 2 0. Wash sieve retainings into
400 mL beaker with hot H 2 and add 7 mL HC1/100 mL H 2 0.
Boil 10 min and pour onto ruled filter paper. Examine mic-
roscopically.
Ref.: JAOAC52, 19(1969).
from third beaker into sink until vegetable material is washed
from sample. If many seeds settle, float them off with hot 15%
NaCl soln, increasing NaCl concn if necessary to complete
flotation. Remove NaCl residue with hot H 2 0. Collect mineral
residue from the 3 beakers on ashless filter paper, and discard
filtrate. Ignite paper in weighed porcelain crucible over me-
dium Bunsen flame and place in furnace 1 hr at ca 600°. Cool,
add 5 mL HC1, and heat to bp. Cool, add 10 mL H 2 0, and
reheat to bp. Filter and wash free from acid. Ignite, ash as
before, and weigh to det acid-insol. residue. Calc. % insol.
residue = wt acid-insol. residue (g) x 100/net wt sample (g).
Ref.: JAOAC 54, 581(1971).
950.90
See 950.86.
950.91
SNACK FOOD PRODUCTS
Filth in Unpopped Popcorn
Procedure
Filth in Popped Popcorn
Procedure
Weigh 50 g sample into 2 L trap flask. Add 500 mL hot
H 2 0, boil 15 min, and cool to room temp. Add 35 mL hep-
tane, 945.75C(1), mix, and let stand 5 min. Fill with H 2 0,
trap off, filter, and examine microscopically.
955.44
Filth in Potato Chips
Flotation Method
Final Action 1988
Weigh 100 g sample into 1.5 L beaker. Crush chips into
small pieces and cover with pet ether. Let stand ca 5 min and
decant thru filter. Add pet ether and decant again thru filter.
Let pet ether evap. from chips. Transfer to 2 L trap flask, add
500 mL 60% alcohol, and boil ca 30 min, replacing alcohol
lost by evapn. Cool, add 35 mL heptane, 945.75C(1), mix,
let stand ca 5 min, and fill with 60% alcohol. Let stand, trap
off twice, and filter as usual. Examine papers microscopically.
984.28
Filth in Corn Chips
Final Action 1988
971.33 Residue (Acid-insoluble) (Soil)
in Fruits and Vegetables (Frozen)
Gravimetric Method
First Action 1971
Final Action 1973
Remove frozen sample from container. Place in weighed
plastic bag, re weigh, and seal tightly with rubber band. Thaw
sample by immersing bag in hot H 2 and transfer contents to
high-speed blender, washing inside of bag. Blend until sample
is disintegrated and transfer to 2 L beaker. Nearly fill beaker
with H 2 and mix contents thoroly by swirling. Let stand 10
min and decant supernate into second 2 L beaker. Refill first
beaker with H 2 and repeat mixing. Fill second beaker with
H 2 and mix by swirling. After 10 min, decant second beaker
into third and first into second. Continue operation, decanting
See 970.71.
SUGARS AND SUGAR PRODUCTS
971.34 Filth in Candy
Flotation Method
Final Action 1988
(a) In hard candy, gum drops, gum, starch, or pectin-base
candies. — Dissolve in boiling HC1 (1 + 70), filter thru rapid
paper on Hirsch funnel, and examine microscopically.
(b) In hard candy difficult to filter by (a) (e.g., licorice
candy).— Proceed as in (c).
(c) All water-insoluble candy except those containing con-
394
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
fectioners corn flakes, wheat bran, or other cereal fillers, and
those whose major constituent, excluding chocolate coating,
consists primarily of finely ground nutmeats (e.g., peanut hut-
ter, almond paste, etc.). — Weigh 225 g sample into 1.5-2 L
beaker. Add 1 L 5% soln of Tergitol, 945.75C(bb)(/), and
heat in steam bath 10 min. Stir 5-10 min on mag. stirrer-hot
plate. Sieve portionwise on No. 230 sieve, 945.75B(r). If res-
idue on sieve is small, transfer directly to ruled filter paper;
otherwise, transfer quant, to 2 L trap flask, using 40% iso-
propanoL Bring vol. to 1 L with 40% isopropanol and add 50
mL HC1. Gently stir on mag. stirrer-hot plate while heating to
full boil. Immediately transfer flask to cool stirring unit and
add 40 mL light mineral oil, 945.75C(p). Stir mag. , 970.66B(c),
2 min. Let stand I min; then slowly fill ilask with 40% iso-
propanol by running liq. down stoppered rod while top of stop-
per is maintained just above liq. After filling flask, gently stir
settled plant material 5-10 sec with stoppered rod. Let stand
undisturbed 2 min and immediately trap off. Add 25 mL light
mineral oil, stir by hand gently 30 sec, and let stand 10 min.
Repeat trapping. Wash flask neck thoroly with isopropanol and
transfer washings to beaker contg trappings. Filter onto ruled
paper and examine microscopically.
(d) Water- insoluble candies containing confectioners corn
flakes, wheat bran, or other cereal fillers, and those whose
major constituent, excluding the chocolate coating, consists
primarily of finely ground nutmeats (e.g., peanut butter, al-
mond paste, etc.). — (Caution: See safety notes on distillation,
toxic solvents, and chloroform.) Proceed as in (c) thru sieving
on No. 230 sieve. Wash residue on sieve with isopropanol.
Form filter paper around 600 mL beaker, 945.75B(j), mois-
tening with H 2 to make paper pliable. Insert paper into 91
mm buchner, wash with isopropanol, and aspirate to near dry-
ness. Quant, transfer residue on sieve to filter paper cup with
isopropanol and add enough isopropanol to cover residue. Af-
ter 1 min, apply vac. until dripping ceases. Place paper cup
contg sieved residue in 1 L beaker, add 200 mL CHC1 3 , and
boil 5 min on steam bath. After few min of cooling, lift paper,
drain, and transfer to 200 mL fresh CHCI 3 . Repeat 5 min boil
and drain. Return paper cup to buchner and apply vac. until
dripping ceases. Cover residue with isopropanol 1 min, reap-
ply vac, and continue to aspirate 5 min after visible dripping
ceases. Proceed as in (c), beginning with "... transfer quant.
to 2 L trap flask, using 40% isopropanol." Continue as in (c),
except after bringing contents of flask to full boil, cool to room
temp, in cold H 2 bath, and use flotation liq., 945.75C(k) s
in place of mineral oil.
(e) In chocolate candy coating. — Heat 400 mL CH 2 C1 2 in
800 mL beaker to 30-35° and keep at this temp. Place portion
of candy in wire basket (ca 8 cm diam. x 3 cm high) made
from No. 8 screen and with wire handles. Move basket up and
down thru CH 2 G 2 until chocolate coating dissolves. Rinse each
candy center with fine stream of CH 2 C1 2 from wash bottle and
save center. Repeat with balance of sample. Stir CH 2 Cl 2 -choc-
olate suspension and pour thru No. 140 sieve. Transfer residue
from sieve to filter paper and examine microscopically. Ex-
amine candy centers by appropriate method, (a), (b), (c), or
(d).
ReL: J AOAC 54, 568(1971).
950.92* Filth in Chewing Gum
Procedure
Surplus 1970
945.79 Filth in Sirups, Molasses,
and Honey
Filtration Methods
Final Action 1988
(a) Mix sample thoroly and dissolve 200 g in 200 mL hot
H 2 acidified with 5 mL HN0 3 . Filter at once thru rapid paper
in Hirsch funnel. Wash with min. amt of hot H 2 and examine
microscopically.
(b) Alternative method. — Dissolve 200 g in 500 mL hot H 2 0.
Filter at once thru 10XX bolting cloth in Hirsch funnel. Wash
with min. amt of hot H 2 and examine microscopically.
945.80
Filth in Sugars
Filtration Method
Final Action 1988
See 40.082, 1 1th ed.
Dissolve 100 g sample in ca 200 mL hot H 2 0. Boil, and
filter at once thru rapid paper in Hirsch funnel. Examine mic-
roscopically.
VEGETABLES AND VEGETABLE PRODUCTS
945.81 Weevils in Beans and Peas
Flotation Method
First Action
Microscopic examination. — If peas or beans are canned and
of normal texture, pour on No. 8 sieve in pan filled with enough
H 2 to stand 2—3 cm above mesh of sieve. Mash peas thru
sieve with fingers. After as much as possible of material has
been worked thru, remove sieve from pan and shake excess
H 2 back into pan. Transfer material retained on sieve to 2 L
beaker. Pour material that passed thru No. 8 sieve onto No.
40 sieve, discarding that which passes thru. Let material on
sieve drain few min, and shake lightly to remove free H 2
from solid material. (If peas are unusually hard, or have tough
skins, pass contents of can thru meat or food chopper directly
onto No. 40 sieve.) Discard any excess H 2 passing thru this
sieve. Cook dried or frozen peas before maceration.
Add material retained on the No. 40 sieve to the beaker.
Add ca 130 mL heptane, 945.75C(1), to this material and mix
thoroly with large spoon. Rinse any material remaining on sieve
into beaker with H 2 0. Stir material in beaker and pick out any
insects that may rise to top of H 2 layer. Repeat stirring and
searching several times until no more larvae are recovered.
Add enough H 2 to bring contents of beaker to within 1-2
cm of top. Pick out any larvae visible at surface. Stir again,
and let mixt. stand ca 5 min; then skim off heptane and upper
part of H 2 layer with spoon and place in trap flask,
945.75B(h)(4), previously filled ca V-i full of H 2 0. Add 90-
100 mL heptane to material remaining in beaker, and stir vig-
orously. Let stand ca 5 addnl min, skim off heptane and upper
part of H 2 layer as before, and add to material already in trap
flask.
Fill flask with H 2 0. Trap off as much heptane as possible
and filter into Hirsch funnel. Lower stopper into flask, and,
to rinse sides of trap flask, apply vac. ca 5 min by fitting large
rubber stopper and glass tube over mouth of flask. (As ordi-
nary erlenmeyer collapses under vac. of 20" of Hg (50 cm;
67.7 kPa), use either less vac. or heavy-wall flask.) Release
vac, add H 2 0, and trap off. Add trapped-off portion to that
already on filter. Examine microscopically.
AOAC Official Methods of Analysis (1990)
Vegetables
395
945.82 3-ight Filth in Broccoli (Canned)
Flotation Method
First Action
(a) Insects. — Transfer contents of can to pan of suitable size
and chop up leaves into pieces 2-5 cm long. Weigh 100 g
well mixed sample into 1 L beaker. Add 500-600 mL H 2
and boil 5 min. Pour H 2 and sample into 2 L trap flask,
945.758(h)(4). Add 35 mL heptane, 945.75C(1), and stir tho-
roly to ensure contact between heptane and all portions of leaves.
Fill flask with deaerated H 2 0, let stand 30 min, trap off hep-
tane layer, filter, and examine microscopically. Add 40 mL
heptane to flask and repeat extn.
If plant tissue rises to interface, place No. 8 sieve, 6-8"
diam., in suitable size evapg dish contg enough H 2 to cover
screen ca I cm. Pour entrapped heptane from trap flask onto
sieve as it is held under the H 2 0. Move sieve gently up and
down to let insects pass thru into the H 2 0. Remove screen and
filter contents of dish. Repeat washing to free any insects left
on greens on screen, and fitter washings. Examine papers mic-
roscopically.
(b) Aphids and Thrips. — Det. drained wt of contents of
canned greens as in 945.62, reserving drained liquor. Chop
drained leaves into pieces 2-5 cm long and weigh 100 g well
mixed sample into I L beaker. Add H 2 to cover adequately,
followed by 25 g neut. Pb(OAc) 2 .3H 2 crystals (orequiv. soln
of Pb(OAc) 2 ) and 10 mL HOAc. Boil on hot plate 5-10 min,
cool, and transfer to 2 L trap flask, 945.75B(h)(4). Add 35
mL heptane, 945.75C(l), and mix thoroly to ensure contact
between heptane and all portions of leaves. Fill flask with
deaerated FLO. Let settle few min for most of vegetable matter
to sink to bottom. To force any tissue that rises (probably held
by entrapped globules of heptane) to sink, pivot lower end of
trap- rod on bottom of flask, and rotate upper part of rod around
neck of flask to knock globules from vegetable tissue without
at same time breaking interface and thus re wetting tissue with
heptane. Again let flask stand, trap off heptane layer, and fil-
ter.
Re-ext with 20 mL heptane, trap off, and filter (usually pos-
sible on same paper). Det. total number of aphids or other light
filth in entire liquor drained from can by subjecting it to hep-
tane flotation as usual . (Normally liquor does not present any
difficulty and use of Pb(OAc) 2 is unnecessary.) Count total
number of aphids and thrips including parts contg heads. Count
cast skins and other insects sep. Calc. on basis of 100 g of
drained material.
973.61 Foreign Matter in Corn (Canned)
Flotation and Macroscopic Methods
First Action 1973
Final Action 1988
(Applicable to canned whole and cream-style corn)
Place 200 g well mixed sample in 1.5 L beaker and add 1
L 40% isopropanol. Bring to bp, stirring mag., 970.66B(c),
add 50 mL mineral oil, 945.75C(p), and boil and stir 3 min
more. Transfer immediately to percolator contg ca 100 mL
40% isopropanol and glass or metal rod for forcing corn thru
spout. Retain stirring bar in beaker. Rinse bar with undild iso-
propanol. Add ca 900 mL 40% isopropanol to beaker, stir, and
add to percolator, 945.758(h) (J). Reserve beaker. After ca 5
min standing, drain percolator to within 8 cm of bottom onto
8" No. 20 sieve nested in large white enamel tray of ca 2 L
capacity. Use rod to force corn thru percolator drain spout.
Withdraw rod after removing corn from percolator and wash
with small amt of undild isopropanol into reserved beaker.
Discard isopropanol collected in tray. Leave sieve in place with
retained corn material. Using reserved beaker, add ca 1.5 L
hot tap H 2 (50-70°) to percolator. Let phases sep. ca 3 min
and make final drain. Discard all but last 5 cm oil-aq. phase.
Drain into 600 mL beaker. Wash sides of percolator with al-
ternate isopropanol and H 2 rinses, and collect in same beaker.
Add rest of corn from can to corn retained on sieve, sieve
portionwise if necessary, and wash with tap H 2 to remove
starch and fine particles. Reverse sieve into white enamel tray.
Wash corn into tray with forceful spray of H 2 (ca 22°) to 3
cm depth in tray. Let corn settle and examine under H 2 for
worm-eaten or rotten kernels and whole worms, heads, or large
fragments. Add these to trappings previously obtained from
percolator. Tip tray and slowly decant H 2 while carefully ob-
serving flowing H 2 for insect fragments. Refill tray with 3
cm H 2 (ca 22°) and repeat decantation, examining closely for
objectionable material. Discard pan contents. Filter beaker
contents thru ruled filter paper and examine microscopically.
If filtration is impeded by excessive starch material, proceed
as in 970.666(d).
Ref.: JAOAC 56, 634(1973).
974.33
A. Light Filth
Filth in Green
Leafy Vegetables
(Applicable to whole or chopped kale, turnip, mustard, and
collard greens.)
(a) Canned {First Action 1979; Final Action 1988). — Drain
can contents on No. 8 sieve 2 min and reserve brine. Imme-
diately take 100 g from sieve. If pieces are <1 cm in length,
place in 2 L trap flask. Cut larger pieces on small cutting board
to this size to avoid interference with mag. stirring. Wash res-
idue from cutting into trap flask with 40% isopropanol. Add
40% isopropanol to fill trap flask to 1 L mark. Add mag. stir-
ring bar (13 x 76 mm). Add 50 mL light mineral oil,
945.75C(p). Place flask on mag. stirrer, 970.66B(c). Slowly
bring stirrer to max. speed at which stirring bar is just visible
at bottom of vortex, avoiding visible or audible splashing. Proper
stirring will produce vertical rolling of contents. Stir 5 min,
remove flask from stirrer, and let stand 1 min. Add 40% iso-
propanol down rod to fill flask. Manually stir material in bot-
tom of flask with rotary motion ca 15 sec. Repeat stirring at
two 5 min intervals. Raise stirring rod from bottom of flask
and clamp. Let stand addnl 10 min and trap off into beaker.
Repeat trapping with 35 mL oil, stirring manually 1 min. Filter
onto ruled papers.
Filter reserved brine on sep. papers. Examine papers for
aphids and other extraneous materials at 10-30X. Det. number
of aphids, etc., in 100 g of drained greens and add to this
number in proportionate amt of drained liq. calcd as follows:
100 g sample
x total number aphids, etc. in liq.
total g drained wt
Ref.: JAOAC 62, 600(1979).
(b) Frozen (First Action 1974; Final Action 1976). — Thaw
and accurately weigh 100 g sample. Chop into ca 2.5 cm (1")
pieces and mix thoroly. Transfer to 2 L trap flask, 945.758(h)(4)
for collard and mustard greens or beaker for other products.
Add /% anhyd. Na 2 S0 4 soln to cover product to depth of 2.5
cm, cover, and boil 30-40 min. Add H 2 as required to main-
tain original vol. Remove from heat, add 1% Na 2 S0 4 soln to
396
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
ca 1.2-1.4 L, and add 35 mL light mineral oil, 945.75C(p).
Stir mag. 10 min, 970.66B(c). Proceed as in (a) or (b).
(/) Percolator method. — Quant, transfer contents to per-
colator, 945.75B(h)(2), and reserve beaker. Stir gently with
long glass stirring rod at 5 and 10 min. Let stand undisturbed
5 min; then drain to ca 250 mL. Add ca 1 L 1% Na 2 S0 4 soln,
and repeat drain and refill cycles until lower aq . phase is clear
and free of suspended material.
After last cycle, drain oil-H 2 interface into reserved beaker.
Immediately wash sides of percolator with 100-200 mL por-
tions hot tap H 2 0, isopropanol or alcohol, and 5% detergent
soln, 945.75C(i), if necessary. Filter thru ruled paper, wash-
ing sides of beaker as above and using rubber policeman if
needed. Examine paper microscopically.
(2) Trap flask method. — After mixing, add trap flask rod
and fill flask slowly with 1% Na 2 S0 4 soln, letting soln run
down rod. Let stand 10 min, stirring gently with rod several
times during first 5 min. Secure rod above settled debris during
last 5 min. Trap as in 970.66B(b), and rinse neck of flask with
H 2 0. Add 25 mL light mineral oil and clamp rod above settled
debris. Stir mag. 30 sec and repeat trapping. Make final trap
after 10 min, rinsing flask neck with alcohol or isopropanol.
Filter thru ruled paper and examine microscopically.
Ref.: JAOAC 57, 693(1974).
B. Heavy Filth
—Final Action 1988
Canned. — Recover heavy filth such as soil, maggots (es-
pecially those of spinach leaf miner), and rodent excreta, that
sink to bottom of trap flask, as follows: Transfer contents of
trap flask, 945.82(a) or (b), by rinsing with H z O into 4-6 L
pail. Add H 2 to pail until ca full. Stir, let stand short time,
and decant ca half pail contents. Refill pail with H 2 and re-
peat operation until most of floating greens are removed. Wash
heavy filth left in pail into black shallow pan and examine
visually for larvae, stones, and other debris, picking material
out with forceps.
975.47
See 971.33.
970.73
Soil in Spinach (Frozen)
Acid-insolubie Residue Method
Final Action
Filth in Pureed
Enfant Food
Final Action 1988
A. Light Filth
Transfer contents of 2 cans or jars (ca 250 g) of food to 1
L trap flask, 945.75B(h)(4), previously rinsed with H 2 0. Tho-
roly mix in ca 20 mL of the oil. Fill with deaerated H 2 either
at room temp, or at 50-70°. Let mixt. stand 30 min, stirring
4-6 times during this period to release filth from layer of food.
Trap off, filter, and examine microscopically.
Use type of oil and temp, indicated in following table:
Food
Oil
Temp.
All fruits
Asparagus
Beets
Carrots
Green beans
Peas
Spinach
Squash
Light mineral oil
Light mineral oil
Castor oil
Room
50-70°
50-70°
B. Fly Eggs and Maggots
Transfer residue in trap flask, 970. 73 A, to 2 L separator.
Add ca 100 mL heptane, 945.75C(I), and shake vigorously.
Let material settle ca 2 hr, occasionally stirring surface layer
to permit any eggs and maggots to settle out. Withdraw ca 200
mL from bottom of separator and filter this material thru 10XX
bolting cloth, 945.75B(d), using several cloths if there is large
accumulation of sediment. Examine microscopically at 15-20x.
967.24
Filth in Mushrooms
(For maggots, mites, etc., in canned, fresh frozen, freeze-dried,
and dehydrate products)
A. Insects
—First Action 1967
—Final Action 1989
(a) Canned mushrooms. — Pour contents of can evenly over
weighed No. 8 sieve. Use 8" sieve, for containers of net wt
<3 lb (L4 kg) and 12" sieve for larger containers. Drain 2
min, and reweigh sieve and mushrooms to det. drained wt
mushrooms.
Rinse container, and use rinsings and several addnl portions
H 2 to rinse mushrooms on sieve (ca 500 mL total). Combine
drained liq. with rinsings and filter thru ruled paper. Examine
residue on paper microscopically and det. total number of
maggots in liq.
Place 100 g drained mushrooms in high-speed blender,
945.75B(c). Add 300 mL H 2 and blend 30-45 sec at ca 3000
rpm. Attain proper speed quickly by using setting of 1.5-2X
final setting on variable transformer for few sec at start. Frag-
ments of mushrooms after blending should be 3-5 mm long.
Pour mixt. into nested set of 8" Nos, 20, 40, and 140 sieves,
945.75B(r). Rinse tissue 2-3 min with spray of tap H 2 from
aerator, 945.75B(a). Discard material on No. 20 sieve. Trans-
fer residue from No. 40 sieve to 600 mL beaker with H 2 and
bring total vol. to ca 100 mL. Add 5 mL sat. aq. crystal violet
soln and heat to bp. Pour stained mixt. into No. 40 sieve.
Wash mushroom tissue, and maggots, if any, to edge of sieve
and remove excess stain with tap H 2 from aerator. Using
wash bottle contg com. 5.25% NaOCl soln, and gentle spray
of tap H 2 from aerator, alternately spray tissue with H 2 and
NaOCl soln until stain has been removed from mushroom tis-
sue. Wash tissue into 600 mL beaker and transfer to ruled pa-
per, using vac. Avoid obscuring maggots with mushroom tis-
sues. (Not more than 2-3 papers should be necessary.)
Transfer residue from No. 140 sieve to 600 mL beaker with
H 2 and repeat staining, bleaching, and filtering as above.
Examine papers for maggots and other extraneous materials
at 10-30X. Maggots are stained dark violet. Det. number of
maggots in 100 g drained mushrooms and add to this value
the number in proportionate amt of drained liq. calcd as fol-
lows:
(100 /total g drained mushrooms) x total number of mag-
gots in liq.
(b) Fresh, frozen, freeze-dried, and dehydrated mush-
rooms. — For fresh and frozen mushrooms weigh 170 g sam-
ple, and for dried mushrooms weigh 15 g sample, into suitable
container, and add enough H 2 to immerse mushrooms. Soften
mushroom tissue by soaking several hours or, alternatively,
by heating on steam bath or simmering \ l k-2 hr as necessary,
followed by cooling 30-60 min to fully rehydrate. Quant.
transfer contents to high-speed blender and proceed as in (a),
beginning "... blend 30-45 sec at ca 3000 rpm."
Refs.: JAOAC 49, 576(1966); 50, 514(1967); 59, 353(1976).
AOAC Official Methods of Analysis (1990)
Spices 397
(For dried (not powdered) products)
B. Light Filth — Procedure
Thoroly mix sample and weigh 15 g portion. Transfer mush-
rooms to trap flask, 945.75B(h)(4), add H 2 0, and let soak
several hr, preferably overnight on steam bath, or boil 30 min.
Cool to room temp., add 30 mL heptane, 945.75C(I), and
churn contents by hard, rapid pounding of mushrooms against
bottom of flask, using vertical movement of rubber plunger.
Trap off twice, filter, and examine microscopically.
972.39 Light Filth in Potato
Products (Dehydrated)
Flotation Method
First Action 1972
Final Action 1988
Weigh 50 g sample into 1.5-2 L beaker. Add 1 L hot HC1
(1 +9) and mag. stirring bar. Boil 10 min with gentle stirring
on mag. stirrer-hot plate, 945.75B(n). Sieve portionwise on
No. 230 sieve, 945.75B(r). Wet residue on sieve with 40%
isopropanol and transfer quant, to 2 L trap flask, 945.75B(h)(4),
using 40% isopropanol. Bring vol. to 1 L with 40% isopro-
panol and add 50 mL HC1. Add mag. stirring bar, heat, and
boil 10 min with gentle mag. stirring. Immediately transfer
flask to cool stirring unit and add 40 mL mineral oil,
945.75C(p). Stir mag., 970.66B(c), 3 min. Slowly fill flask
with 40% isopropanol by letting liq. flow down stoppered rod
while top of stopper is held just above liq. After filling flask,
gently stir settled plant material by hand 5-10 sec with stop-
pered rod. Let stand undisturbed 5 min and immediately trap
off. Add 25 mL mineral oil, gently stir by hand 30 sec, and
let stand 10 min. Repeat trapping. Wash flask neck thoroly
with undild isopropanol and transfer washings to beaker contg
trappings. Filter onto ruled paper and examine microscopi-
cally.
Ref.: J AOAC 55, 71(1972).
955.45
Filth in Sauerkraut
Sieving Method
Final Action 1988
Use entire contents of container of <2 lb (900 g). Use 24
oz (700 g) well mixed sample from larger containers. Wash
small portion at time on nested 8" Nos. 8, 20, and 140 sieves,
945.75B(r). Wash material remaining on No. 20 sieve with
washings passing thru No. 140 sieve. Transfer material on No.
20 sieve to paper and examine at ca 10x for whole insects or
large body parts. Transfer material remaining on No. 140 sieve
to paper and examine microscopically.
955.46 Filth in Tomato Products
Final Action 1988
A. Fly Eggs and Maggots
(a) Comminuted products . — Thoroly mix sample and trans-
fer 100 g to 2 L separator. Add 20-30 mL heptane and shake
thoroly, releasing pressure as necessary. Fill separator with H 2
in such manner as to produce max. agitation. Place separator
in ring stand and let settle; at 15 min intervals during 1 hr,
drain 15-20 mL from separator, and gently shake separator
with rotary motion to facilitate settling out of fly eggs and
maggots. If drained liq. contains seeds, pass it thru No. 10
sieve, and thoroly rinse seeds and sieve, recovering both liq.
portion and rinse H 2 in beaker. Filter thru 10XX bolting cloth,
pretreated and dyed as in 945.75B(d), in Hirsch funnel. Ex-
amine for eggs and maggots at ca 10 X . If fly eggs or maggots
are found in this examination, continue sepg and draining, as
above, addnl hr.
(b) Canned tomatoes. — Pulp entire contents of can in such
way that min. number of eggs and maggots are crushed or
broken. (This may be done by passing material thru No. 6 or
No. 8 sieve and adding seeds and residue remaining on sieve
to pulp.)
Place 500 g of the well mixed pulped tomatoes in 6 L sep-
arator. Add 125-150 mL heptane, 945.75C(1), and ca 1 L H 2
and shake vigorously, releasing pressure as necessary. Fill
separator with H 2 0. Place separator in ring stand and let layers
sep. At 15 min intervals during 1 hr, drain 25-30 mL from
bottom of separator, and gently shake separator with rotary
motion to facilitate settling of fly eggs and maggots. Each por-
tion may be examined at once or combined with subsequent
portions. Pass drained portions thru No. 10 sieve and thoroly
rinse seeds and sieve, recovering both liq. portion and rinse
H 2 in beaker. Filter thru 10XX bolting cloth in Hirsch fun-
nel. Examine cloth for eggs and maggots at ca 10x. If fly
eggs or maggots are found in this examination, continue sepg
and draining, as above, addnl hr.
a Light Filth
(a) Comminuted products. — Place 200 g of any tomato
product except paste (where 100 g is used) in trap flask,
945.75B(h)(4), with 20 mL castor oil and mix well. Add enough
hot tap H 2 (ca 70°) to fill flask. (At first, bubbles of air tend
to bring up tomato tissues, but after several stirrings these be-
gin to settle out, leaving H 2 layer near oil fairly clear.) Let
stand with occasional gentle stirring 30 min; then trap off into
beaker. Wash out neck of flask with heptane to remove ad-
hering castor oil. Add little more hot H 2 to flask, stir, let
stand 10 min, and then trap off again. (Occasionally it may
be necessary to transfer trapped-off material to another trap
flask and rewash to eliminate tomato tissue.) Filter trapped-
off portion; thoroly wash beaker, sides of funnel, and paper
with heptane to dissolve oil and speed filtration. Examine pa-
per at 20-30X.
(b) Canned tomatoes (Procedure). — Drain entire can on No.
6 sieve, saving drained juice. (For cans contg <3 lb use 8"
sieve; use larger sieve for larger cans or drain and rinse por-
tionwise.) Rinse portion on sieve with hot H 2 (ca 70°) from
wash bottle and transfer drained juice, fragments, and wash-
ings to 1 or more 2 L Wildman trap flasks (max. 900 mL/
flask; No. 10 cans require S:2). Bring vol. in flasks to ca 900
mL with H 2 (70°) and add 20-25 mL castor oil. Tilt flask
to ca 45° and mix 1 min with brisk rotary motion (200-250
strokes/min). Avoid splashing thru surface with stopper. Add
hot H 2 to bring oil layer into neck and let stand 30 min with
occasional stirring. Trap off into beaker oil-H 2 layer and any
debris that rises. Wash out oil in neck with heptane. Add ca
10 mL hot H 2 to flask, stir, let stand 10 min, and trap into
same beaker. Add 25-30 mL heptane to beaker and stir to
dissolve oil. Filter thru paper (use hot H 2 or heptane if nec-
essary) and examine paper microscopically.
SPICES AND OTHER CONDIMENTS
945.83* Filth in Spices (Whole)
Flotation Method
First Action
Surplus 1970
(Applicable where no specific method exists)
See 40.115, 11th ed.
398
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
965.40
Filth in Spices
Flotation Method
First Action
(Applicable to whole, cracked, or pieces of allspice, anise,
caraway, celery seed, cloves, coriander, cumin, dill seed, fen-
nel seed, fenugreek, ginger, mace, mixed pickling spice, mus-
tard, nutmeg, black pepper, white pepper, poppy seed, and
turmeric)
Weigh 25 g sample into 400 mL beaker and proceed as in
975.48(a) and (b), except use more reagent, and where nec-
essary, 2 L trap flask, also use 35 mL heptane, instead of 25
mL. For whole, cracked, or pieces of marjoram, and savory,
add 400 mL hot H 2 and 20 mL HCl.
960.51 Foreign Matter in Spices
and Condiments
Sieving Method
First Action
(Applicable to ground allspice, anise, curry powder, dill seed,
fennel, fenugreek, poppy seed, savory, and condiments; heavy
filth only: caraway seed, cardamon, celery seed, cloves, cori-
ander, cumin, ginger, mace, marjoram, mustard, oregano,
rosemary, sage, and thyme)
Sift 200-400 g ground spice thru No. 20 sieve. Transfer any
insects or other filth retained on sieve to suitable dish and ex-
amine with wide.fie.ld stereoscopic microscope.
975.48 Heavy and Light Filth in Spices
and Condiments
Flotation Method
First Action 1975
(Heavy and light filth for products lacking specific method)
{Caution: See safety notes on distillation, flammable solvents,
toxic solvents, chloroform, and petroleum ether.)
(a) Heavy filth and sand.— Weigh 10 g sample into 250 mL
beaker. Add ]50 mL pet ether and boil gently 1 5 min on steam
bath in hood. Occasionally add pet ether to keep vol. const.
Decant pet ether onto smooth 7 cm paper in buchner. Add 1 50
mL CHQ3 to beaker and let stand 30 min with occasional stir-
ring. Decant spice and CHC1 3 onto funnel, leaving heavy res-
idue of sand and soil, if any, in beaker. If appreciable spice
tissue remains on bottom of beaker, add successive portions
of CHCI3 mixed with CC1 4 to give increasingly higher sp gr
until practically all spice tissue is floated off. Transfer residue
from beaker to ashless paper and examine microscopically. If
there is appreciable amt of residue, place paper in weighed
crucible, ignite, and weigh sand and soil.
(b) Light filth. — Thoroly dry material in buchner and trans-
fer, including fine material that must be scraped from paper,
to I L trap flask, 945.756(h)(4). Add ca 150 mL H 2 0, heat
to bp, and simmer 15 min, with stirring; wash down inside of
flask with H 2 0; and cool to <20°. Add 25 mL heptane,
945.75C(S), stir mag., and let stand 5 min; then fill flask with
H 2 and let stand 30 min. Stir every 5 min, trap off, and filter.
Add ca 15 mL heptane and mix thoroly; trap off and filter
second time after 15 min. If second extn yields appreciable
amt of filth, decant most of liq. from flask, add 15 mL hep-
tane, and make third extn. Examine papers microscopically.
975.49 Light Filth in Spices
and Condiments
Flotation Method
Final Action 1988
(See Table 975.49 for applicability and parameters
for specific spices.)
A. Pretreatment
(Caution: See safety notes on chloroform.)
Form filter paper cup, 400 mL- I L, 945.75B(j), and weigh
sample into cup.
(a) Isopropanol extraction. — Add 400 mL isopropanol to
sample beaker contg cup, and boil gently on hot plate 10 min.
Transfer cup to buchner and aspirate to slow drip. Repeat twice
with 400 mL isopropanol. Proceed with isolation step speci-
fied in Table 975.49.
(b) Chloroform-isopropanol extraction. — Add 400 mL
CHCI3 to cup in sample beaker, and boil gently on hot plate
in fume hood 10 min. Transfer cup to buchner and aspirate to
slow drip. Repeat twice with 400 mL CHCI3. Turn off vac,
cover sample with isopropanol, and let stand 1 min. Aspirate
to slow drip. Repeat isopropanol extn. Proceed with isolation
step specified in Table 975.49.
(c) For crushed red peppers. — Weigh 25 g sample into fil-
ter paper cup formed in 250 mL beaker. Place in 80-85° H 2
bath or on top of steam bath. Add 100 mL isopropanol and
heat 5 min. Lift paper cup and let drain. Discard drainings,
avoiding contact with liq.
Add 100 mL isopropanol and repeat extn and draining. Place
cup in Hirsch funnel and wash with ca 100 mL isopropanol.
Aspirate to near dryness.
(d) For ground mace and ground caraway seed. — Add 400
mL CHCI3 to cup in sample beaker, and boil gently on hot
plate in fume hood 10 min. Transfer cup to buchner and as-
pirate to slow drip. Return cup to empty beaker, add 400 mL
isopropanol, and boil gently 5 min. Transfer cup to buchner
and aspirate to slow drip.
(e) Alternative solvent saver (reflux) technic for ground tur-
meric. — Weigh sample into 1 L beaker. Add 400 mL isopro-
panol and boil gently on hot plate 30 min with sol v. saver app.
inserted into beaker top. Sol v. saver app. consists of support
stands with clamped 1 L r-b flasks which are stoppered with
2-hole rubber stoppers. Each hole has glass tube and rubber
hose attached. One hose is connected to cold H 2 source, the
other to drain outlet. With cold H 2 circulating thru flask,
which is inserted into I L beaker contg sample, sol v. is heated
to boil and allowed to reflux back into sample for set period
of time. Multiple units should be set up in parallel, using "T"
connectors, rather than in series, because increased temp, at
end of series may affect efficiency of sol v. reflux process. See
Fig. 975.49.
Pour sample into No. 230 sieve and wash with gentle stream
of hot tap H 2 0. Proceed with extn as in 975.496(b).
B. Isolation
(a) Mineral oil-n-heptane (85 + 75). — Quant, transfer
sample to 2 L trap flask, 945.756(h)(4), with 40% isopro-
panol. Dil. to 400 mL with 40% isopropanol and boil gently
10 min with mag. stirring. Cool in H a O bath to 20-25°. Add
mixt. 50 mL Tween 80-40% isopropanol soln, 945.75C(x),
and 50 mL Na 4 EDTA-40% isopropanol soln, 945.75C(z),
slowly down stirring rod. (Omit for parsley, rosemary, and
bay leaves.) Hand stir 1 min, using gentle rotary motion, and
let stand 5 min. Dil. to 800 mL with 40% isopropanol, add
AOAC Official Methods of Analysis (1990)
Spices
399
Table 975.49 Methods for Spices, Herbs, and Botanicals; for Those Not Listed, Use 975.48(a) and (b) for Ground Form of Product
Spice
Form
Sample,
9
Pretreatment
975.49A
Isolation
975.49B
Method
Heavy Light Filth
Alfalfa leaves
Whole
10
Allspice
Ground
10
Anise
10
Annato
25
Basil
Whole (1)
25
Bay leaves
" (D
25
Capsiciums 3
Ground
25
Caraway seed
I (4)
10
Cardamon
10
Celery leaves
Whole (1)
25
Seed
Ground
10
Chervil
Whole (1)
10
Chives
" (1)
5
Cinnamon
Ground
see method
Unground
100
Cloves
Ground (1)
10
Condimental seeds
Whole
200
Coriander
Ground
10
Cumin
" (1)
10
Curry powder
Powder
10
Fennel
Ground
10
Fenugreek
10
Dill seed
10
Weed
Whole (1)
25
Garlic
Powder
50
Ginger
Ground
10
Mace
Ground (4)
10
Marjoram
Ground (1)
10
Unground
10
Mint
Flakes (1)
25
Mustard seed
Ground (1)
10
Nutmeg
"
10
Reconditioned
see method
Onion
Powder
50
Oregano
Ground (1)
10
Unground
10
Papaya leaves
Whole
10
Paprika
Ground
25
Parsley
Whole (1)
10
Pepper
Black
Ground
see method
White
"
see method
Red
Crushed (2)
25
Peppermint leaves
Whole
5
Poppy seed
Ground
10
Rosemary
" (1)
10
Whole (1)
25
Sage
Ground (1)
10
Rubbed
25
Rubbed and
10
ground
Savory
Ground
10
Spearmint leaves
Whole
5
Tarragon
Whole (1)
10
Thyme
Ground (1)
10
Whole (1)
25
Turmeric
Ground (3)
10
Vegetables
Flakes (1)
25
a
a
a
a or e
a
975.48(a) 985.37
975.48(a) 981.21
975.48(a) 975.48(b)
978.20
978.21 978.22
975.48(a) 977.24
975.48(a) 977.24
968.38(a)
968.38(b)
969.43
945.84 (Excreta)
975.48(a) 977.24
975.48(a) 975.48(b)
975.48(a) 975.48(b)
975.48(a) 975.48(b)
975.48(a)
975.50(a)
975.48(b)
975.50(b)
977.24
985.39
979.26
971.35
975.50(a)
975.50(b)
969.44
985.37
977.25B
972.40B
972.40B
972.40A
977.24
975.48
985.37
975.48(b)
979.25
985.38
975.48(a)
975.48(b)
985.37
a Excluding paprika.
Refs.: JAOAC (1): 58, 447(1975); (2): 58, 445(1975); (3): 58, 451(1975); (4): 59, 27(1976).
50 mL flotation liq., 945.75C(k), and stir mag., 970.66B(c),
5 min. Fill flask with 40% isopropanol, and let stand 30 min
with intermittent stirring.
Trap off, rinsing neck of flask with 40% isopropanol. Add
35 mL flotation liq. Hand stir solids on bottom with vigorous
rotary motion. Fill flask with 40% isopropanol. Let stand 20
min. Trap off, rinse neck with isopropanol, and filter onto ruled
paper. Examine microscopically at 30 x.
(b) Mineral oil. — Place filter cup with sample in No. 230
sieve, 945.75B(r), and wash sample into sieve with gentle
stream of hot tap H 2 0. Sieve with forceful spray of hot (55—
70°) tap H 2 until rinse is clear. Wet residue on sieve with
40% isopropanol and transfer quant, to 2 L trap flask,
945.75B(h)(4), using 40% isopropanol. Dil. to 400 mL with
40% isopropanol and boil gently 10 min with mag. stirring.
Remove from heat and immediately add mixt. 50 mL Tween
80-40% isopropanol soln, 945.75C(x), plus 50 mL Na 4 EDTA-
40% isopropanol soln, 945.75C(z), slowly down stirring rod.
Hand stir 1 min with gentle rotary motion. Let stand undis-
turbed 5-10 min. Dil. to 800 mL with 40% isopropanol, add
400 Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
FIG. 975.49— Solvent saver apparatus
50 mL mineral oil, 945.75C(p), and stir mag., 970.66B(c), 3
min. Fill flask with 40% isopropanol, and let stand 30 min
with intermittent stirring. Trap off, and repeat as in (a), using
35 mL mineral oil for second trapping.
(c) For crushed red peppers. — Transfer bulk of sample di-
rectly to 2 L trap flask, 945.756(h)(4), by scraping from paper
with spatula. Complete transfer by rinsing paper with 40% iso-
propanol, and finally dil. to ca 800 mL. Stir and heat to vig-
orous boil for ca 5 min {Caution: Watch for excessive foam-
ing! Control with cold H 2 from wash bottle.). Transfer to
cooling bath until temp, drops to 20-25°.
Add 40 mL flotation liq., 945.75C(k), and stir mag.,
970.66B(c), 5 min. Let stand 5 min while mixing 50 mL Tween
80-40% isopropanol soln, 945.75C(x), and 50 mL Na 4 EDTA-
40% isopropanol soln, 945.75C(z), with 200 mL of 40% iso-
propanol. Slowly add mixt. down rod with top of stopper held
just below top of liq. Gently swirl upper portion of liq. using
particular care not to disturb settlings at this time.
Let stand 5 min. Raise rod and spin stopper with gentle ro-
tary motion to free suspended material. With top of stopper
just above oil phase, slowly fill flask with 40% isopropanol.
Swirl top portion of liq. gently, avoiding any disturbance of
settled material.
Clamp rod and stopper about midpoint of flask. Let stand 5
min, spin stopper to dislodge material on it, and let stand 20-
30 min undisturbed. Trap off and rinse neck with 40% iso-
propanol .
Add 35 mL flotation liq. and swirl rapidly to suspend plant
material without incorporating air. Let stand ca 20 min. Trap
off and filter onto ruled paper. Examine microscopically at
30x.
(d) For ground mace and ground caraway seed. — Proceed
as in (b), except after adding 50 mL mineral oil, stir mag. 5
min.
Refs.: JAOAC 58, 445, 447, 451(1975); 59, 827(1976); 62,
419(1979); 65, 1089(1982).
985.37 Light Fifth in Whole Leaves
of Alfalfa, Papaya, Peppermint, and Spearmint
Flotation Method
First Action 1985
A. Apparatus and Reagents
(a) Reflux apparatus. — 975.49A(e).
(b) Tween 80-40% isopropanol.— 945.75C(x).
(c) Tetrasodium EDTA-40% isopropanol. — 945.75C(z).
B. Pretreatment
Add 5 g (peppermint, spearmint) or 10 g (alfalfa, papaya)
sample and 500 mL isopropanol to 1 L beaker. Boil 10 min
with mag. stirrer, using reflux app.
C. Isolation
Wet-sieve product on No. 230 sieve with hot H 2 until
washings are clear. Quant, transfer residue to 2 L trap flask
with 40% isopropanol. Fill flask to 400 mL with 40% isopro-
panol. Boil on hot plate for 10 min with slow mag. stirring,
945.75B(n). Cool to 20-25° in H 2 bath. Add 100 mL pre-
mixed Tween 80-Na 4 EDTA (1 + 1) down stirring rod. Hand-
stir gently 1 min. Let stand 5 min. Fill flask to 800 mL with
40% isopropanol. Add 50 mL flotation liq., 945.75C(k), down
stirring rod. Mag. stir, 970.66B(c), 5 min. Let stand 5 min.
Fill flask successively to 1200 mL, 1600 mL, and into neck
with 40% isopropanol down stirring rod, agitating contents
vigorously (up-down) with rod and wafer for 3-4 s after each
fill. Let stand 5 min. Again vigorously agitate flask contents.
Let stand 25 min. Trap into 400 mL beaker, washing neck,
wafer, and rod with 40% isopropanol and add rinse to beaker.
Add 35 mL flotation liq. Hand-stir (up-down) for 30 s. Let
stand 20 min. Trap into original beaker, washing neck, wafer,
and rod with isopropanol and add rinse to beaker. Filter onto
ruled filter paper, wash beaker with isopropanol, and filter
washings. Examine papers microscopically at 30 X.
Refs.: JAOAC 68, 697(1985); 70, 997(1987)
981.21 Light Filth in Ground Allspice
Flotation Method
First Action 1981
Final Action 1989
A. Determination
Weigh 10 g sample into 1 L beaker. Add 500 mL 40% iso-
propanol-HCl (93 + 7). Simmer on steam bath 10 min. Quant.
transfer to No. 230 plain weave sieve, 945.75B(r), using gentle
stream of hot (50-70°) tap H 2 0. Sieve with forceful stream of
hot tap H 2 until rinse is clear. Wet residue on sieve with 40%
isopropanol and quant, transfer to 2 L Wildman trap flask,
945.75B(h)(4), using 40% isopropanol. Bring vol. to 400 mL
with 40% isopropanol and boil gently 10 min on hot plate,
945.75B(n), with mag. stirring. Remove from heat and im-
mediately add, by pouring down stirring rod, 100 mL 1 + 1
mixture of Tween 80-40% isopropanol soln, 945.75C(x), and
Na 4 EDTA-40% isopropanol soln, 945.75C(z). Hand-stir 1 min,
using gentle rotary motion. Let stand 10 min. Bring vol. to
800 mL with 40% isopropanol and add 50 mL mineral oil,
945.75C(p). Mag. stir 3 min, 970.66B(c), at speed that results
in vortex where only upper surface of stirring bar is visible.
Fill to top with 40% isopropanol, let stand 30 min with inter-
mittent stirring, and trap off into beaker, rinsing neck of flask
with isopropanol. Repeat extraction, using 35 mL mineral oil.
Hand-stir bottom contents with gentle rotary motion. Add
enough 40% isopropanol to bring oil into neck of flask. Let
stand 30 min. Trap off, rinse, and filter entire beaker contents
AOAC Official Methods of Analysis (1990)
Spices
401
through ruled filter paper(s). Examine for hairs at 30x. If ex-
cessive plant tissue is present, bleach as in 965.38B(d). Ex-
amine microscopically.
Ref.: J AOAC 63, 1266(1980).
978.20
See 978.22.
978.21
Filth in Ground Annatto
First Action 1978
Final Action 1988
Heavy Filth
in Capsicums (Ground)
Sedimentation Method
First Action 1978
Final Action 1988
(Applicable to red and cayenne pepper, chili powder, etc.)
{Caution: See safety notes on toxic solvents and carbon
tetrachloride.)
For heavy filth and sand. — Isolate gross filth such as large
larvae, adult insects, clumps of webbing, and insect and rodent
excreta pellets by sifting pepper thru No. 10 sieve.
Weigh 50 g sifted sample into 600 mL beaker and add 400
mL pet ether. Boil gently 30 min, occasionally adding pet ether
to keep vol. constant. Decant pet ether onto smooth 15 cm
paper in buchner. Add 400 mL CC1 4 and let stand 30 min with
occasional stirring. Decant pepper and solv. onto same 15 cm
paper in buchner, leaving heavy residue of sand and soil in
beaker. Repeat decantation with CC1 4 if necessary to secure
practically complete sepn of spice tissues from heavy residue.
Transfer residue from beaker to ashless paper and examine for
filth. If there is appreciable residue, place paper in weighed
crucible, ignite, and det. sand and soil.
978.22 Light Filth
in Capsicums (Ground)
Flotation Method
First Action 1978
Final Action 1988
(Not applicable to paprika. Complete analysis without over-
night interruptions.)
A. Pretreatment
Proceed as in 975.49A(a), using 25 g sample.
0. Isolation
Wet-sieve on No. 230 sieve with warm tap H 2 until drain-
ings are clear. Transfer sample with spoon thru wide-stem fun-
nel into 2 L trap flask, 945. 756(h)(4). Rinse remaining ma-
terial to edge of sieve and transfer quant, to trap flask with
60% alcohol. Dil. to 600 mL with 60% alcohol.
Simmer 10 min on hot plate. Alternatively, place on pre-
heated hot plate, bring to boil, and then transfer to 3-4 cm
deep boiling H 2 bath for 10 min and simmer. This technic
avoids severe frothing encountered in hot plate boiling. Cool
to >:20° but <25°. Remove from bath and add 40 mL flotation
liq., 945.75C(k). Dil. to 800 mL with 60% alcohol and stir
mag. 5 min, 970.66B(c). Set ajide, add 100 mL 1 + 1 mixt.
Tween-80 plus 60% alcohol, 945.75C(w), and Na 4 EDTA-al-
cohol soln, 945.75C(y), and mix thru liq. by gently swirling
stopper (wafer) I min. Let stand 3 min. Slowly add 60% al-
cohol down trap rod, maintaining stopper above oil layer, until
oil just reaches neck of flask. Swirl stopper thru lower portion
of trap flask to suspend settlings (dark material may rise half-
way up in trap flask). Add 60% alcohol down rod to bring
bottom of oil layer to level 1 cm above fully raised stopper.
Clamp rod with stopper at ca midpoint of flask. Let stand
15 min; then gently swirl stopper thru upper half of liq. to
hasten rising of oil droplets. Let stand 15 min undisturbed and
trap off into beaker, rinsing neck of flask with 60% alcohol.
Filter onto labeled ruled paper.
Add 30 mL flotation liq. and stir manually 1 min. Clamp
rod at midpoint and let stand 10 min. Swirl gently thru upper
half of liq. and adjust oil level. Let stand 15 min and trap off.
Rinse neck of flask with 95% alcohol or isopropanol. Filter
onto second ruled paper and examine papers at 30 x .
Ref.: J AOAC 61, 900(1978).
977.24 Light Filth in Cardamon,
Celery Seed, Coriander,
Ginger, and Pepper (White)
Flotation Method
First Action 1977
(Applicable to the ground spices)
Weigh 10 g sample (25 g for white pepper) into 800 mL
beaker contg 400 mL isopropanol. Add mag. stirring bar, place
on stirrer, 945.75B(n), and stir mag. 6 min, keeping all solids
in motion. Pour mixt. onto No. 230 sieve, 945.75B(r), and
wash residue with H 2 until washings are clear. Transfer res-
idue from sieve with 40% isopropanol into 2 L trap flask,
945.75C(h)(4). Add 760 mL 40% isopropanol and 40 mL HCI.
Bring to vigorous boil on hot plate with mag. stirring. Cool
to 20-25° in cold H 2 bath. Add 40 mL flotation liq.,
945.75C(k), and stir mag., 970.66B(c), 5 min. Let stand 5
min and then slowly fill flask with 40% isopropanol by letting
liq. flow down stirring rod with top of stopper just below oil
layer. Resuspend material at bottom of flask without disturb-
ing oil (upper) layer. Let stand 20 min, stirring bottom oc-
casionally, and trap off. Add 30 mL flotation liq., and stir
mag. 30 sec while pushing oil into aq. (lower) layer; continue
stirring 4.5 min. Let stand 15 min, trap off, filter onto ruled
paper, and examine microscopically at 30 x . If filtering action
slows, use new filter paper.
Ref.: J AOAC 60, 117(1977).
968.38 Filth in Cinnamon (Ground)
Flotation Method
First Action 1968
(a) Heavy filth and sand. — Weigh 2 g sample into 50 mL
centrf. tube and add ca 45 mL CCl 4 . Centrf. 5 min at 800 rpm.
Stir layer at top of liq. and repeat centrf g. Decant ca 2 /s of liq.
and floating layer, and add fresh CCl 4 up to 45 mL. Mix tho-
roly and again centrf. Decant as much of liq. and floating layer
as possible without disturbing residue in centrf. tube. Wash
residue onto 11 cm ashless paper with CC1 4 . Examine under
low-power microscope for filth. If there is appreciable residue,
place paper in weighed crucible, ignite, and weigh sand and
soil .
(b) Light filth. — (Where alcohol and 60% alcohol are spec-
ified, isopropanol and 40% isopropanol, resp., may be sub-
stituted. Use same alcohol thruout method.) Weigh 50 g sam-
402
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
pie into 800 mL beaker. Add 500 mL hot (55-70°) tap H 2
and 50 mL HCl. Stir several min with stirring bar at high speed
on mag. stirrer-hot plate, 970.66B(c), holding temp, without
boiling until geJ is dispersed (suspension will become less vis-
cous and vortex will become more pronounced). Sieve por-
tion wise onto No. 230 sieve, 945.75B(r), with forceful stream
of hot tap H 2 0, using aerator, 945.75B(a). After fine material
has passed thru sieve, wash residue alternately with alcohol
and hot tap H 2 until most foam and color have passed thru.
Transfer residue to 1.5 L beaker with 60% alcohol, using
spoon to transfer bulk of material. Dil. to 1 L with 60% al-
cohol. Add 50 mL HCl and heat (do not boil) while stirring
with mag. stirrer to prevent charring. When mixt. is hot (ca
55°), add 50 mL mineral oil, 945.75C(p), and stir mag. 4 min.
Transfer beaker contents to 2 L percolator, 945.75B(h)(2), rinse
beaker well with 60% alcohol, and add rinsings to percolator.
Bring vol. in percolator to ca 1.7 L with 60% alcohol. Re-
suspend material in percolator by vigorously stirring with glass
rod, and rinse rod into percolator with 60% alcohol. Let settle
3 min, and immediately drain material in percolator to within
several cm of bottom of mineral oil layer. Refill percolator
with hot tap H 2 0, adding H 2 rapidly to thoroly resuspend
material in percolator. Let settle 3 min, and drain again. Re-
peat hot H 2 rinses until aq. medium is practically free of
suspended matter with max. of 7 rinses. Discard hot H 2
rinses. Drain mineral oil layer into 800 mL beaker and rinse
sides of percolator with alternate rinses of 95% alcohol and
hot (55-70°) tap H 2 (use rubber policeman if necessary). Pour
mineral oil and final rinses onto ruled paper and examine mi-
croscopically.
Ref.: JAOAC 51, 518(1968).
969.43 Light Filth in Cinnamon
(Unground) (Crude and Reconditioned)
Sieving Method
First Action 1969
If sample is reconditioned or if pieces are not rolled and are
8 cm (3") long, weigh 100 g sample directly into 1 .5 L beaker.
If sample consists of quills, break open quills into lengths of
<8 cm and transfer broken pieces, including dust and small
particles, to "1.5 L beaker. Add 1 L hot tap H 2 and 50 mL
HCl. Heat on hot plate to ca 60°. Pour portionwise onto No.
6 over No. 230 sieve, 945.75B(r), and rinse well with forcible
stream of hot tap H 2 0, using aerator, 945.75B(a), while turn-
ing larger pieces with glass rod. Discard material on No. 6
sieve and transfer residue on No. 230 sieve to 2 L trap flask,
945.75B(h)(4), with hot tap H 2 0, using spoon if necessary.
Fill trap flask to 1 L with H 2 and add 50 mL HCl. Heat with
stirring to ca 60-70°. Add 50 mL mineral oil, 945.75C(p),
and stir mag., 970.66B(c), 2 min. Fill with H 2 0, let stand 5
min, and trap. Add 25 mL mineral oil, gently stir with stopper
1 min, let stand 5 min, and again trap. Rinse neck of flask
with alcohol or isopropanol. Filter trappings onto ruled filter
paper and examine for insects and other arthropods, hairs, ex-
creta, etc.
Ref.: JAOAC 52, 469(1969).
945.84 Excreta (Rodent and Insect)
in Condiment Seeds
Sedimentation Method
First Action
Prep. liq. with sp gr of 1 . 16-1 . 19 by mixing CHC1 3 or CC1 4
with alcohol or pet ether. Mix 200 g sample with 500-700 mL
of the liq. in 1 qt (1 L) drug percolator. Let stand 30 min,
stirring at ca 5 min intervals. Trap sediment in lower end of
percolator with cork plug and remove lower cork so as to de-
liver all sediment into beaker. Lift upper cork slightly and rinse
tube and cork by letting small amt of liq. pass. After stirring
top layer, make 2 more sepns at 5 min intervals. Transfer con-
tents of beaker to filter paper, drain liq., and examine. Sep.
rodent excreta and insect excreta, air dry, and weigh each sep.
to nearest mg.
975.50 Filth in Garlic Powder
and Ground Onion
Sedimentation/ Flotation Method
First Action
(Caution: See safety notes on toxic solvents and carbon
tetrachloride.)
(a) Heavy filth and sand. — Weigh 50 g sample into 250 mL
hook-lip beaker. Add 200 mL CC1 4 , stir thoroly, and let stand
30 min with occasional stirring. Decant plant tissue onto 15
cm paper in buchner, add 100 mL CC1 4 , and repeat decantation
until practically no plant tissue remains with sand and soil on
bottom of beaker. Transfer residue in beaker to ashless paper
with stream of CC1 4 from wash bottle and examine for filth.
If there is appreciable residue, place paper in weighed cruci-
ble, ignite, and weigh sand and soil.
(b) Light filth. — Dry residue of plant tissue from buchner,
(a), overnight or in oven 1 hr at 80°, and transfer to 2 L trap
flask, 945.75B(h)(4). Add 250 mL Tween 80-60% alcohol
soln, 945.75C(w), mix well, and let stand 15-30 min. Add
60% alcohol to 800 mL and trap off twice in 60% alcohol with
75 and 35 mL heptane, 945.75C(1), resp., as in 970.66B(b).
Let stand 1-1.5 hr for each extn and avoid stirring except for
few circular upward strokes immediately after filling flask with
60% alcohol. Filter, and examine microscopically.
979.25 Light Filth in Sage (Rubbed)
Flotation Method
First Action 1979
A. Pretreatment
Form 32 cm filter paper cup over 400 mL beaker and insert
into 1 L beaker, 945.75B(j). Place 25 g sample into cup. Add
400 mL CHC1 3 , ca equally between cup and beaker. Boil gently
10 min in hood. (Alternatively, bring to bp on hot plate and
continue heating 10 min total on steam bath.) Transfer cup to
buchner and aspirate to slow drip. Return cup to beaker and
repeat extn with two 400 mL portions CHC1 3 . After third CHC1 3
aspiration, turn off vac, cover sample with isopropanol, let
stand 1 min, and aspirate to slow drip. Repeat isopropanol extn
once.
B. Isolation
Transfer sample from paper cup to No. 230 sieve, 945.75B(r),
with gentle stream of hot tap H 2 0. Sieve with forceful spray
of hot (55-70°) tap H 2 until rinse is clear. Wash residue to
edge of sieve and let drain momentarily. Rinse sieve with ca
100 mL of alcohol delivered from wash bottle. Let stand 1
min. Again wash sieve residue with hot tap H 2 until drain-
ings are colorless; then wet well with 40% isopropanol.
Add mag. stirring bar, 945.75B(n), to 2 L trap flask,
945. 75B (h)(4). Place wide stem funnel in flask opening and
transfer bulk of sample with spoon. Rinse remaining material
to edge of sieve with aerator spray and quant, transfer to flask
with total of 400 mL 40% isopropanol. Stir gently while boil-
ing 10 min on hot plate. (Alternatively, bring to boil on hot
AOAC Official Methods of Analysis (1990)
Spices
403
plate and continue heating 10 min in ca 40 mm boiling water
bath.) Remove from heat and immediately add 100 mL 1 +
1 mixt. Tween 80 plus isopropanol soln, 945.75C(x), and
Na 4 EDTA, plus 40% isopropanol soln, 945.75C(z), slowly
down rod and mix by gentle swirling 1 min.
Cool in H 2 bath at 20-25°. Add 50 mL mineral oil,
945.75C(p). Dil. to 800 mL with 40% isopropanol added slowly
down stirring rod to avoid mixing or agitation of flask con-
tents.
Stir mag,, 970,66B(c), 5 min, and let stand 3 min. Add 100
mL premixed Tween 80-Na 4 EDTA and very gently swirl thru
top of liq. 1 min.
Fill flask with 40% isopropanol , added slowly down stirring
rod to minimize agitation of liq. Let stand 20 min undisturbed.
Trap off, rinsing neck of flask with 40% isopropanol, and add
to trappings in beaker.
Add 35 mL mineral oil and hand stir 1 min. Let stand ca 1
min. Slowly fill flask with 40% isopropanol. Let stand 7 min,
spin stopper to free of settlings, adjust oil level to ca 1 cm
above fully raised stopper and let stand 8 min. Trap off into
beaker, rinsing neck of flask well with isopropanol. Transfer
trappings to ruled filter paper, rinsing beaker well with iso-
propanol. Examine papers at 30X.
Ref.: JAOAC 62, 597(1979).
985.38 Light Filth in Sage
(Rubbed and Ground)
Brine Saturation Method
First Action 1985
A. Reagents
(a) Brine. — Prep, by dissolving ca 360 g NaCl/L H 2 0. Less
expensive sources of salt which were found by use and by
comparative study to be equiv. to AR grade NaCl were: (I)
Morton Rock Salt for making ice cream, Morton Salt Div. of
Morton Thiokol, Inc., Chicago, IL 60606; or, (2) Sterling salt
crystals for water softeners, Internationa] Salt Co., AKZO,
Abington Executive Park, Clarks Summit, PA 18411. Filter
brine before use.
(b) Olive Oil, NF. — Less expensive sources of olive oil
which were found to be equiv. to NF grade oil by use and by
comparative study and which were more readily com. avail-
able were: (J) James Plagnoil Pure Olive Oil, Marseille, France;
(2) BertoJli Pure Olive Oil, Lucca, Italy; (3) Vigo Spanish Vir-
gin Olive Oil imported by Vigo Import Co., Tampa, FL; and
(4) Flag Brand Pure Sicilian Olive Oil, Product of Italy, packed
for Progresso Foods Div., Imasco Corp., Harahan, LA.
B. Extraction
Rubbed and ground sage, — Weigh 10 g sample into 600 mL
beaker. Add mag. stirring bar, 945.75B(n), and 300 mL iso-
propanol. Cover with watch glass and boil ca 3 min with const
stirring on mag. stirrer hot plate. Transfer to 230 mesh sieve,
945.75B(r), with isopropanol, and wash with ca 100 mL iso-
propanol from wash bottle, then with hot tap H 2 to remove
isopropanol, and then with brine to remove tap H 2 0. Transfer
to original 600 mL beaker and bring vol. to ca 350 mL with
brine. Cover and bring to boil with const stirring. (Note: Do
not boil. Product loss will occur from boil-over.) Transfer beaker
to cold H 2 bath (use plastic or glass bowl filled with cold
H 2 on mag. stirrer). Uncover, wash down insides of beaker,
and cool to touch with const stirring. Repeat heating and cool-
ing to complete brine satn, and then transfer to 2 L trap flask,
945.75B(h)(4), with ca 100 mL brine, fill flask ca 3 / 4 full with
32° (± 2°) tap H 2 0, and insert trap rod. (Note: Temp, variation
below that specified could result in loss of oil trapped due to
adhesion to sides of flask.) Add 50 mL olive oil, and then fill
flask to normal (neck) level with tap tLO. Place on mag. stir-
rer with trap rod to one side of flask bottom to avoid inter-
fering with stirring bar. Stir rapidly for 5 min so that no sig-
nificant oil layer is visible in neck of flask but not so rapidly
that vortex extends more than I in. into flask. Remove from
stirrer and let oil layer rise. Stir bottom of flask to release any
trapped oil; repeat after 5 min. Wait ca 20 min, and then briefly
and gently stir entire flask contents, particularly at the neck,
to release and sink any product that has accumulated at inter-
face. After 10 min, stir interface only with pivoting rotary mo-
tion of trap rod, keeping plunger relatively stationary at bottom
of flask. Wait 10 min and repeat previous stirring if product
has accumulated at interface. Then wait 10 more min and trap
oil layer into 250 mL beaker, rinsing neck of flask and trap
thoroly several times with isopropanol and finally with H 2 0.
Filter on ruled paper and examine microscopically, 970.66B(g),
Ref.: JAOAC 68, 894(1985).
985.39 Light Fifth
in Unground Marjoram
Flotation Method
First Action 1985
A Sample Preparation
Form filter paper cup, 400 mL into 1 L, 945.75B(j), and
weigh 10 g sample into cup.
Place cup into 1 L beaker contg 400 mL 95% EtOH. Boil
gently on hot plate 10 min, with mag. stirring. 970.66B(c).
Place cup in buchner funnel. Draw off excess EtOH with vac.
Discard EtOH in beaker. Repeat operation twice more; then
proceed with extn.
Quant, transfer material to 2 L Wildman trap flask.
945.75B(h)(4). Bring to 600 mL with 60% EtOH. Add mag.
stir bar, 945.75B(n). Boil gently 10 min with mag. stirring at
minimal rate. If material accumulates on walls of flask, rinse
side with minimal amt of 60% EtOH. Remove from heat and
add 100 mL Tween 80-EDTA mixt. [945.75C(w)-(y)] (1 +
1). Hand-stir contents, using gentle rotary motion; then cool
in H 2 bath to 25 ± 2°. Bring vol. to 800 mL with 15% EtOH.
Add 50 mL mineral oil, 945.75C(p). Stir mag. 3 min at speed
resulting in vortex where only the upper surface of stirring bar
is visible. Add enough 15% EtOH to bring interface into neck
of trap flask and let stand. Stir bottom of flask gently at 10
and 20 min intervals. Let stand addnl 10 min. Trap off, rinsing
neck of flask with 15% EtOH. Repeat extn using 35 mL min-
eral oil. Vigorously hand-stir trap flask contents to mix oil
with aq. phase. Add enough 15% EtOH to bring oil to neck
of flask. Trap off, and rinse with 95% EtOH followed by 60%
EtOH. Filter entire beaker contents thru ruled filter paper(s).
Examine microscopically at 30 X.
Ref.: JAOAC 68, 699(1985).
979.26 Light Fifth in Nutmeg (Ground)
Flotation Method
First Action 1979
Final Action 1988
A. Pretreatment
Form 32 cm filter paper defatting cup, 400-800 mL,
945.75B( j). Weigh 10 g sample into cup, add 400 mL CHCl 3s
and simmer 10 min. Drain or aspirate and discard CHC1 3 . Re-
turn cup to beaker, add 400 mL isopropanol, and heat to vig-
orous boil.
404
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
B. Isolation
Immediately wet- sieve on No. 230 sieve, 945.75B(r), until
washings are clear. Rinse material on sieve with 40% isopro-
panol and let drain. Quant, transfer to 2 L trap flask,
945.758(h)(4), with 40% isopropanol and dil. to 800 mL. Add
stirring bar, 945.75B(n), and heat to vigorous boil while stir-
ring. Add 40 mL mineral oil, 945.75C(p), and continue heat-
ing to vigorous boil.
Transfer flask to cool stirrer, and mag. stir, 970.66B(c), 5
min. Set aside and fill with 40% isopropanol down stirring rod
to bring oil just into neck of flask. Stir to suspend settlings.
Stir after 5 min and clamp wafer at ca midpoint of flask. After
5 min adjust oil level to 1 cm above fully raised stopper, and
swirl interface. Let stand 5 min and trap off into beaker. Rinse
neck with 40% isopropanol and add rinse to trappings.
Add 30 mL mineral oil and hand stir 1. min. Let stand 10
min and trap off. Rinse neck of flask with isopropanol and add
rinse to beaker. Filter onto ruled paper(s). Examine papers at
30X.
Ref.: JAOAC62, 595(1979).
971.35 Light Filth
in Nutmeg (Reconditioned)
Flotation Method
First Action 1971
Final Action 1988
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Weigh 100 g sample into 1.5 L beaker (50 g if finely ground
product is used). Add 400 mL CHC1 3 and boil 5 min. Prep.
32 cm folded S&S 588 filter paper, by moistening with H 2
and forming around base of 1 L beaker. Place 7 cm disk of
bolting cloth (mesh size not critical) in 10 cm plate diam.
buchner, insert paper, apply vac, and press moistened paper
until good seal is obtained. Rinse paper with isopropanol, and
aspirate until nearly dry. Quant, transfer nutmeg to paper, and
aspirate off CHC1 3 . Transfer paper contg spice tissue back to
original beaker, add 400 mL fresh CHC1 3 , and boil 5 min.
After cooling few min, lift paper, and drain and discard CHC1 3 .
Replace paper in beaker, add 400 mL CHC1 3 , and repeat 5
min boiling third time.
Replace paper in buchner and aspirate off CHC1 3 , maintain-
ing suction ca 5 min after visible dripping ceases. Release vac.;
add isopropanol until spice is covered, let stand few min, and
reapply vac. until visible dripping ceases. Repeat isopropanol
wash step and aspirate ca 5 min after visible dripping ceases.
Quant, transfer retained spice to 8" diam. No. 230 sieve,
945.75B(r), with copious rinses of hot tap H 2 0. Wash ma-
terial on sieve with forceful stream of hot H 2 from aerator,
945.756(a), until no more spice tissue passes sieve. Transfer
most of sieve contents with spoon and quant, transfer remain-
ing material to 2 L trap flask with 60% alcohol-CaCl 2 soln,
945.75C(c), from wash bottle. Add mag. stirring bar, bring
vol. to 1 L with 60% alcohol-CaCl 2 soln, add 50 mL HC1, and
place flask on mag. stirrer-hot plate, 945.75B(n). Heat to full
boll with gentle stirring. Immediately transfer flask to cool
stirring unit and add 40 mL light mineral oil, 945.75C(p), by
pouring down stirring rod. Stir 2 min with mag. stirrer,
970.66B(c). Fill with 60% alcohol-CaCI 2 soln and gently stir
5-10 sec with stirring rod. Let stand 2 min and trap off. Add
35 mL light mineral oil, stir by hand gently 30 sec, and let
stand 10 min. Repeat trappings. Wash flask neck thoroly with
isopropanol and transfer washings to beaker with trappings.
Filter onto ruled paper and examine microscopically.
Ref.: JAOAC 54, 575(1971).
969.44 Light Filth in Oregano (Unground)
Flotation Method
First Action 1969
A. Reagent
} 5% Alcohol. -
-Prep, ca 1700 mL/sample prior to analysis.
B. Determination
Weigh 10 g sample into 2 L trap flask, 945.75B(h)(4), add
400 mL 60% alcohol, and boil gently 10 min, occasionally
swirling flask gently and/or using plunger to prevent material
from accumulating on wall of flask above surface of liq. Im-
mediately add 100 mL 1 + 1 mixt. Tween-80 plus 60% al-
cohol soln, 945.75C(w), and Na 4 EDTA plus alcohol soln,
945.75C(y), and swirl few sec, again using plunger to clear
material from wall of flask. Let stand 10 min in cold FLO bath.
Dil. to 800 mL with 15% alcohol. Add 50 mL mineral oil,
945.75C(p), and stir mag., 970.66B(c), 2 min. Fill with 15%
alcohol and hand stir every 2-3 min for 20 min. Clamp stirring
rod in place so that plunger is held above sediment at bottom
of flask. Leave flask undisturbed 10 min. Trap, filter onto ruled
paper, and examine microscopically.
Ref.: JAOAC 52, 21(1969).
977.25
Filth in Paprika
First Action 1977
Final Action 1988
A. Gross Contamination
See 960.51
Light Filth in Ground Paprika
B. Pretreatment
Form 32 cm filter paper (rapid flow) cup around 400 mL
beaker, as in 945.75B(j). Remove paper, place in 1 L beaker,
and add 25 g sample.
Pour 400 mL isopropanol into the 1 L beaker, distributing
liq. equally inside and outside cup. Place on preheated hot
plate and boil gently exactly 10 min. (Alternatively, bring to
boil on hotter hot plate; then set aside in steam bath opening
for 10 min boil.) Remove cup from beaker without delay, and
let drain or place on buchner and aspirate to slow drip. Discard
liq. Replace cup in 1 L beaker and repeat twice with 400 mL
isopropanol.
C. Isolation
Wash sample from cup into No. 230 sieve, 945.75B(r), with
gentle H 2 stream, avoiding splashing and loss of sample. Wet
sieve with forceful stream of warm H 2 from aerator until
washings are clear. (Ignore foam or froth produced by action
of strong spray on paprika.)
Add 400 mL 40% isopropanol to wash bottle. Place wide-
stem powder funnel in trap flask. Transfer bulk of sieved sam-
ple to trap flask, using portion of 40% isopropanol. Wash re-
maining material on sieve to edge with warm H 2 and com-
plete quant, transfer to trap flask with 40% isopropanol. W r ash
walls of flask with 40% isopropanol. Pour remainder of 400
mL 40% isopropanol into trap flask.
Place on not plate and boil gently 10 min; swirl to rinse
material from walls of flask. (Do not allow material to accu-
AOAC Official Methods of Analysis (1990)
Spices
405
mulate and dry on flask walls.) Remove from hot plate and
immediately add 100 mL 1 + 1 mixt. Tween-80 plus 40%
isopropanol soln, 945.75C(x), and Na 4 EDTA plus 40% iso-
propanol soln, 945.75C(z). Stir gently ca 1 min. Let stand 10
min. Dil. to 800 mL with 40% isopropanol added slowly down
stirring rod, positioned with stopper just above liq. level. Add
50 mL mineral oil, 945.75C(p), and stir mag., 970.666(c), 3
min, with stopper located above liq. level.
Add 40% isopropanol slowly down stirring rod to bring oil
into neck of flask. Let stand ca 10 min. Raise stopper to mid-
dle of flask and swirl gently to cause movement of upper liq.
and hasten rising of oil droplets. Rinse rod with 40% isopro-
panol and clamp so that stopper is at midpoint of flask. Add
40% isopropanol down rod to bring bottom of oil layer to level
1 cm above fully raised stopper. Let stand 10 min and swirl
gently again. Let stand 10 min undisturbed and trap off into
beaker.
Add ca 35 mL mineral oil and hand stir 1 min at speed
sufficient to keep oil moving thru trap flask. Add ca 20 mL
40% isopropanol, stir gently at ca 5 min intervals for 20-25
min, then let stand undisturbed 5-10 min. Trap off into second
beaker and rinse neck of trap flask with alcohol or undild iso-
propanol.
Filter solns from both beakers onto sep. ruled filter papers
in Hirsch funnel, rinsing each beaker carefully with isopro-
panol. Examine microscopically at 30 X.
Ref.: J AOAC 60, 114(1977).
972.58* Light and Heavy Filth in Peppers
(Unground, Fermented, Crushed)
972.40
Light Filth in Pepper
First Action 1972
Final Action 1988
A. Light Fifth (Ground Black Pepper Only)
Weigh 50 g sample (or use floated material from 972.40B
before ignition) into 400 mL beaker and add enough hot H 2
(55-70°) to make thin slurry. Pour slurry onto No. 230 sieve,
945.74B(r), and wash residue with forceful stream of hot H 2 0,
using aerator, 945.758(a), until effluent is clear. Wash sieve
residue with ca 100 mL isopropanol and let drain. Transfer
residue from sieve with 40% isopropanol into 2 L trap flask.
Dil. to 800 mL with 40% isopropanol. Bring to rolling boil
with mag. stirring. Cool to room temp, in H 2 bath. Add 40
mL flotation liq., 945.75C(k), and stir mag. 3 min, 970.66B(c).
Let oil phase sep. 5 min and then fill flask with 40% isopro-
panol by letting liq. flow down stirring rod. Let stand 20 min,
with gentle stirring at 5 min intervals, and trap off. Repeat
trapping with 20 mL flotation liq.; stir gently after addn to
avoid disturbing bottom layer. Let stand 10 min and trap off.
Filter onto ruled paper and examine microscopically.
Ref.: JAOAC55, 83(1972).
B. Heavy Filth and Sand (Ground Black and White Pepper)
(Caution: See safety notes on toxic solvents and carbon
tetrachloride.)
Weigh 50 g sample into 600 mL beaker. Add 400 mL CC1 4
and let beaker stand ^ 1 hr with occasional stirring. Decant
pepper and sol v. onto 15 cm paper in buchner, leaving heavy
residue of sand and soil in beaker. Repeat decantation with
CC1 4 if necessary to secure practically complete sepn of spice
materials from any heavy residue. Transfer residue from beaker
to ashless paper and examine for filth. If there is appreciable
residue, place paper in weighed crucible, ignite, and weigh
sand and soil.
First Action
Surplus 1970
See 40.121, 11th ed.
972.59* Light and Heavy Filth in Pepper Sauce
First Action
Surplus 1970
•See 40.122, 11th ed.
945.85
Filth in Pickles
First Action
A. Whole Pickles
Pour entire contents of jar onto No. 8 sieve nested in No.
140 sieve, 945.75B(r). Wash jar thoroly to remove any filth
adhering to sides, and pour washings thru sieves. Wash pickles
thoroly with stream of hot H 2 0, turning from time to time.
Transfer material on No. 140 sieve directly to ruled paper and
examine microscopically.
B. Chopped Pickles and Relish
Add 200 mL H 2 to 100 g sample in trap flask or beaker,
boil 15 min, and cool. If boiling is done in beaker, transfer to
trap flask, 945.75B(h)(4). Trap off twice, using 25 and 15 mL
heptane, 945.75C(I). Filter, and examine microscopically.
945.86 Filth in Dressings for Food
Filtration Method
First Action
(Applicable to salad dressing, trench dressing, and related
products)
Weigh 200 g sample into 800 mL beaker, stir in 50 mL
H3PO4, and mix thoroly. Thin with ca 600 mL H 2 0, and again
mix thoroly. If possible, filter thru S&S No. 8 ruled paper with
suction; otherwise thru No. 140 sieve, 945.75B(r), and trans-
fer to ruled paper. Examine papers microscopically.
973.62 Filth in Horseradish (Prepared)
Flotation Method
First Action 1973
Final Action 1988
Weigh 100 g sample into 600 mL beaker. Add 200 mL H 2
and transfer to 2 L trap flask with H 2 0. Dil. to 1 L, add 50
mL HC1, and stir few sec. Add mag. stirring bar, 945.75B(n),
and 50 mL flotation liq., 945.75C(k), and stir mag.,
970.66B(c), 3 min. Slowly fill flask with distd H 2 by running
liq. down stoppered rod while stopper is maintained just above
liq. After filling flask, gently stir settled material 5-10 sec
with stoppered rod. Let stand undisturbed 5 min; then trap off.
Add 35 mL flotation liq., stir gently by hand 30 sec, and let
stand 10 min. Repeat trapping. Wash flask neck thoroly with
isopropanol and transfer washings to beaker contg trappings.
Filter onto ruled paper and examine microscopically.
Ref.: JAOAC 56, 629(1973).
406
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
968.39 Light Filth in Mustard (Prepared)
Flotation Method
First Action 1968
Final Action 1988
form second trapping. Rinse neck of flask with 95% aJcohol
and pour trappings onto ruled filter paper. Examine at 30 X .
Ref.: JAOAC 52, 17(1969).
Weigh 100 g weJJ mixed sample into 1 L beaker, and slowly
add 400 mL HC1 (3 + 97) and 20 mL mineral oil, 945.75C(p),
with constant stirring until smooth slurry forms. Place on hot
plate and bring to rolling boil; hold at rolling boil ca 10 min.
Transfer quant, to Kilborn funnel, 945.75B(h)(7); retain beaker
and stirring rod for rinsing. Fill separator to ca 1 cm from top
with cold H 2 0.
After 1.5—2 min, gently stir contents of separator; let oil
layer sep. again ca 1.5-2 min, and slowly drain and discard
lower layer until interface is ca 5 cm above constriction. Fill
separator with cold H 2 to ca 1 cm from top; let oil sep. 1 .5-
2 min, and slowly drain and discard lower aq. layer until in-
terface is ca 5 cm above constriction. Repeat H 2 wash until
lower layer is clear.
Filter mineral oil and H 2 retained in separator thru ruled
paper, 945.75B(i), using Hirsch funnel. After mineral oil layer
has passed thru paper, rinse all glassware thoroly with alcohol,
followed by H 2 0, then 5% detergent soln, 945.75C(i), and
cold H 2 0. Filter each rinse sep. thru same paper. Rinse final
papers with enough alcohol to remove yellow color. Examine
papers at 30 x.
Ref.: JAOAC 51, 522(1968).
945.87*
Filth in Whole
Tamarind Pulp
First Action
Surplus 1970
See 40.125, 1 1th ed.
MISCELLANEOUS
969.45 Light Filth in Gums (Plant, Crude)
Flotation Method
First Action 1969
Final Action 1988
(if av. particle is <5 mm, proceed with method. If particle
size is >5 mm, break into pieces by hand or by dropping small
amts at a time into high-speed blender until desired size is
reached. Where 95% and 40% alcohol are specified, isopro-
panol and 30% isopropanol, resp., can be substituted.)
Weigh 50 g sample into 2 L beaker, add 1.2 L H 2 and 15
mL HC1, and stir well. Autoclave 1 hr at 121°. Slow vent.
(Arabic and guar gums will completely dissolve in 15-30 min
in 1.2 L H 2 + 25 mL HC1 when placed on mag. stirrer-hot
plate or in steam bath.) Sieve portion wise on No. 230 sieve,
945.75B(r), using forcible stream of hot (55-70°) tap H 2 from
aerator, 945.75B(a), until all gum has passed thru. Transfer
directly to ruled filter paper if negligible amts of plant tissue
remain on sieve. If large amts of plant debris remain on sieve,
transfer to 1 L trap flask with 40% alcohol. Bring vol. to 500
mL with 40% alcohol and add 25 mL HCJ. Heat to ca 60° on
mag. stirrer-hot plate with stirring. Add 25 mL mineral oil,
945.75C(p), and stir mag. 2 min, 970.66B(c) Fill flask with
40% alcohol and gently swirl contents with stopper. Let stand
10 min and perforin first trapping. Add 25 mL mineral oil,
and gently stir with stopper 1 min. Let stand 5 min and per-
970.74 Foreign Matter
in Drugs (Leafy, Crude)
First Action
A. Gross Contamination
See 960.51.
B. Heavy and Light Filth
See 975.48.
971.36 Light Filth in Papain
(Crude and Refined)
Flotation Method
First Action 1971
Final Action 1988
Weigh 50 g sample into L.5 L beaker. (Caution: Weigh un-
der effective fume removal device to avoid breathing dust. Avoid
skin contact.) Add 1 L hot H 2 and small amt of Antifoam A
spray. Boil 20 min with stirring. Wet sieve forcefully on No.
230 sieve until all whitish material passes thru sieve and only
plant debris and extraneous materials remain. If small amt of
material remains on sieve, transfer directly to ruled filter pa-
per. If large amt of plant tissue remains, transfer to 1 L trap
flask with 40% isopropanol or 55% alcohol (use same alcohol
thruout method). Bring vol. to 500 mL with alcohol and add
25 mL HC1. Boil 5 min, add 25 mL mineral oil, 945.75C(p),
stir mag. 2 min, 970.66B(c), fill with alcohol, and trap off
after 10 min. Perform second trapping, using 15 mL mineral
oil, stir by hand 1 min, and trap off after 10 min. Filter trap-
pings thru ruled paper and examine microscopically.
Ref.: JAOAC 54, 565(1971).
973.63 Insect Penetration
thru Packaging Materials
Microscopic Examination Method
First Action 1973
Final Action 1988
(a) Entrance Characteristics
(/) Kraft paper, paper box.
(a) Surface fraying. — Consists of paper fibers cut and lifted
from surface of packaging material by mandibular activity.
Represents first activity of hole formation. May occur at ran-
dom on "entrance" surface of packaging materials. See Fig.
973.63(B)
(b) Terraced depression. — Consists of "step effect" formed
when secondary depression is superimposed on initial depres-
sion; see Fig. 973.63(D). This terracing may be present around
entire perimeter of final hole or at one or more points around
it.
(c) Tapered hole. — Diam. of hole is greater on entrance side
than exit side. This feature is most obvious on thicker pack-
aging materials. See Fig. 973.63(E).
(2) Foil, Cellophane, polyethylene plastic, waxed paper.
(a) Mandibular scratches. — Found on Al foil, Cellophane,
and polyethylene plastic. Consists of small, short surface
scratches or grooves formed by pincerlike action of mandibles.
AOAC Official Methods of Analysis (1990)
Animal Excretions
407
(A)
(B)
(Q
(D)
(E)
Sound
packaging
Fraying
initial
depression
Secondary
depression
s a
Completed
hole
(F)
Foil
Cellophane
Polyethylene
(G)
(H)
l't
Foil
Polyethylene
1*t
(I)
^c£
Laminates
FIG. 973.63 — Insect penetration of packaging
Frequently observed around perimeter of hole or in localized
groups at random on entrance surface. See Fig. 973.63(F).
(b) Upturned edges.— Present around perimeter of holes in
A.l foil and polyethylene plastic materials. Appear as contin-
uous irregularly upturned edge in foil (Fig. 973.63(G)) and
generally as upturned fraying of plastic (Fig. 973.63(H)) in
polyethylene materials. Not observed on waxed paper and Cel-
lophane materials.
(c) Roughened surface . — Observed around perimeter of holes
or randomly on surface of polyethylene plastic and waxed pa-
per. Consists of surface fraying or pulled up tufts resulting
from mandibular action on material. Distinct mandibular
scratches may be observed around or in roughened areas.
(3) Foil/paper, foil /plastic, or other laminates.
(a) Entrance characteristics. — See specific materials.
(b) Terracing of laminates. — Quite common on entrance side
of these materials. Observed as larger hole bored in laminate
material on entrance surface and smaller hole in exit side of
material. See Fig. 973.63(1).
(b) Exit Characteristics
All types of packaging materials.
(/) Clean-cut hole perimeter.
(2) Diam. of hole smaller than on entrance side.
(3) No surface fraying, scratches, or depressions (see Fig.
973.63(E)).
Ref.: JAOAC 56, 640(1973).
ANIMAL EXCRETIONS
(Liquid Excretions: See Fig. 945.88.)
(Solid Excretions: See Fig. 981.22.)
945.88 Urine Stains
on Foods and Containers
Ultraviolet Light Examination
First Action
(Caution: See safety notes on hazardous radiations.)
(Applicable to suspect urine stains on all materials except seeds)
Examine suspected stains in dark room under long wave UV
light (366 nm). (Dried urine on textiles usually fluoresces blue-
white, but color varies somewhat, depending upon natural color
of textile and type of lamp and filter used.) Run check patches
with known types of urine. For microehem. analysis, outline
stained area with pencil under the UV light. When odor of
urine is detected, report this.
942.24 Urine Stains on Foods
and Containers
Urease Test for Urea
First Action
(Applicable to urine residues on materials with significant amts
of interfering substances, i.e., fats and oils)
Cut out portion of stained area and transfer 1 or 2 threads
to 5 niL crucible or beaker. Save balance of cloth to confirm
urine by 973.64. Leach 10 min in just enough warm H 2 to
cover material. Remove threads and squeeze out as much liq.
as possible with clean, flat-tip forceps.
Transfer 2 or 3 drops to mieroculture slide with deep cylin-
drical depression. Add small drop urease mixt. (suspension of
l U of 25 mg urease tablet in 0.5-0.7 niL H 2 0). Place small
drop 10% H 2 PtCl 6 soln on cover slip and invert over the
depression, with hanging drop at center of depression opening.
(Cover slip may be sealed on with petrolatum if only minute
amts of urea are suspected.)
With evolution of NH 3 , brilliant, highly refractive, octahed-
ral crystals of (NH 4 ) 2 PtCl 6 are formed in hanging drop. Time
408
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
1) Use this test if fats, oils, or other interferences are expected to be present.
2) Use this test if interfering substances are not expected to be present.
3) If only minimal sample is available, proceed directly to TLC confirmation.
FIG. 945.88 — Methods for urine: method selection and results interpretation guide
AOAC Official Methods of Analysis (1 990)
Animal Excretions
409
MammaBian Feces
981.22 and 988.17
Bird Droppings and Insect Excreta:
Insect Excreta
Suspect Bird
Dropping Stains
Suspect Particulate
Bird Droppings
Obvious Particulate
Bird Droppings
< + ) Uric Acid =
Probable Bird
Droppings/Insect
Excreta
-) Uric Acid = Bird
Dropriings/lnsect
Excreta not
Confirmed
( + ) Uric Acid =
Probable Bird
Droppings
{-) Uric Acid = Bird
Droppings not
Confirmed
Spectrophotometric Test
for Uric Acid in Flour
969.46
FIG. 981.22— Method selection guide: methods for solid excretory materials
required for crystals to form varies from few sec to 30 min,
or even longer in some instances, according to conditions.
Crystals may be visible to naked eye and are readily detected
under microscope at 100x. Certain org. compds that are vol-
atile and H 2 0-sol. may yield crystals in the hanging drop, and
if reagent soln is too coned, H 2 PtCl 6 may crystallize. How-
ever, crystal habits of these substances are different from those
of (NH 4 ) 2 PtCl 6 . (Stained patches of the food material can be
tested by method similar to above.)
Ref.: JAOAC 25, 772(1942).
CAS-57-13-6 (urea)
959.14 Urine Stains on Foods
and Containers
Xanthydrol Test for Urea
First Action
(Not applicable in presence of dried skim milk. Applicable to
fluorescing urine residues on materials without significant amts
of interfering substances.)
Place portion of stained cloth, ca 3 mm sq (stain located by
fluorescence) on microscope slide. Add drop of HO Ac (2 +
1) and stir. (Or instead of cutting out a patch of cloth, rinse
stained material with H 2 or other suitable sol v. such as HO Ac,
acetone, or hot alcohol, evap. soln to dryness, dissolve residue
in little HO Ac (2 + 1), and place drop on slide.)
Transfer droplet with stirring rod to another place on slide
and dil. with drop of HO Ac (2 + 1). To both drops add very
small amt of xanthydrol and stir into soln. If urea is present,
crystals of dixanthylurea form very shortly. Examine with
magnification of ca 1 00-1 20 x (higher power may be used for
closer examination if crystals formed are quite small). Use of
polarizing microscope is desirable but not essential.
Crystals may be either or both of 2 kinds, depending on
concn of urea present: (a) most prevalent are clusters of narrow
feather-blades of low birefringence which form thruout soln at
ca 1:200 to 1 :25,000 concn (under low power they may appear
to be needles or threads); (b) straight needles, often in sheaves
or clusters, of much greater birefringence, forming chiefly at
or near edge as drop evaps, at conens from 1:50 to 1:1,000.
Both kinds have neg. elongation (observed with polarizing mi-
croscope, using red plate). Crystals should be noted before
drop dries, but remain when it dries. Response is given by
fresh urine solids content of >:4 jxg in drop. Test material from
portion of sample other than fluorescent spot as blank.
Ref.: JAOAC 42, 473(1959).
CAS-57-13-6 (urea)
410
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
963.28 Urine on Grain
Magnesium Uranyl Acetate Test
First Action 1963
(Applicable to grains and seeds)
A. Principle
Grain is sprayed with Mg uranyl acetate soln. If rodent urine
is present, its Na content reacts to cause greenish fluorescence
on kernel when sample is viewed under short-wave UV light.
B. Reagents
(a) Magnesium uranyl acetate soln. — Prep, reagents
929.03A(a) and (b) in Vio amis, mix, add 22 mL glycerol,
mix, and filter thru washed, dried paper.
(b) Urease soln. — Wet 0.2 g urease powder with small amt
of H 2 0, stir into paste, and dil. to 10 mL with H 2 0.
(c) Bromothymol blue soln. — Rub 0. 15 g indicator powder
in mortar with 2.4 mL QAN NaOH soln. After indicator dis-
solves, wash mortar and pestle with H 2 0, and dil. to 50 mL
with H 2 0. Soln should be green; pH ca 7.0.
(d) Urease-bromothymol blue test paper. — Mix 10 mL in-
dicator soln, (c), with 10 mL urease soln. Pour mixt. into watch
glass. Using clean tweezers, dip pieces of heavy filter paper
(Whatman No. 5, S&S No. 598, or 589 green ribbon have
been found satisfactory) in soln. (To avoid uneven distribution
of indicator and enzyme, wet entire paper at once by laying it
on surface of soln.) Hang paper to dry in place free from NH 3
fumes, strong air currents, or heat. Paper should be orange
when dry. Store dry paper in well -stoppered, dark glass bottle
in cool place.
(e) Xanthydrol. — Eastman Kodak Co. No. 1559, crystals.
C. Apparatus
(a) Ultraviolet lamp. — Short wave, 253.7 nm, with filters
to eliminate most visible light.
(b) Chromatographic sprayer. — 250 mL, to deliver fine
spray from air supply (Kontes Glass Co., K-422500, or equiv.).
Hand-operated atomizer is satisfactory if it delivers fine spray.
D. Ultraviolet Test
Spread 50 g grain in shallow tray, or on sheet of waxed
paper on tray. Place in hood or well ventilated area, and spray
evenly with Mg uranyl acetate reagent, making several sweeps
horizontally and vertically across sample. Let stand 1-3 min,
and examine under short-wave UV light. With clean tweezers,
transfer kernels showing greenish fluorescent areas to spot plate.
(Avoid prolonged exposure to UV light and do not touch grains
with bare fingers (use gloves). Perspiration may cause false
fluorescence.)
Use as blanks 1 or 2 kernels showing no green fluorescence
under UV light.
E. Urease-Bromothymol Blue-Paper Test
Add 1-4 drops H 2 to each suspect kernel on spot plate.
Let stand 3~5 min. Place strip of test paper, (d), on glass
microscope slide, transfer drop of ext to paper with stirring
rod, and cover with second slide. Blue spots, slowly devel-
oping over 2-4 min, indicate urea. (As reagent is slightly acid,
color may not appear for several min, depending on how heavily
grain was sprayed.)
F. Confirmatory Test
Transfer 1—2 drops aq. ext of suspect kernels to microscope
slide and evap. to dryness. Add drop of HO Ac (2 + 1) and
very small amt of xanthydrol crystals. If urea is present, char-
acteristic crystals of dixanthylurea form quickly, and are vis-
ible at 60x or lower with wide-field stereoscopic microscope.
Ref.: JAOAC 46, 685(1963).
972.41 Urine on Grain
Urease-Bromothymol BIue-Agar Test
First Action 1972
(Applicable to grains, seeds, and packaging materials that do
not show fluorescent residues or where background fluores-
cence is present)
A. Apparatus
(a) Disposable trays. — Microtiter© plates, flexible vinyl,
flat-bottom (Dynatech Laboratories Inc. , 14340 SuJlyfield Cir-
cle, Chantilly, VA 22021), or equiv.
(b) Culture tubes. — 6 (od) x 50 mm.
B. Reagents
(a) Bromothymol blue {BTB) indicator soln. — Sol. form
(Fisher Scientific Co., B-100, or equiv.). Transfer 50 mg BTB
to 20 mL g-s test tube and add 10 mL H 2 0. Add 1 drop H 3 P0 4
(1 + 9) and dissolve completely by stirring. Add ca 0. \N NaOH
dropwise to dark green (pH 5.8-6.0). (This prepn is enough
for 1 batch of test agar.)
(b) Urease suspension. — Grind 0.30 g urease in small mor-
tar, add few mL H 2 0, and continue grinding. Slowly dil. to
15 mL with stirring.
(c) Test agar. — Add 0.75 g bacteriological grade agar and
0.30 g Na benzoate to 300 mL cold H 2 with vigorous stir-
ring, and place on mag. stirrer- hot plate, 945.75B(n); heat with
stirring and boil gently 1 min. Cool to 45-48°, add BTB soln,
(a), and adjust to pH 5.5 (yellow-green), using 0.9-1 AN NaOH
or H 2 S0 4 . Add urease suspension to pH -adjusted, 45-48° agar
and readjust pH, if necessary. Divide agar into 2 equal por-
tions in beakers. To one portion add 0.5 mL 0AN AgN0 3 . To
other portion add 0. IN H 2 S0 4 (ca 0.2 mL) to adjust to match
color shade or pH of first portion. Mix each portion thoroly
and let stand ca 5 min.
Agar sensitivity test. — Prep, known urea-contaminated seeds
by spotting each seed with 0.5 \xL 0.25% aq. soln of urea and
let dry. Test these known samples as in 972. 41C. If no color
response is obtained with known grains, either obtain new lot
of urease or increase urease content after checking activity as
in 941.04A(b) and 941. 04B so that suspension contains enough
urease to convert 0.80 g urea, and repeat sensitivity test.
C. Test
Initially check sample for free alkali, using test agar contg
AgN0 3 soln (45-48°), by (a) or (b) below:
(a) For immediate use. — Add grain, or unknown sample,
to wells of disposable tray, 972.41A(a), and, using dropping
pipet, add agar until object is covered.
(b) For storage and /or intermittent use. — Add agar to cul-
ture tubes, 972.41A(b), to V2 ht. Transfer tubes to heat- seal-
able plastic bags, seal, and store at 4 ± 1°. Agar can be stored
<120 days. To use, let stand 1 hr at room temp. Use small
glass rod to force test object below surface of agar. Remove
rod and shake down agar. Observe frequently for color change
near surface of object.
If test for free alkali is neg. (no color change of indicator),
proceed with AgN0 3 -free test agar, analyzing sample in sim-
ilar manner. (Grains must be totally immersed during test.)
Color change of indicator is yellow — » green — » blue, de-
AOAC Official Methods of Analysis (1 990)
Animal Excretions
411
pending on concn of NH 3 produced. Reaction usually requires
1-3 min to give detectable color. Time varies inversely with
urea concn. Spots from higher levels of urea continue to de-
velop and enlarge for 10-12 min and then fade gradually.
Ref.: JAOAC55, 76(1972).
980.28 Urine Stains on Foods
and Containers
Thin Layer Chromatographic Method I
First Action 1980
(Applicable to fluorescing urine residues on materials not ex-
pected to have significant amts of extractable interfering
substances)
A. Apparatus and Reagent
(a) Thin layer apparatus. — See 945.75B(s).
(b) Sandwich chamber. — With 1.4-2.0 mm spacer. See Fig.
980.28.
(c) Blender. — High speed with stainless steel semimicro jar
(Thomas Scientific, No. 3392-G15, or equiv.). See 945.756(c).
(d) Tube heater. — Nine tube heater with control and cir-
cular gas manifold (Kontes Glass Co., Cat. Nos. K-720000
and K-655800, or equiv.).
(e) Surface thermometer. — Range —10° to 150°.
(f) Developing solvent.— n-BuOH-MeOH-H,0 (2 + 2 +
1).
B. Preparation of Thin Layer Plates
(a) Quickfit plate leveler and spreader. — Add 12 g cellu-
lose powder, 945.75C(g), to 15 mL H 2 in blender operated
at 0.75 x line voltage. Work powder into H 2 0, using small
spatula. Apply line voltage, add 15 mL H 2 0, and blend ^1.5
min. Apply slurry to 5 plates, using 0.50 mm slot, and air dry
overnight.
(b) Brinkmann-Desga. — Prep, slurry as in (a), using 15 g
cellulose powder and 100 mL H 2 (total). Apply slurry to 5
plates, using 0.375 mm layer, and air dry overnight.
(c) Commercial plates. — 250 |xm, precoated with MN 300
cellulose. Plates from Analtech, Inc., have been found satis-
factory. Use 500 fxm plates for "dirty" extns.
^:
<5-7MM
I CM
N^
-20 CM-
-*>|
FIG. 980.28 — Sandwich chamber with three spacer strips along
three edges. Snug fit is necessary at top corners. (Not to scale.)
(d) P re-cleaning Analtech MN 300 cellulose plates. — De-
velop unused plate in either std developing tank or sandwich
chamber using developing sol v. 980. 28 A. Develop to ht of 15
cm above lower edge of plate. Thoroly dry plate using either
hair dryer, forced draft oven at <80° for about 15 min, or
overnight in fume hood. Plate must be at room temp, and com-
pletely free of sol v. odor before use.
C. Determination
(a) Preliminary examination. — Check samples as in 945.60
for fluorescent areas. Mark fluorescent spots and nearby equiv.
nonfluorescent areas as controls. Transfer selected spots to 30
mL beakers. For intense fluorescent spot, use ca 8 mm diam.
(b) Extraction and concentration. — Add 10 mL acetone to
selected spots in beakers and simmer 5 min on steam bath,
avoiding excessive loss of solv. Preheat 13—1.5 mL conical
tubes in tube heater or beaker of warm H 2 0, add portion of
acetone ext, and evap. to dryness before very slowly adding
next portion. (Tubes must be preheated and acetone added very
slowly to avoid bumping.) To hasten evapn, use air manifold
or 4-6 SiC No. 60 chips per tube. Repeat extn and evapn
twice, keeping tubes hot to avoid bumping. Chromatograph
final residue without delay. If chromatgy must be delayed, add
50-75 (xL acetone, stopper, and store in dark.
(c) Preheating plates. — Scrape ca 1 cm layer from 3 sides
of plate. Place plate on heated surface, reading ca 87° on sur-
face thermometer or 70° on 3" (76 mm) immersion thermom-
eter inserted thru hole in stopper until tip touches bottom of
250 mL conical flask contg 125 mL glycerol.
(d) Spotting plate. — Along unscraped side, 2 cm up from
edge of plate and ca 1 cm from each scraped edge, spot at
single point, drying between applications, 1 |xL urea,
945.75C(aa), 20 |xL allantoin, 945.75C(d), and 1 jjlL indoxyl
sulfate, 945.75C(m), std solns. Spot individual stds near cen-
ter of plate and spot sample exts between stds ca 10—12 mm
apart. Rinse sample tubes with two 50 p,L portions acetone
and transfer to sample spots, keeping spot size to min.
(e) Cleanup. — If there is appreciable color and/or other ex-
traneous material present, clean up with benzene as follows:
Line TLC tank with heavy blotting paper, or equiv. Add 40-
60 mL benzene to bottom of tank, add glass solv. trough, and
cover tank. (For frequent use, coat ground glass surfaces with
high viscosity silicone grease.) Add 25-30 mL benzene to trough
in tank. Place plate in trough and let front rise ca 25 mm above
spotting line, remove plate, and fan dry. Repeat benzene de-
velopment and fan dry. Place plate in trough third time with
tank closed and let benzene rise >13 cm. Dry plate in forced
draft oven 5 min at 80°.
(f) Development of plate. — Scribe line across plate 10 cm
above spotted stds. Form sandwich chamber with spotted plate,
spacer, and uncoated plate. If com. spacers are not available,
fabricate sandwich by cutting 3 strips cardboard or Teflon 5-
7 mm wide, 1.4-2.0 mm thick, one strip 20 cm long and two
strips ca 18.5 cm long. Position strips as in Fig. 980.28, leav-
ing ca 1 cm gap at bottom. Snug fit at top corners is necessary.
Clamp, place in trough, add developing solv. to ca 1 cm of
spotting line, cover with opaque material, and develop 10 cm.
Dry plate 5 min in 80° forced draft oven.
(g) Color development. — Spray plate with pDMAB soln,
945.75C(r), until urea and allantoin stds appear. Heat 5 min
in forced draft oven at 80°. Strong yellow to orange area, R f
ca 0.65, is urea. Pale yellow smaller spot, /? f ca 0.43, is al-
lantoin. Using soft (No. 1) pencil, lightly outline each spot as
color develops. Place under long wave UV light in well dark-
ened room, check for pale yellow indican fluorescent area, R f
ca 0.79, and outline as above. Protect urea and allantoin spots
412
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
from fading due to NaOAc spray, 945.75C(s), by masking
with plate glass. Spray satd NaOAc soln into indican area until
damp. Let air dry in hood ca 10 min (do not heat).
High level of indican gives deep pink to red in room light.
If no visual color is present, check with longwave UV light
for fluorescent pink to orange on very pale blue background.
This color is stable several days in dark.
Refs.: JAOAC 63, 189(1980); 66, 394(1983).
973.64 Urine Stains on Foods
and Containers
Thin Layer Chromatographic Method II
First Action 1973
Final Action 1977
(Applicable to residues on materials with significant amts of
extractable interfering substances)
A. Apparatus
(a) Soxhlet extractor. — 250 mL extn flask; extn chamber
39 (id) X 115 mm with top joint I 45/50 and bottom joint
124/40; 35 x 90 mm thimbles; condenser joint 145/50 (Thomas
Scientific, No. 4406-E34, or equiv.).
(b) Kuderna-Danish concentrator.
(c) Thin layer apparatus. — See 945.75B(s).
B. Reagents
(a) Tryptophan soln. — 1 mg/mL 50% aq. acetone (used as
longwave fluorescent marker with R f approx. that of urea in
developing solv.).
(b) Developing solvent.— /7-BuOH-HOAc-H 2 (10 + 5 +
5); prep, fresh daily.
C. Determination
(Caution, See safety notes on distillation, flammable solvents,
and acetone.)
On previous day prep, plates for overnight drying. Equili-
brate tanks ca 1 hr before extg samples.
(a) Soxhlet extraction. — Ext 18 g sample with 60 mL ace-
tone 1 hr at 3-4 min/siphon. Transfer ext to 100 mL g-s grad-
uate, dil. to vol., and take aliquot equiv. to 9 g sample. Cone,
as in (c).
(b) Alternative extraction. — Place 18 g sample in 50 mL
beaker or erlenmeyer. Add 1 mL acetone/g sample and boil
gently 5 min, avoiding excessive loss of acetone. Decant thru
glass wool pad into 100 mL g-s graduate. Repeat acetone boil
and decanting 3 addnl times, and dil. to 100 mL. Cone, aliquot
equiv. to 9 g as in (c).
(c) Concentration.— Cone, aliquot to ca 0.2 mL in Ku-
derna-Danish concentrator as follows: Evap. to ca 5 mL on
steam bath in Kuderna-Danish concentrator fitted with 3-ball
Snyder column and vol. flask or graduated collection tube; 20
mesh boiling chip is necessary. Remove collection tube from
concentrator and fit tube with 2-ball micro-Snyder or micro-
Vigreux column. Evap. to slightly less than desired vol., per-
mit condensate to drain into tube, and remove column. Alter-
natively, place empty 13 mL graduated conical centrf. tube in
beaker of boiling H 2 0. When tube is hot, slowly transfer por-
tions ext, using syringe fitted with long needle, to evapn tube.
Let each portion evap. before next is added. Evap. to c a 0.20
mL. Chromatograph without appreciable delay.
(d) Preparation ofTLC tank. — Add ca 150 mL satd NaHS0 4
soln to lined tank; then add ca 15 g addnl solid NaHS0 4 to
bottom of tank. Place empty solv. trough in bottom of tank
and cover tank.
(e) Preparation of thin layer plates. — (J) Brinkmann-
DeSaga.— Add 15 g cellulose, 945.75C(g), to 100 mL H 2
in blender operating at ca 90 v setting of variable transformer.
Use small spatula to work powder into H 2 0. Turn variable
transformer to 120 v (line voltage) and blend 5:1 .5 min. Apply
slurry as 0.375 mm layer to 5 plates and air dry plates over-
night.
(f) Spotting of plates. — Spot sample cone, as band ca 25
mm long on line 15 mm up and 15 mm in from edge of plate.
Wash sides of evapn tube with ca 50 jjlL acetone and transfer
wash to sample band area. Repeat 50 jxL washings and trans-
fers until last transfer is colorless (ca 4 transfers). Spot 1 |jlL
each of urea std soln, 945.75C(aa), allantoin std soln,
945.75C(d), and indoxyl sulfate std soln, 945.75C(m), and
tryptophan soln, 973.64B(a), ca 10 mm apart along line 15
mm to left of center of plate and 15 mm from bottom of plate.
(g) Development of plates. — Place plate in trough contg ether
in lined tank presatd with ether. Let ether travel to top of plate.
Remove plate and let air dry. Immediately draw intersecting
lines to divide plate into 4 equal sqs. Dry plate 5 min in 80°
forced-draft oven. Remove plate from oven and promptly place
in dry solv. trough in TLC tank with spotted band down. Close
tank and let stand 20 min. Slide top aside just enough to in-
troduce long-stem funnel into solv. trough. Slowly add 20 mL
developing solv., (b), to trough. Close lid and develop in dark
to line of first direction. Dry plate 5 min in 80° forced-draft
oven.
Rotate warm plate to place chromatographed stds in upper
left quarter of plate and promptly place in dry trough in tank.
Let stand 20 min without touching any Jiq. in closed tank.
Then slide cover aside just enough to introduce long-stem fun-
nel into solv. trough and slowly add 20 mL developing solv.
Let front move to line in this second dimension. Dry plate 5
min in 80° forced-draft oven.
(h) Color development. — Spray plate with pDMAB re-
agent, 945.75C(r), until distinctly moist but not shiny wet and
again heat 5 min in 80° forced-draft oven. Strong yellow-to-
orange area at R { 0.75-0.80 is urea. Pale yellow smaller spot
at R f 0.45-0.50 is allantoin. Mark each area as color develops,
since colors fade from one step to next. Place under longwave
black light in darkened room and check for pale yellow flu-
orescent area between urea and allantoin. Spray satd NaOAc
soln, 945.75C(s), (ca 1—2 mL /plate) in space between urea
and allantoin until yellow of both has faded. Let plate air dry
ca 10 min in hood (do not heat), and check plate under long-
wave black light. Weak fluorescent pink-to-orange color against
very pale blue fluorescent background confirms presence of
urinary indican.
Refs.: JAOAC 56, 637(1973); 57, 689(1974).
981.22
Mammalian Feces
Alkaline Phosphatase Test
First Action 1981
A. Principle
Intestinal tract of most mammals contains alk. phosphatase
isoenzyme. Isoenzyme, at test pH and temp., splits phosphate
radical from phthln diphosphate to produce reddish free phthln.
B. Apparatus
(a) Cups. — Plastic, 4 mL (Technicon Instruments Corp.,
S 127-0018-01, or equiv.).
(b) Plates. — Tissue culture, 4x6 wells, 3 mL capacity
(Thomas Scientific, No. 9383-C15, or equiv.).
(c) Pipet. — Cornwall, adjustable to deliver 1 mL (Fisher
Scientific Co., No. 13-689, or equiv.).
AOAC Official Methods of Analysis (1990)
Animal Excretions
413
(d) Spatula. — Curved on one end, knob on the other end
(Thomas Scientific, No. 8340-H10, or equiv.).
C. Reagents
(a) Magnesium chloride soln. — Dissolve 0.406 g
MgCl 2 .6H 2 in H 2 and dil. to 1 L.
(b) Stock test reagent. — Dissolve 9.5 g borax (Na2B4O 7 10
H 2 0) and 3 .14 g anhyd. Na 2 C0 3 in 500 mL H 2 with stirring.
Add 0.47 g phthln diphosphate and stir while adding 1 mL
MgCl 2 soln. Prepn is stable.
(c) Work test media (WTM). — It is recommended that only
small amts of this reagent be prepd. Measure equal vols stock
test reagent and H 2 into sep. beakers. Place beaker of cold
H 2 on stirring hot plate, add stirring bar, and, with rapid
stirring, add sufficient agar to yield 2% agar dispersion. Con-
tinue to stir while heating to boil (watch for foam-over). When
agar foams, add stock test reagent and stir rapidly ca 1 min.
Add ca 1 mL portions of WTM to cups before cooling to
40-41°. WTM must be cooled to 40-41° before contacting
samples.
Short term storage: WTM may be held ca 48 h at 40-41°
if covered snugly with foil or plastic.
Long term storage: Gelled plugs, in cups, may be stored up
to 4 months if sealed in plastic bag, held at room temp., and
protected from direct sunlight. Discard any gells showing pink
color and/or vol. loss.
D. Determination
Transfer suspect feces, ca 1-3 mm diam. x 3-5 mm long,
to moistened spot of filter paper in petri dish. Moisten with
min. amt of addnl H 2 if needed. Cover with small piece of
clean Al foil and crush. Check for hairs or other diagnostic
indicators. If no such evidence is seen, proceed as follows:
Air dry paper and crushed particles. Cut out stained area and
transfer paper and adhering particles to cup contg 1 mL gelled
WTM. Cover with addnl 1 mL cool (40-41°) WTM or, al-
ternatively, plug of gelled WTM. Use clean spatula to manip-
ulate covering plug of WTM and to press plug into close con-
tact with sample; place in 40-41° H 2 bath. Check for
development of red color near particles.
Blank preparation. — Autoclave crushed particle 15 min at
15 psi. Alternatively, place 100 mL beaker in 800 mL or 1 L
beaker and add ca 25 mm depth H 2 to each. Place small test
tube with 1 mL WTM in smaller beaker. Heat H 2 to boiling
ca 2 min. Remove small tube and quickly transfer crushed pel-
let material to tube. Return tube to rapidly boiling H 2 ca 2
min. Remove tube and with small glass rod work all particles
from sidewall of tube down into liq. Replace tube in small
beaker, cover large beaker with watch glass, and continue to
boil ca 5 min. Remove small tube, mix contents quickly, and
transfer to test cup position designated as blank (negative test)
for test plate.
Positive control preparation. — Using calf intestine alkaline
phosphatase (AKP) (Calbiochem Corp. No. 52457 or equiv.),
prep. 1 mg/mL soln in borate buffer (Stock test reagent
981.22C(b)) without phthln diphosphate. Add 20 )aL AKP soln
to 1 cm diam. filter paper disks (Whatman No. 1, or equiv.).
Use pos. control disks with either liq. WTM or alternative gelled
plug WTM. Note: Pos. control disks may be stored up to 4
months if held at room temp, and protected from light.
Test response. — Time of color development varies widely,
depending in part on species variation in levels of alk. phos-
phatase and on temp, of WTM. Time varies from 2 to 3 min
for most mouse samples, up to 4 h for samples from some
grass eaters, such as deer or rabbit.
Refs.: JAOAC 64, 196(1981); 66, 394(1983).
988.17 Mammalian Feces
Thin Layer Chromatographic Method
for Coprostanol
First Action 1988
(Applicability includes identification of feces in heat-
processed materials.)
A. Principle
Suspected fecal material is extd with hexane. Coprostanol,
characteristic sterol of mammalian feces, is resolved from other
sterols in ext by TLC, and produces blue spot when heated
with phosphomolybdic acid.
0. Apparatus
(a) Thin layer plates. — Glass, 20 x 20 cm, precoated with
250 |xm layer of silica gel. Prechanneled, with preadsorbent
zone. (Whatman, or equiv. plates.)
(b) Dipping tank and accessories. — Glass (Kontes Cat. No.
K416160, or equiv.).
(c) Chromatographic tank with lid. — Glass (Kontes Cat. No.
K416180, or equiv.).
(d) Spotting pipets. — 20 jxL, glass (Drummond Scientific
Co., 500 Parkway, PO Box 700, Broomall, PA 19008, or
equiv.).
C. Reagents
(a) Alcoholic phosphomolybdic acid (PMA ) . — Dissolve 50 g
PMA (Fisher Certified ACS, or equiv.) in 500 mL alcohol,
filter soln, and dil. to 1 L with alcohol. Store in dark. Discard
if greenish tinge appears.
(b) Developing solvent. — Ether-heptane (55 + 45).
(c) Coprostanol std soln. — 5 jxg coprostanol (Supelco std)/
mL hexane.
(d) Cholesterol std soln. — 5 p-g cholesterol (Supelco std)/
mL hexane.
D. Preparation of Sample
Weigh sample of suspected fecal material to nearest 0. 1 mg.
If origin of feces is unknown or if herbivore dung is suspected,
use >5 mg sample to reduce possibility of false neg. conclu-
sions. Analyses may be made on much smaller test portions
of samples, such as those from reconditioning operations of
rodent-contaminated products, where more is known about
sample.
Transfer sample to glass vial (ca 1-2 mL). Gently crush
sample with glass rod if particle is >3-4 mm diam. Add hex-
ane at ratio of 10 fxL/mg feces, but not less than 30 p,L per
sample. Cap vial; let stand 1 h.
E. Preparation of TLC Plates
Prep, plates >24 h before use to ensure evapn of alcohol.
To prevent contamination, wear vinyl gloves when handling
plates. Dip plates, top edge leading, into 5% ale. PMA until
soln is 2-3 mm below preadsorbent-silica gel juncture. Do not
let PMA diffuse into preadsorbent zone. Hold plate 10-15 s,
then remove plate from soln and let it stand vertically ca l / 2
h, preadsorbent edge up, on paper toweling. Do not let dust
settle on damp surface. Store plates in clean, dark container
with gel surface free of any contact. Store plates <3 months.
F. Determination
Use No. 1 (soft) pencil to lightly draw sol v. front line 10
cm from preadsorbent-silica gel line. Spot 20 jjlL vols of stds
and samples and at least 1 hexane reagent blank individually
onto preadsorbent regions of plates. After std or sample has
been applied, number lane with soft pencil and record lane no.
414
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
and sample. If all lanes are not required, avoid lanes nearest
edges. Let hexane evap. 10 min.
Work in hood. Apply thin bead of silicone grease around
chromatgc tank top to seal lid. Pour 55 mL ether and 45 mL
heptane into 100 mL mixing cylinder. Invert cylinder 3-4 times
to mix and pour contents into TLC tank. Immediately cover
tank and let system equilibrate 10 min. Place TLC plate in tank
and cover tank. Develop to solv. front line (ca 20 min). Re-
move plate and air-dry in hood ca 5 min. Place plate in 120°
forced-draft oven 20 min. Remove plate and circle spots with
soft pencil. Record R { for coprostanol and cholesterol stds.
Record /? r for any spots that appear in coprostanol and cho-
lesterol regions. Cholesterol and coprostanol should be com-
pletely sepd.
Feces are indicated by presence of 2 spots: at R f ca 0.30
(cholesterol std) and at/? f ca 0.40 (coprostanol std). For small
samples (<2 mg) all spots may be faint and effort should be
made to detect indicator spots. For larger samples, spots will
be distinct. In addn to distinct cholesterol spot, cockroach ex-
creta may present trace reaction in coprostanol region. There-
fore, coprostanol spot should be approx. same or even greater
in intensity than cholesterol spot to conclude that material is
mammalian or bird feces rather than insect excreta. Copros-
tanol may occur in feces of some birds; therefore, if there is
no evidence to eliminate possibility that material could be bird
feces, pos. results should be reported as "fecal material from
mammal or bird."
Colors fade in light. Photocopy machine may be used to
make permanent record of plate.
Ref.: JAOAC 70, 499(1987).
962.20 Excrement (Bird)
on Food and Containers
Microchemical Test for Uric Acid
Final Action
(Not suitable for minute residues from suspect stained areas of
food containers)
Transfer white, amorphous, grainy particles to depression
of spot plate preheated to ca 100° on hot plate or in oven. Add
small drop of HN0 3 (1 + 1) to sides of depression so that it
will run down to wet particles; then evap. to dryness in 0.5-
1.0 min. Heat 1-3 min. If particles turn orange-red to deep
red with heat, uric acid and /or its salts may be present.
To confirm: Cool plate until there is no perceptible heat to
back of hand; then streak across colored area with small glass
rod wetted with 50% NaOH soln. Intense purple will develop
almost immediately.
Modification for particles about J mg. — Position micro-
scope or strong magnifying glass to observe 18 mm No. 2
cover glass placed on metal surface heated to ca 110-120°.
Place suspect particle on glass, add 10 |xL HN0 3 , evap. to
dryness, and heat in oven 5-7 min at 135-140°. Remove to
cool white surface under magnifier and observe baked reaction
residue. Pos. reaction shows yellow-orange to orange-red ring.
To confirm: With I mm glass rod place small drop 50%
NaOH soln on edge of cover glass. Wipe rod and transfer small
portion of drop to edge of baked residue. Do not flood. Purple-
violet color develops promptly with uric acid or its salts.
Refs.: JAOAC 45, 659(1962); 47, 516(1964).
CAS-69-93-2 (uric acid)
986.29 Excrement (Bird and insect)
on Food and Containers
Thin Layer Chromatographic Method for Uric Acid
First Action 1986
Final Action 1989
(Applicable to suspect material not suitable for detn by 962.20
and /or to confirmation of 962.20 when adequate material is
available.)
A. Apparatus and Reagents
(a) Thin layer cellulose plates. — See 980. 28B. E. Merck
cellulose plates, 0.10 mm (EM Science No. 5716-7) have also
been found satisfactory.
(b) Cellulose powder.— See 945.75C(g).
(c) Detection spray.— (1) Soln A. — 1% K 3 Fe(CN) 6 . (2) Soln
B. — 2% FeCJ 3 (calcd as anhyd.). Refrigerate both solns. Pro-
tect soln A from light. Solns are stable ca 2 weeks. (3) Spray
reagent. — To 18 mL H 2 0, add 1 mL each of solns A and B;
mix. Prep, immediately before use.
(d) Developing solvent.— n-BuOH-MeOH-H 2 (4 + 4 +
3). Measure vols sep. and mix well to form stable single phase.
To 30 mL of this soln, add 1 mL HOAc; mix well. Prep, fresh
daily.
(e) Dye mixture. — Dissolve 16 mg amaranth (formerly FD&C
Red No. 2) and 32 mg FD&C Yellow No. 6 in 50 mL H 2 0;
mix well.
(f) Lithium carbonate soln. — 1 mg/mL.
(g) Uric acid std soln. — (/) Stock soln. — .1 mg/mL. Dry
105 mg uric acid in 100° oven overnight and cool to room
temp, in desiccator. Accurately weigh 60 mg Li 2 C0 3 and transfer
to 100 mL vol. flask. Accurately weigh 100 mg cool uric acid
and transfer quant, to the 100 mL flask with ca 50 mL H 2 0.
Place in 60° H 2 bath and agitate until soln clears. Cool im-
mediately under tap H 2 to room temp, and dil. to vol. with
H 2 0. For short term use (<3 days), store in refrigerator; for
extended use, place portions in small containers and store hard-
frozen. (2) Working soln. — 100 (Jtg/mL. Pipet 10 mL stock
soln into 100 mL vol. flask and dil. to vol. with FLO. Prep,
fresh daily.
B. Preparation of Sample
(a) Insect excreta. — Transfer material to small test tube, crush
with glass rod, and add 0.05-0.10 mL Li 2 C0 3 soln, (f). Let
soak ca 10 min and centrf. Obtain clear supernate and proceed
as in 986.29C.
(b) Paper bags or cartons. — Cut 5-6 mm diam. portion
from suspect area. Cut another 5-6 mm portion from nearby
unstained area as neg. control. Place individually in small test
tubes. Add ca 0.1 mL Li 2 C0 3 soln, (f), to each tube; agitate
with small stirring rod. Let soak ca 10 min and proceed as in
986.29C.
(c) Other suspect material. — Transfer small portion to test
tube, add ca 0.1 mL Li 2 C0 3 soln, (f), and stir with glass rod.
Let soak ca 10 min; centrf. Obtain clear supernate and proceed
as in 986.29C.
C. Determination
(a) Spotting of plates. — Place coated plate on heated metal
slab reading ca 87° on surface thermometer or 70° on 3 in. (76
mm) immersion thermometer inserted through hole in stopper
until tip touches bottom of 250 mL conical flask contg 125
mL glycerol. (Note: Plates tend to crack, particularly pre-
scored plates, unless heated evenly.) Place infrared lamp or
forced hot air source (e.g., hair dryer) above plate to speed
AOAC Official Methods of Analysis (1990)
Insect Excretions
415
drying of spots. Spot 1 fxL uric acid working std soln, (g)(2),
at each edge and at center of plate ca 15-20 mm up from
bottom. Spot 1 p/L dye mixt., (e), to side of each working std
spot. These dyes serve as visual markers during development,
with R { for amaranth at 0.38-0.40; uric acid, 0.41-0.43; and
Yellow No. 6, 0.65, using Analtech plate and sandwich cham-
ber. R f values are lower on Merck plates, with R f for amaranth
approx. equal to that of uric acid. Spot samples and neg. con-
trols along same line at >10 mm intervals. Keep spots at min.
size by drying well between successive small addns.
(b) Development of plates. — Scribe horizontal line, ca 1 mm
wide, across plate exactly 10 cm above origin, completely re-
moving cellulose layer. Develop to this line in conventional
satd tank without pre-equilibration or, alternatively, form
sandwich chamber with uncoated plate [see 980.28A(b) and
980.28C(c) and (f )] and develop. Dry plate on heated metal
slab or in forced draft oven ca 5 min at 75-80°.
(c) Examination of UV light. — Observe plate under short-
wave (254 nm) UV light in darkened room, marking each
quenching (dark) spot with penciled dots at top, bottom, left,
and right edges. Shortwave lamps in fluorescent tube-style have
integral filters with transmission characteristics that change with
use. Some UV viewing cabinets have label attached calling
attention to this fall-off of transmittance of 254 nm. High lev-
els of uric acid should appear as dark spots at R f = 0.40 ±
0.05, depending on conditions of development.
(d) Color development. — Spray plate evenly in hood, con-
centrating on horizontal zone between upper (yellow) dye spots
and ca 2 cm below lower (red) dye spots, only until blue uric
acid spots clearly appear at /? f stated in (c). Immediately out-
line spots with soft (No. 1) pencil, marking weakest spots first.
Continue spraying only until background begins to darken.
Immediately outline any addnl spots that appear (again, weak-
est ones first). (Note: Excessive spraying accelerates plate
darkening.)
Refs.: JAOAC 61, 903(1978); 66, 394(1983); 69, 499(1986).
CAS-69-93-2 (uric acid)
969.46 Excrement (Insect) in FSour
Spectrophotometry Method for Uric Acid
First Action 1969
Final Action 1970
(Applicable to levels >4 mg/100 g)
A. Apparatus
(a) Spectrophotometer. — Beckman Model DU, or equiv.
(b) Centrifuge. — Desk centrf. with multiple head to hold
15 mL polyethylene test tubes.
(c) Incubator or water bath. — Capable of maintaining temp,
of 37 ± 1°.
B. Reagents
(a) Uric acid std soln. — 100 |jLg/mL. Dissolve 100 mg uric
acid in 1 L 5% NaOAc soln. (If necessary, warm in H 2 bath
at 60-70°.) Filter and store in brown bottle; discard after 1
week. (Do not use com. uric acid std solns, as they may con-
tain uricase inhibitors.)
(b) Sodium borate buffer.— 0.01M, pH 9.2. Dissolve 3.8
g Na 2 B 4 7 . 10H 2 O in H 2 and dil. to 1 L.
(c) Sodium acetate soln. — 5%. Dissolve 100 g anhyd.
NaOAc in H 2 and dil. to 2 L. If necessary, adjust pH to 8.8-
9.2 with HO Ac or NaOH.
(d) Glutathione soln. — 10 mg/mL in H 2 0. Use within 30
min.
(e) Uricase soln. — Prep, suspension of 10 mg dried uricase
in 50 mL 0.01 M Na borate buffer. Use within 1 hr. (Clean all
glassware that comes in contact with uricase enzyme with
chromic acid soln; adsorbed uricase on glass surface produces
low results.)
C. Preliminary Tests
(a) Test for purity of reagents. — Dil. 5.0 mL uric acid std
soln to 25 mL with 5% NaOAc soln. Place 5 mL in each of
3 test tubes. To 1 tube add 5 mL Na borate buffer, invert
several times, and measure A at 292 nm. A should be >0.72,
which corresponds to 0.072 A unit/|ULg uric acid/mL final soln.
Test std uric acid soln daily.
(b) Test for efficiency of uricase soln. — Label remaining 2
tubes in (a) as No. 1 and No. 2; label a third test tube No. 3.
Add 5 mL uricase soln to tubes No. 1 and No. 3. Close mouth
of tube No. 1 with piece of cellophane sheet under thumb and
invert. Stopper all 3 tubes with clean rubber stoppers and in-
cubate 2 hr at 37°. After incubation, mix contents of tubes No.
2 and No. 3 by repeatedly pouring (6 times) from one tube to
other, and immediately (within 60 sec) read A of combined
solns at 292 nm, using soln in tube No. 1 as blank. A should
be > 0.648 for ^90% of theoretical efficiency of uricase. If
efficiency is <90%, incubate 4 hr. If increased incubation does
not increase efficiency to 90%. discard uricase sample.
D. Preparation of Standard Curve
Pipet 0.0, 2.5, 5.0, 10.0, and 15.0 mL uric acid std soln
into 5 beakers (corresponds to 0.0, 1.0, 2.0, 4.0, and 6.0 |mg
uric acid/mL in final soln, resp.), and perform all steps as in
969. 46E, except omit flour.
E. Determination
Add 25 mL 17V HC1 and 5 mL glutathione soln to 4 g flour
in 250 mL beaker. Mix well with glass rod and let stand over-
night (>16 hr). Add 25 mL 17V NaOH with stirring and adjust
pH to 9.0-9.3 with IN NaOH or \N HC1. Transfer to 100 mL
g-s graduate, carefully scraping all material sticking to sides
of beaker with glass rod. Rinse beaker with 6 small portions
5% NaOAc and dil. to 100 mL with 5% NaOAc. Shake gently
by inverting graduate several times every 10 min for 1 hr.
(Vigorous shaking tends to produce turbid soln.) Transfer ali-
quot to 15 mL polyethylene test tube and centrf. 30 min at
3000 rpm. Decant supernate into small erlenmeyer, mix well,
and pipet 4 mL into each of 2 test tubes, No. 1 and No. 2.
To each tube, add 1 mL Na borate buffer and mix by rotating
between palms of hands. (Mix soln with Na borate buffer within
15 min to avoid turbid soln.) Label third tube as No. 3. Add
5 mL uricase soln to tubes No. 1 and No. 3. Mix contents of
tube No. 1 as in 969.46C(b). Stopper all 3 tubes with rubber
stoppers and incubate 2 hr at 37°. Combine solns in tubes No.
2 and No. 3, as in 969.46C(b), and read A immediately (within
60 sec) at 292 nm against soln No. 1 (blank). (If flour ext
appears very turbid after centrfg, dil. centrfd ext 1 + 4 with
Na borate buffer and pipet 5.0 mL into each of 2 test tubes,
No. 1 and No. 2. Add 5 mL uricase to each tube (No. 1 and
No. 3) and proceed with detn as above.)
Reading, A, corresponds to amt of uric acid present in 4 mL
portions of centrfd soln; amt of uric acid obtained from std
curve X diln factor — amt of uric acid in sample.
Refs.: JAOAC 49, 899(1966); 50, 776(1967); 52, 833(1969).
CAS-69-93-2 (uric acid)
416 Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1 990)
984.29
MOLD AND ROT FRAGMENTS
(See Fig. 984.29A.)
Howard Mold Counting
General Instructions
A. Diagnostic Characteristics of Mold
Before attempting to make mold count, analysts should be
familiar with cellular structure of product. They can do this
thru direct microscopic examination of healthy tissue excised
from raw product or thru study of ref . books. Analysts should
assume that any product can be contaminated with a variety
of vegetable, animal, and synthetic fibers. Presence of such
materials greatly increases probability for misidentification of
molds.
It is essential that analyst be able to distinguish hyphae and
look alikes with which they might be confused. Although many
filaments can be easily and accurately recognized as mold, others
require more careful appraisal before they can be reported as
mold. Only rarely are all mold characteristics given here pres-
ent at one time; usually 2 or 3 are absent. Features mentioned
below are observed at 1 00-450 x .
(a) Parallel walls. — Mold hyphae are tubular. In most in-
stances, diam. of filament is uniform throughout its length.
Thus, hyphal walls usually look like parallel lines under mi-
croscope. This is one of the most useful characters for rec-
ognizing mold and for differentiating it from other fibers. Ex-
ceptions do occur, however. In some larger molds, walls may
be collapsed or twisted. In certain molds, hyphae may have
swellings along sides so that walls are not parallel. Hyphae of
Mucor and Geotrichum are often tapered.
(b) Septation. — Many types of mold filaments are divided
into segments or sections by cross walls. Some plant hairs also
look segmented, but their walls are frequently convergent,
forming sharp-pointed apex. Mucor and a few other molds
generally have no cross walls.
(c) Granulation. — Thin- walled, tubular hypha contains
protoplasm which shows thru cell walls and appears granular
or stippled under high magnification. This is most clearly seen
in some large mucors. In some fine molds, such as the one
causing anthracnose, granulation of protoplasm is not evident.
It disappears in some molds, such as those occasionally found
in butter, leaving thin- walled, clear, almost invisible tubes.
This empty mold is extremely difficult to count. At times it
may become twisted, resembling a cotton fiber. Often the pro-
toplasm separates intermittently, forming line of short links or
chains connected by almost invisible hyphal walls.
(d) Branching. — If mold fragments are not too short, many
of them may show an abundance of branching. Branches and
main trunk are almost always same diam. When present,
branching is one of the most reliable characteristics for rec-
ognizing mold.
(e) Ends of filaments . — Natural end of filament is usually
bluntly rounded, much like a fingertip. Filaments are rarely
sharply pointed, except in fertile (reproductive) hyphae. Oc-
casionally, they are expanded into a ball or head, especially
when mold is forming a fruiting body. Broken end of a fila-
Geotrichum
Mold Count
945.95, 974.34, 980.30,
982.34, 984.30
Fruit and Vegetable
Products
Ground Spices
1
' ^"^
Decomposed Raw
Material
Tomato
Products
]
1 J*
1
r
Sample
Preparation
Sample
Preparation
i
r
1
'
Howard Mold
Count
937.13 •, 945.89,945.90,
945.91,945.92, 945.93,
945.94, 955.47, 965.41,
970.75, 970.76, 972.42,
975.51,982.33, 984.29
Tomato Rot Fragment
Count
952.23
FIG. 984.29A— Method selection guide: methods for mold and rot.
AOAC Official Methods of Analysis (1 990)
Rot and Mold
417
ment is normally square. That portion of hypha adjacent to
broken end may be collapsed and may contain no protoplasm.
(f ) Nonrefractile appearance. — Hyphae do not strongly re-
fract light. Some objects seen in mold preparation may resem-
ble hyphae but have highly refractile appearance, such as un-
rolled spiral thickenings from walls of plant vessels. These
refract light as a solid glass or plastic rod might do.
B. Determination
Clean Howard celt, 945.75B(m)(/), so that Newton's rings
are produced between slide and cover glass. Remove cover
and with knife blade or scalpel, place portion of well mixed
sample on central disk; with same instrument, spread evenly
over disk, and cover with glass so as to give uniform distri-
bution. Use only enough sample to bring material to edge of
disk. (It is of utmost importance that portion be taken from
thoroly mixed sample and spread evenly over slide disk. Oth-
erwise, when cover slip is put in place, insol. material, and
consequently molds, may be more abundant at center of mount.)
Discard any mount showing uneven distribution or absence of
Newton's rings, or liq. that has been drawn across moat and
between cover glass and shoulder.
Place slide under microscope 945.75B(o)(7) and examine
with such adjustment that each field of view covers 1.5 sq
mm. (This area, which is essential, may frequently be obtained
by so adjusting draw-tube that diam. of field becomes 1.382
mm. When such adjustment is not possible, make accessory
drop- in ocular diaphragm with aperture accurately cut to nec-
essary size. Diam. of area of field of view can be detd by use
of stage micrometer. When instrument is properly adjusted,
vol. of liq. examined per field is 0.15 cu mm.) Use magni-
fication of 90-125X. In those instances where identifying
characteristics of mold filaments are not clearly discernible in
std field, use magnification of ca 200 x (8 mm objective) to
confirm identity of mold filaments previously observed in std
field. See Fig. 984.29B.
"l$**i % ' : % .1 ■;»* **V'^8&* ."'»■''■*■ v; i ' ' '* j **'*'•' ■■ T * -jj*- ■■■■ > ', m * ,- , "■' , ! i
"+flKWKif; |£** ?§pf' ^w't^S. .?".>.*. , • ,.*• "* .* ■ . ' . : ' - - ■' ** ->■■ ■ ■ ■ '
FIG. 984.29B — Mold filaments in tomato products (100x). 1, branching mold and tomato cells. 2, coarse mold showing nonparallel
and parallel walls, branching, granulation, and blunt tips. 3, very fine mold. 4, mold showing beginning of sporulation at end of
hypha. 5, Geotrichum mold showing cross walls and feathery appearance characteristic of slimy machinery. 6, Alternaria spore
with attached hypha.
418
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
From each of >2 mounts examine >25 fields taken in such
manner as to be representative of all sections of mount.
A field is scored either pos. or neg. No field can be scored
pos. more than once. Method requires that field be counted as
pos. when aggregate lengths of not >3 filaments of mold pres-
ent exceed one-sixth diam. of field. One-sixth diam. of field
is not enough to be counted as pos.; aggregate length must
exceed one-sixth diam. of field.
Analyst must decide whether field is pos. Most pos. fields
qualify as such on basis of single mold filament which, in-
cluding length of branches, exceeds one-sixth of field diam.
Field may be qualified as pos. if any one of following lengths
exceeds one-sixth of field diam.
@ Length of single unbranched filament.
® Length of single filament plus lengths of branches (aggre-
gate length).
©Aggregate length of 2 mold filaments.
©Aggregate length of 3 mold filaments (no more than aggre-
gate lengths of 3 filaments of mold can be counted).
@ Aggregate length of all filaments in a clump of mold (a clump
of mold is considered a single piece, and aggregate lengths of
all filaments are counted).
C. Calculations
Calc. proportion of pos. fields from results of examination
of all observed fields and report as % pos. fields.
FRUBT AND FRUIT PRODUCTS
975.51 Mold in Apple Butter
Howard Mold Count
Final Action
Without braking, let centrf. come to complete stop before
removing tubes and read vol. sediment in centrf. tube. Re-
move tube and decant supernate without disturbing sediment.
With pineapple juice, add 0.5 mL HCJ (to dissolve oxalate
crystals). Add H 2 to tube to bring level to 10 mL mark and
then add 5 mL stabilizer soln, 945.75C(v). Thoroly mix sed-
iment, H z O, and stabilizer soln and pour into small beaker.
Mix by pouring back and forth between beaker and tube >6
times. Stir mixt. thoroly in beaker. Make Howard Mold count
as in 984.29. Sep. record those fields pos. due to Geotrichum
candidum; see 984. 30 A.
Ref.: JAOAC 66, 393(1983).
970.76 Mold in Cranberry Sauce
Howard Mold Count
Final Action 1974
(a) Strained sauce, — Immerse unopened can of sauce in
boiling H 2 bath 30-45 min to facilitate breaking gel. Re-
move can from bath and open carefully to avoid loss of sauce
thru sudden release of pressure. Transfer contents into beaker
(1 L for No. 2 can). Stir sauce to break gel. (Slow-speed mech.
mixer (350-450 rpm) may be used.) Thoroly mix 50 g stirred
sauce with 50 g stabilizer soln, 945.75C(v). Proceed as in
984.29.
(b) Whole sauce (seeds and skins included). — Pulp con-
tents of container (if considerably >1 lb (500 g), such as No.
10 can, remove well-mixed aliquot of 1 lb) thru cyclone to
remove skins and seeds, and prep, homogeneous pulp. Mix 50
g of this pulp with 50 g stabilizer soln, 945.75C(v). Proceed
as in 984.29.
Dil. 50 mL well-mixed sample with 50 mL stabilizer soln,
945.75C(v). Make Howard mold count as in 984.29.
955.47 Mold in Drupelet Berries
Howard Mold Count
Final Action 1974
(Applicable to blackberries, loganberries, raspberries, and other
drupelets; fresh, canned, and frozen)
(a) Frozen with or without sugar. — Pulp berries thru cy-
clone, 945.75B(g), and mix thoroly. Mix 25 g pulp with 50
mL stabilizer soln, 945.75C(v). Proceed as in 984.29.
(b) Frozen in sirup, canned in sirup or water. — Drain ber-
ries 2 min on No. 20 sieve. Pulp, dil., as in (a). Make Howard
mold count as in 984.29.
970.75 Mold in Citrus
and Pineapple Juices (Canned)
Howard Mold Count
Final Action 1974
(Applicable to single strength juices)
Pour contents of can into beaker and mix thoroly by pouring
back and forth between beaker and can >12 times. After mix-
ing, transfer 50 mL juice to graduated 50 mL conical-bottom
centrf. tube. Centrf. 10 min at 2200 rpm, 945.758(f). Check
speed with tachometer, since rheostat does not necessarily in-
dicate speed in rpm.
982.33 Mold in Fruit Nectars.
Purees, and Pastes
Howard Mold Count
First Action 1982
Final Action 1988
A. Sample Preparation
(a) Fruit nectars. — Measure 40 mL well mixed sample into
40 mL graduated, thick- wall centrf. tube (Corning, Pyrex No.
8340, or equiv.) and proceed as in 982. 33B.
(b) Fruit purees with no added starch. — Dil. sample 1 +
1 with H 2 0, measure 40 mL well mixed sample into 40 mL
graduated, thick-wall centrf. tube, and proceed as in 982. 33B.
(c) Fruit purees with added starch. — Weigh 50 g fruit puree
into beaker and add 50 mL HC1 soln (5 + 45). Mix well and
heat on steam bath 15 min. Measure 40 mL well mixed, hy-
dro I yzed sample into 40 mL graduated, thick- wall centrf. tube
and proceed as in 982. 33B.
(d) Fruit pastes. — Disperse 1 part paste in 3 parts H 2 0. If
necessary, warm gently to break gel. Measure 40 mL well mixed
sample into 40 mL graduated, thick- wall centrf. tube and pro-
ceed as in 982.33B.
B. Centrifugation and Concentration Adjustment
Centrf. 10 min at 2200 rpm as in 970.75. Gradually let centrf.
come to complete stop. Remove tubes and immediately decant
supernate without disturbing sediment. Gently tap centrf. tube
to level top of sediment. Dil. sediment with stabilizer soln,
945.75C(v), as follows: (J) peach, apricot, mango, and pa-
paya: 1 + 1; (2) pear and guava: 1 + 3; (3) strawberries,
blackberries, raspberries, and blueberries: 1+6.
AOAC Official Methods of Analysis (1990)
Rot and Mold
419
Proceed with Howard mold count as in 984.29. For products
dild 1 + J in 982.338(7), divide number of pos. fields by 2
before calcg % mold count.
Refs.: JAOAC 65, 1093(1982); 66, 393(1983).
945.89 Mold in Pureed Infant Food
Howard Mold Count
Final Action 1974
Proceed as follows: Add ca 0.2 g NaOH to ca 6 g product
before counting, and stir thoroly until NaOH is dissolved. Pro-
ceed as in 984.29.
952.22 Mold in Strawberries (Frozen)
Howard Mold Count
Final Action
945.91
Mold in Tomato Soup
Howard Mold Count
Final Action 1974
Place the unopened, punctured can in hot H 2 and heat until
contents are thoroly warmed; then open. Transfer 10 mL tho-
roly mixed soup to 50 mL centrf. tube and add 3 mL NaOH
soln (1 + 1). If starch is absent, omit the NaOH. Stir until starch
dissolves and tissues clear. Add enough H 2 to fill tube, and
centrf. (Time required to centrf. sample varies greatly. With
centrf. arm length of 5 1 // and speed of ca 1600 rpm, ca 20
min is required for av. sample. In heavy soups, gelatinizing
of much starch sometimes interferes with proper settling out
of solids during centrfg. If liq. remains cloudy, it may be nec-
essary to discard sample and start again by adding 3 mL NaOH
soln to only 5 mL soup.) When supernate is clear, pour off;
if not entirely clear, check supernate for mold before discard-
ing. Add enough H 2 to residue in tube to bring to original
vol. of soup, mix, and count mold as in 984.29.
Pulp thawed berries thru cyclone and mix thoroly. (Pour juice
thru cyclone last.) If necessary, remove air bubbles with suc-
tion or by mixing ca 100 g pulp with 3-5 drops 2-octanol.
Again mix thoroly and make mold count as in 984.29.
VEGETABLES AND VEGETABLE PRODUCTS
945.90 Mold in Tomatoes
(Canned)
Howard Mold Count
Final Action 1974
(a) Packing medium. — Drain contents of can 2 min on No.
2 sieve. For containers of <3 lb net wt, use 8" diam. sieve;
for containers of ^3 lb net wt, use 12" sieve. Make Howard
mold count, as is, as in 984.29.
(b) Whole drained tomatoes. — Examine drained tomatoes
and record number and size of any rotten portions present.
Pass drained tomatoes thru laboratory cyclone, 945.75B(g).
Make mold counts on pulped tomatoes, as for juice, as in
984.29.
945.92
Mold in Tomato Sauce
Howard Mold Count
Final Action 1974
(Applicable to sauce in pork and beans, spaghetti, ravioli, chili
con came, tamales, etc.)
Place unopened, punctured can in hot H 2 and heat until
contents are thoroly warmed. Open can and transfer contents
onto No. 6 sieve. Drain until major portion of liq. passes thru.
(With some products, sauce runs thru at once, but in case of
some beans and spaghetti, >10 min may be required.) Mix
sauce thoroly, place 10 mL in centrf. tube, and proceed as in
945.91, beginning "add 3 mL NaOH." Note: Use care in
counting products contg meat so as not to confuse mold fila-
ments and muscle fibers that superficially resemble each other;
muscle fibers are usually much thicker and striations are often
visible.
945.93 Mold in Tomato Sauce Packing
Medium on Fish
Howard Mold Count
Final Action 1974
965.41 Mold in Tomato Products
Howard Mold Count
Final Action
(Applicable to tomato juice, sauce, catsup, paste, and puree.
Not applicable to dehydrated products)
In making mold counts of tomato products, use juice and
sauce as they come from container. For catsup, place 50 mL
stabilizer soln, 945.75C(v), in 100 mL graduate, add 50 mL
well mixed catsup sample by displacement, and mix thoroly.
In case of puree and paste, add H 2 to make mixt. with tomato
sol. solids content that gives refractive index of 1.3448-1.3454
at 20° (1.3442-1.3448 at 25°). Add 2-6 drops 2-octanol to
each 100 mL mold count prepn to reduce or eliminate air bub-
bles on Howard mold counting slide. Proceed as in 984.29.
Refs.: Bur. Chem. Circ. 68 (1911). Food and Drug Adm.
Leaflet, July 1942. Am. Can Co. Bull. (1954). Natl.
Canners Assoc. Tomato Products Tables, 2nd rev. (Feb.
1966). JAOAC 49, 572(1966); 53, 366(1970).
Place unopened, punctured can in hot H 2 (ca 90-95°) until
contents are thoroly warmed. Open can and drain contents on
No. 6 sieve until major portion of sauce and oil passes thru.
Mix liq., place up to 50 mL in 50 mL centrf. tube, and centrf.
as in 945.91. Record vol. of lower oil-free sauce layer, and
discard oil and part of mold-free aq. layer. Add H 2 to bring
to recorded vol., mix, and count mold as in 984. 29A, remov-
ing bits of fish tissue from slide, if necessary, before counting.
See caution in 945.92.
972.42 Mold in Tomato
Powder (Dehydrated)
Howard Mold Count
First Action 1972
Final Action 1988
(Tomato powder is produced by dehydrating coned tomato
pulp. In prepg powder for mold counting, moisture content is
disregarded and diln with H 2 is made to give mixt. with ap-
420
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
prox. tomato solids content of stdzd prepn for mold count of
tomato puree or paste, i.e., 8.5%.)
A Microscopic Identification as Spray-Dried Product
Mold counts of spray-dried tomato powder show signifi-
cantly higher counts than paste from which it is made because
of breakage of mold hyphae aggregates. Use following pro-
cedure to det. whether powder represents spray-dried product.
Suitably mount a small portion of product on microscope
slide in mineral oil or other non-aqueous mounting medium
and examine microscopically at 100-200X. Spray-dried par-
ticles are translucent and contain air bubbles and numerous
small granules within the particles. Shape of particles ranges
from spherical to elongate to irregular with rounded outlines
and essentially no sharp angles. In rehydrated powder, prac-
tically no intact tomato cells are evident. Drum-dried or sim-
ilarly processed powder or flakes are characterized by irreg-
ular-shaped particles with angular outlines and practically no
embedded air bubbles.
B. Determination
Weigh 17.0 g thoroly mixed sample into high-speed blender,
945.75B(c), contg 150 mL H 2 to produce mixt. equiv. to
tomato puree. Blend 30 sec at ca 3200 rpm and, with rubber
policeman, rub down any material adhering to walls. Rinse
walls with 50.0 mL H 2 to bring total vol. to 200 mL, and
blend 1 min. Add 2 drops 2-octanol to break foam and count
mold as in 984.29.
Refs.: JAOAC 55, 73(1972); 61, 992(1978).
984.30 Geotrichum Mold Counting
A Diagnostic Characteristics
Geotrichum mycelial fragments have the same characteris-
tics of mold hyphae as described in 984. 29A. In addition,
Geotrichum mold filaments are septate, tend to taper toward
the tip, and branch at a 45° angle, giving the mold a feathery
appearance as shown in Fig. 984.30.
B. Determination
Using pipet, 945.75B(p), take up 0.5 mL of well mixed
sample and apply as streak ca 4 cm long to rot fragment count-
ing slide, 945.75B(q). Blow out last drop, if necessary. Prep,
addnl slides in same way.
Examine each slide at 30-45 X, using transmitted diffused
bottom illumination. Count Geotrichum mycelial fragments (with
3 or more characteristic hyphae branches, Fig. 984.30) on 2
entire slides.
C. Calcufations
Calc. mycelial fragments/500 g product:
N = (S/V{slides}) x (500/W) x V{diln}
where S = total mycelial fragments counted; V{ si ides} — total
vol. counted (0.5 mL/slide); W - net wt of sample, g; and
Vjdiln} = vol. after final diln with stabilizer soln.
945.95
Mold in Soft Drinks
Geotrichum Mold Count
Final Action 1988
937.1 3*
Mold in Butter
Final Action
Surplus 1970
See 40.024, 11th ed.
Det. net wt, W, of container and transfer contents to 3" No.
230 sieve, 945.75B(r). Transfer residue from sieve to 50 mL
graduated centrf. tube. Dil. to 10 mL. Add 1 drop crystal vi-
olet staining soln, 945.75C(h), and mix thoroly. Add 10 mL
stabilizer soln, 945.75C(v), and bring total vol., V, to 20 mL.
Make Geotrichum mold count on 2 slides as in 984.30.
Ref.: JAOAC 57, 957(1974).
945.94
Mold in Ground Spices
Howard Mold Count
Final Action 1974
(Applicable to garlic powder, paprika, red and cayenne
pepper, chili powder, and other ground capsicums)
Weigh 10 g thoroly mixed sample of ground spice and trans-
fer to high-speed blender. Add 200 mL 1% NaOH soln in 3
or 4 successive portions, stirring after each addn, washing down
with final portion any material that may stick to walls of blender.
Agitate mixt. in blender I min at ca 13,000 rpm. With rubber
policeman, rub down into mixt. any material sticking to walls
and repeat blending 2 min longer. Add 2 or 3 drops 2-octanol
to break foam. Mix 100 g of this mixt. with 50 g stabilizer
soln, 945.75C(v), and count as in 984.29.
Occasionally blended mixt. contains particles of seed tissue
that make it difficult to obtain Newton's rings in prepg slide
for mold counting. Clamp devised for holding cover slip in
place to obviate this difficulty consists of metal plate with cir-
cular opening, 2.5 cm diam., in center of plate; 2 clips at-
tached to edge of plate hold cover slip in position when slide
is placed on plate. Seed particles may also be picked off How-
ard slide with micro-forceps.
Refs.: JAOAC 55, 78(1972); 61, 475(1978).
980.29 Mold in Citrus Juices
Geotrichum Mold Count
First Action 1980
Final Action 1988
Use single strength juice as is; dil. cone, to single strength.
Pour vol. <250 mL (record vol. and wt used) onto 5" No. 40
sieve, 945.75B(r), resting in 2 L beaker. Wash container with
H 2 from wash bottle and transfer washings to sieve. Save
residue, liq., and washings. Quant, transfer liq. and washings
onto 5" No. 140 sieve resting in second 2 L beaker. Wash first
beaker and transfer washings onto No. 140 sieve. Save resi-
due, liq. and washings. Quant, transfer liq. and washings from
No. 140 sieve onto 5" No. 230 sieve. Wash second beaker into
sieve. Discard liq. and washings but save residue.
With wash bottle and spatula, quant, transfer residues from
Nos. 40 and 140 sieves to No. 230 sieve. Tilt No. 230 sieve
at ca 30° and wash residue to lower edge of sieve with H 2 0.
With wash bottle and spatula, transfer residue on No. 230 sieve
to 50 mL graduated centrf. tube. If vol. transferred is <20
mL, dil. to 20 mL with H 2 0. Add 5 drops crystal violet soln,
945.75C(h), mix well, and dil. to 40 mL with stabilizer soln,
945.75C(v). Mix well and proceed as in 984.30.
If vol. transferred is >20 mL, add 5 drops crystal violet soln
AOAC Official Methods of Analysis (1990)
Rot and Mold
421
A» x v
■'.'■'■%.'■■
FIG. 984.30— Geotrichum mold fragments
and centrf. 6 min at 2200 rpm (see 970.75). Dil. pellet to 20
mL with H 2 0, mix well, and dil. to 40 mL with stabilizer soln.
Mix well and proceed as in 984.30.
Ref.: J AOAC 63, 483(1980).
974.34 Mold in Vegetables,
Fruits, and Juices (Canned)
Geotrichum Mold Count
First Action 1974
Final Action 1988
(Applicable to products where mold is not masked
by large amts of tissues)
Det. net wt (g) of can contents. Drain contents 3 min on 8"
No. 8 sieve, 945.75B(r), in pan. Remove fruit from sieve with
spoon and discard. Wash can and sieve with ca 300 mL H 2
from wash bottle, saving liq. and washings. Quant, transfer
combined liq. and washings onto 5" No. 16 sieve resting in
2 L beaker. Wash residue on sieve with ca 50 mL H 2 0, and
discard residue. Quant, transfer combined liq. and washings
onto 5" No. 230 sieve, tilted at ca 30° angle, and discard liq.
and washings. Wash tissue to lower edge of sieve with H 2 0.
With wash bottle and spatula, transfer residue from sieve to
50 mL graduated thick- walled centrf. tube with min. vol. H 2 0.
For vols <10 mL, use (a); >10 mL but <30 mL, use (b); for
>30 mL, use (c).
(a) Dil. to 10 mL. Add 1 drop crystal violet staining soln,
945.75C(h), and mix thoroly. Add 10 mL stabilizer soln,
945.75C(v), to bring total vol. to 20 mL. Proceed as in 984.30.
(b) Dil. to 40 mL. Add 3 drops crystal violet soln. Mix
well. Centrf. ca 6 min at ca 2200 rpm, 970.75. Decant and
discard supernate. Bring vol. of sediment in centrf. tube to
422
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
nearest 5 mL graduation by adding H 2 0. Note combined vol.
of sediment and H 2 7 add equal vol. stabilizer soln, and mix
thoroly but gently. Record mL total vol. of mixt. in centrf.
tube, (V). Proceed as in 984.30.
(c) Transfer to g-s graduate. Dil. to >100 mL (Vjprepn})
and mix well. Quickly pour off two 25 mL aliquots (Vjaliq.}
= sum of aliquots taken) into sep. centrf. tubes and proceed
as in (b). Keep final vols equal after dilg with stabilizer soln;
Vjdiln} = sum of vol. in both tubes. Proceed as in 984.30,
using pipet to prep. 1 slide from each dild aliquot. Calc. my-
celial fragments/500 g product:
N - (S/V{slides}) x (500/ W) x Vjdiln} X (V{prepn}/V{aliq.})
where S = total mycelial fragments counted; Vjslides} = total
vol. counted (0.5 mL/slide); W = net wt of sample, g; V{diln}
= sum of vol. in both centrf. tubes after final diln with sta-
bilizer soln; Vjprepn} = vol. before aliquots removed; and
V{aliq.} - sum of vol. of aliquots taken.
Refs.: JAOAC 57, 957(1974); 62, 390(1979); 63, 287(1980).
982.34 Mold in Comminuted Fruits
and Vegetables
Geotrichum Mold Count
First Action 1982
Final Action 1988
A. Sample Preparation
(a) Fruit nectars. — Add 40 mL nectar and 10 drops of crys-
tal violet stain, 945.75C(h), to 40 mL centrf. tube (Corning,
Pyrex No. 8340, or equiv.). Mix well and proceed as in
982.34B.
(b) Purees with no added starch. — Add 20 mL puree and
10 drops of crystal violet stain, 945.75C(h), to 40 mL centrf.
tube. Mix well. Bring vol. to 40 mL with H 2 and mix well.
Proceed as in 982.34B.
(c) Purees with starch added. — Add 50 mL HCJ soln (5 +
45) to 50 g fruit puree. Mix well and heat with mag. stirring
until starch clears. Neutze soln with 50% KOH or 50% NaOH
to pH 7.0 ± 1.0. Transfer 40 mL soln to 40 mL centrf. tube
and add 20 drops of crystal violet stain, 945.75C(h). Mix well
and proceed as in 982. 34B.
(d) Pastes. — Disperse 3 part paste in 3 parts H 2 0. If nec-
essary, warm gently to break gel. Transfer 40 mL soln to 40
mL centrf. tube and add 10 drops of crystal violet stain,
945.75C(h). Mix well and proceed as in 982.34B.
B. Centrifugation
Centrf. 10 min at 2200 rpm, 945.75B(f ). Immediately after
centrf. comes to rest, decant aq. layer and read vol. of sedi-
ment. Dil. sediment 1 + 3 (v/v) with stabilizer soln,
945.75C(v). Proceed as in 984.30. Express results in mycelial
fragments per 100 mL prepn. TV = S x 100, where S — total
mycelial fragments/mL sample prepn countd (0.5 mL/slide).
Ref.: JAOAC 65, 1095(1982).
980.30 Mold in Cream Style Corn
Geotrichum Mold Count
First Action 1980
Final Action 1988
Weigh 250 g product onto 5" No. 16 sieve, 945.75B(r),
resting in container. Wash can and residue on sieve with ca
1.5 L hot (44-55°) H 2 0, saving liq. and washings. Discard
residue and rewash sieve with ca 300 mL hot H 2 0. Quant.
transfer combined liq. and washings onto 5" No. 70 sieve. Wash
residue on sieve with ca 1 L hot H 2 0, saving liq. and washings
but discarding residue. Quant, transfer combined liq. and
washings onto 5" No. 230 sieve tilted at ca 30°. Wash residue
with ca 500 mL hot H 2 and discard liq. and washings. Wash
tissue to lower edge of sieve with hot H 2 0. With wash bottle
and spatula, transfer residue from sieve to 100 mL graduate,
keeping vol. <50 mL. Dil. to 50 mL with H 2 0. Add 20 drops
crystal violet soln, 945.75C(h), and mix well. Dil. to 100 mL
with stabilizer soln, 945.75C(v). Mix well and proceed as in
984.30, making count on 2 slides.
Ref.: JAOAC 63, 481(1980).
952.23 Rot in Tomato
Products (Comminuted)
Rot Fragment Count
Final Action 1974
Weigh sample directly into 400 mL beaker, using elec. top-
loading balance with readability of 0.1 g, precision (std dev.)
of ±0.05 g, and 1200 g capacity (Sartorius No. 3706,
Sartorius Gmbh, Weender Landstr 94/108, D-3400 Gottin-
gen, West Germany, or equiv.). In case of puree or paste, add
H 2 to make mixt. of tomato sol. solids content that gives
refractive index of 1.3448-13454 at 20° (1.3442-1.3448 at
25°). Use 5.0 g catsup, sauce, or dild puree or paste; and 10.0
g juice.
Add ca 100 mL H 2 to sample in beaker and stir thoroly
using glass rod until sample material appears well dispersed.
Add 12 drops crystal violet soln, 945.75C(h), stir, and let stand
5 min. Add 200 mL H 2 and pour directly from beaker onto
a 3 in. diam. No. 60 sieve. Rinse beaker with addnl 200 mL
H 2 and pour H 2 directly from beaker over sieve as before.
Spread samples evenly over sieve. Tilt sieve to ca 30° angle
and carefully wash tissue to lower edge, using stream of H 2
from polyethylene wash bottle (500 mL Nalgene No. 2402-
0500 wash bottle with delivery tube molded on side, Nalge
Co., 75 Panorama Creek Dr, PO Box 20365, Rochester, NY
14602, or equiv.). Let tissue drain. If necessary, repeat wash-
ing and draining steps until tomato tissue is coned at lower
edge of sieve. Transfer tissue, portion- wise, with micro-spoon
(Scientific Products No. S1571, or equiv.), or small scoop-
style spatula to bottom of graduated tube ca 12 X 3 cm. Tissue
remaining on sieve should be washed to lower edge as before.
Hold sieve at ca 80-90° angle so that some H 2 and tissue is
retained at edge of sieve. Immediately take up H 2 and 'issue
with eyedropper having tip cut off at 2 mm id, and transfer to
graduated tube. Repeat process of washing tissue to lower edge
of sieve with wash bottle and transferring with dropper until
all tissue has been transferred. Bring vol. of H 2 and tissue
to 10 mL with H 2 0. Add stabilizer soln, 945.75C(v), to bring
vol. to 20 mL and mix well using micro-spoon or spatula.
Pipet 4 sep. 0.5 mL portions using pipet, 945.75B(p), stirring
sample prepn with pipet before drawing up each portion and
pipetting from ca center of prepn. Spread 0.5 mL portion evenly
over each of 4 counting slides, 945.75B(q), letting material
flow slowly, and spread uniformly in center of slide to cover
area ca 6 x 2 cm. Touch lower end of pipet to slide several
times to ensure complete removal of material. Blow out last
drop if necessary. Examine each slide at 40-45 x , using trans-
AOAC Official Methods of Analysis (1990)
Rot and Mold 423
■ • ',.
FIG. 952.23 — Rot fragments from tomato puree
mitted light. Rot fragment is defined as particle of tomato cel-
lular material with one or more mold filaments attached. Some
may appear as almost solid masses of mold. See Figs. 984. 29B,
984.30, 952.23.
Count number of rot fragments on each of 4 slides. Add
results from 4 slides to obtain number of rot fragments/g juice.
Add results from 4 slides and multiply by 2 for 5.0 g samples
(catsup, sauce, or dild puree or paste). Report number of rot
fragments/g product.
Refs.: JAOAC 35, 337(1952); 53, 366(1970); 68, 278,
896(1985). Natl. Canners Assoc. Tomato Products Ta-
bles, 2nd rev. (Feb. 1966).
945.96* Yeasts and Spores
in Tomato Products
Final Action
Surplus 1970
(Not applicable to dehydrated products)
5<?e 40.086, 11th ed.
GENERAL REFERENCES
(J) Principles of Food Analysis for Filth, Decomposition, and
Foreign Matter. AOAC, 2200 Wilson Blvd, Arlington,
VA 22201 (1981).
424
Extraneous Materials: Isolation
AOAC Official Methods of Analysis (1990)
(2) Annotated Bibliography of Methods for Examination of
Foods. JAOAC 29, 420(1946); 38, 1016(1955).
(3) Insect Contaminants of Foods. JAOAC 33, 898(1950).
(4) Insect Setae. JAOAC 37, 960(1954).
(5) Radiographic Applications. JAOAC 37, 148(1954).
(6) "Identification of Stored Products by the Micromorphol-
ogy of the Exoskeleton." A series published in JAOAC
(reprints are no longer available): Elytral patterns, 38,
776(1955); Adult antennae, 39, 879(1956); Larval frag-
ments, 39, 990(1956); Adult legs, 40, 973(1957); Adult
and larval beetle mandibles, 41, 460(1958); Adult labral
characteristics, 41, 472(1958); Head, thorax, abdomen,
41, 828(1958); Adult moths, 43, 444(1960); Larvae of
moths, 41, 704(1958); Cockroach fragments, 41,
886(1958); Miscellaneous insects, 41, 206(1958).
(7) "Micro-Analytical Entomology for Food Sanitation Con-
trol." AOAC, 2200 Wilson Blvd, Arlington, VA 22201
(1962) (out of print).
(8) Winton, A. L. and Winton, K. B., "The Structure and
Composition of Foods," John Wiley & Sons (4 Vols:
1932-1939).
(9) "Food Microscopy." A series published in Food 25(1956)-
28(1959).
(10) "Training Manual for Analytical Entomology in the Food
Industry." AOAC, 2200 Wilson Blvd, Arlington, VA
22201 (1977).
17. Microbiological Methods
Wallace H. Andrews and James ft/lesser, Associate Chapter Editors
Food and Drug Administration
(When preparing culture media, use distd or deionized H 2 such
as Purified Water USP XXII, found to be free from
traces of
dissolved metals, and bactericidal
or inhibitory compds. Use anhyd. salts unless otherwise specified.)
970.77 Cross Reference Tables
Salmonella sp., Escherichia coli, and
other Enter ohacteriaceae in foods
989.12
A. Methods for Examination of Foods
Staphylococcal enterotoxin
Beef, ground
Extraction and separation
980.32
Virus
975.56
Microslide gel double diffusion
976.31
Candy and candy coatings
Staphylococcus aureus
Salmonella
975.54D
Most probable number
987.09
Casein
Surface plating
975.55
Salmonella
967.26
Total coliforms, fecal coliforms, and
Cheese powders
Escherichia coli
983.25
Salmonella
985.42
Foods, canned
Chocolate, milk
Clostridium botulinum and its toxins
977.26
Salmonella
Foods, canned, low-acid
Culture
967.26
Commercial sterility
972.44
Hydrophobic grid membrane filter
985.42
Microleak detection
984.36
Coconut
Sporeformers
985.41
Salmonella
975.54D
Foods, chilled, frozen, precooked, or prepared, anc
Dairy products
nutmeats
Coliforms
Aerobic plate count
966.23C
Dry rehydratable film
989.10
Coliform organisms
966.24
Pectin gel
989.11
Escherichia coli
966.24
Egg, powdered
Foods, chilled or frozen
Salmonella
985.42
Escherichia coli
988.19
Eggs and egg products
Foods, low-moisture
Aerobic plate counts
Salmonella
Standard
940.37B
Colorimetric monoclonal EIA screening
987.11
Spiral
977.27
Foods, outbreak
Coliform organisms
940.37C
Clostridium perfringens
Direct microscopic count
940.37F
Alpha toxin estimation
974.38
Fungi
940.37E
Plate count for isolation and enumeration
976.30
Salmonella
Foods and cosmetics
Culture
967.26
Aerobic plate count
977.27
Fluorescent antibody
975.54D
Frog legs
Staphylococci, hemolytic
940.37D
Salmonella
976.54D
Streptococci
940.37D
Garlic powder
Fish meal
Salmonella
967.26
Salmonella
975.54D
Mammalian cells
Food ingredients, raw; and nonprocessed food
Escherichia coli, invasiveness
982.36
Staphylococcus aureus
980.37
Meat and meat products
Foods
Salmonella
975.54D
Aerobic plate count
Mild, fluid
Hydrophobic grid membrane filter
986.32
Dry rehydratable film
Pectin gel
988.18
Bacterial count
986.33
Bacillus
980.31
Coliform count
986.33
Bacillus cereus
983.26
Somatic cell count 973.68, 978.25, 978.26, 980.33
Salmonella
Milk, nonfat dry
Biochemical identification kit
978.24
Salmonella
Colorimetric monoclonal EI A screening
986.35
Culture
967.26
Colorimetric polyclonal EIA screening
989.14
Fluorescent antibody
975.54D
DNA hybridization screening
987.10
Hydrophobic grid membrane filter
985.42
Fluorogenic monoclonal EIA screening
989.15
Milk products, dried
Salmonella, motile
Salmonella
Immunodiffusion screening
989.13
Culture
967.26
425
426
Microbiological Methods
AOAC Official Methods of Analysis (1990)
Fluorescent antibody
975.54D
Onion powder
Salmonella
967.26
Oysters
Poliovirus 1
985.43
Vibrio cholerae
988.20
Pepper
Salmonella
985.42
Poultry, raw
Salmonella
985.42
Sugars
Thermophilic bacterial spores
972.45
Waters, shellfish-growing
Medium A-l
978.23
Yeast
Dried active
Salmonella
967.26
Dried inactive
Salmonella
975.54D
B. Methods for Examination of Organisms
Aerobic plate count
Dry rehydratable film
Milk, fluid
986.33
Hydrophobic grid membrane filter
Foods
986.32
Pectin gel
Foods
988.18
Spiral
Foods and cosmetics
977.27
Standard
Eggs and egg products
940.37B
Foods, chilled, frozen, precooked.
or prepared
,
and nutmeats
966.23C
Bacillus
Foods
980.31
Bacillus cereus
Foods
983.26
Clostridium, botulinum and its toxins
Foods, canned
977.26
Clostridium perfringens
Alpha toxin estimation
Outbreak foods
974.38
Plate count for isolation and enumeration
Outbreak foods
976.30
Coliform organisms
Dairy products
Dry rehydratable film
989.10
Pectin gel
989.11
Eggs and egg products
940.37C
Foods
Hydrophobic grid membrane filter
983.25
Foods, chilled, frozen, precooked, or
prepared,
and nutmeats
966.24
Milk, fluid
986.33
Direct microscopic count
Eggs and egg products
940.37F
Escherichia coli
Enterotoxin
DNA colony hybridization
984.34
DNA colony hybridization using synthetic
oligodeoxyribonucleotides
986.34
Foods, chilled, frozen, precooked, or
prepared,
and nutmeats
966.24
Foods, chilled or frozen
988.19
Mammalian cells 982.36
Waters, shellfish-growing 978.23
Fecal coli forms
Waters, shellfish-growing
Fungi
Eggs and egg products
Microleak detection
Low -acid canned foods
Poliovirus 1
Oysters
Salmonella
Candy and candy coatings
Casein
Cheese
Chocolate, milk
Coconut
Egg, powdered
Eggs and egg products
Fish meal
Foods
Colorimetric monoclonal El A screening
Colorimetric polyclonal EIA screening
DNA hybridization screening
Fluorogenic monoclonal EIA screening
Foods, low-moisture
Colorimetric monoclonal EIA screening
Frog legs
Garlic powder
Meat and meat products
Milk, nonfat dry
Milk products, dried
Onion powder
Pepper
Poultry, raw
Yeast, dried
Active
Inactive
Salmonella, motile
Foods
Salmonella cultures
Foods
Salmonella sp., Escherichia coli, and other
Enter obacteriaceae cu I tures
Foods
Somatic cells
Milk, fluid 973.68, 978.25, 978.26, 980.33
Sporeformers
Low-acid canned foods
Spores, thermophilic bacterial
Sugars
Staphylococcal enterotoxin
Foods
Extraction and separation
Microslide gel double diffusion
Staphylococci, hemolytic
978.23
940.37E
984.36
985.43
975.54D
967.26
985.42
967.26, 985.42
975.54D
985.42
967.26, 975.54D
975.54D
986.35
989.14
987.10
989.15
987.11
975.54D
967.26
975.54D
975.54D, 967.26, 985.42
967.26, 975.54D
967.26
985.42
985.42
967.26
975.54D
989.13
978.24
989.12
985.41D
972.45
980.32
976.31
Eggs and egg products
Staphylococcus aureus
Most probable number
Most probable number,
Surface plating
Sterility, commercial
Low -acid canned foods
Streptococci
Eggs and egg products
Vibrio cholerae
Oysters
with pyruvate
940.37D
980.37
987.09
975.55
972.44
940.37D
988.20
AOAC Official Methods of Analysis (1990)
Egg Products
427
Virus
Beef, ground
975.56
EGGS AND EGG PRODUCTS
939.14 Sampling of Eggs
and Egg Products
Microbiological Methods
Final Action
("Compendium of Methods for the Microbiological Exami-
nation of Foods/' 2nd ed. Prepd by the APHA Intersociety/
Agency Committee on Microbiological Methods for Foods.
1984. Marvin L. Speck, Ed., should be used as guide for fur-
ther study of microorganisms obtained in culturing technics
described.)
A. Equipment
(a) Liquid eggs. — Sampling tube or dipper, sterile sample
containers with tight closures (pt (500 mL) Mason jars or fric-
tion top cans are most practical), alcohol, alcohol lamp or other
burner, absorbent cotton, clean cloth or towel, and H 2 pail.
(b) Frozen eggs. — Elec. (high-speed) or hand drill with 1
x 16" auger, hammer and steel strip (12 x 2 X 0.25"), or
other tool for opening cans; tablespoon, hatchet or chisel, pre-
cooled sterile containers, etc., as in (a).
(c) Dried eggs. — Grain trier long enough to reach to bot-
tom of containers to be sampled. Clean sample containers with
tight closures (pt (500 mL) Mason jars or paperboard cartons),
clean cloth or towel, and tablespoon.
B. Methods
Take samples from representative number of containers in
lot, 925.29. Sterilize sampling tube or dipper, auger, spoon,
and hatchet by wiping with alcohol-soaked cotton and flaming
over alcohol lamp or other burner. Between samplings, thoroly
wash instruments, dry, and resterilize. Open and sample all
containers under as nearly aseptic conditions as possible.
(a) Liquid eggs. — Thoroly mix contents of container with
sterile sample tube or dipper, and transfer ca 400 mL (0.75
pt) to sterile sample container. Keep samples at <5° but avoid
freezing. Observe and record odor of each container sampled
as normal, abnormal, reject, or musty.
(b) Frozen eggs. — Remove top layer of egg with sterilized
hatchet or chisel. Drill 3 cores from top to bottom of container:
first core in center, second core midway between center and
periphery, and third core near edge of container. Transfer drill-
ings from container to sample container with sterile spoon.
Examine product organoleptically by smelling at opening of
fourth drill-hole made after removal of bacteriological sample.
(Heat produced by elec. drill intensifies odor of egg material,
thus facilitating organoleptic examination.) Record odors as
normal, abnormal, reject, or musty. Refrigerate samples with
solid C0 2 or other suitable refrigerant if analysis is to be de-
layed or sampling point is at some distance from laboratory.
(c) Dried, eggs. — For small packages, take entire parcel or
parcels for sample. For boxes and barrels, remove top layer
with sterile spoon or other sterile instrument, and with sterile
trier remove >3 cores as in (b). (Samples should consist of
ca 400 mL (0.75 pt).) Aseptically transfer core to sample con-
tainer with sterile spoon or other suitable instrument. Store
samples under refrigeration or in cool place.
Ref.: J AOAC 22, 625(1939).
940.36 Culture Media for Eggs
and Egg Products
Microbiological Methods
Final Action
A. Standard Methods Media
(a) Dilution water. — To prep, stock soln, dissolve 34 g
KH 2 P0 4 in 500 mL H 2 0, adjust to pH 7.2 with 1/V NaOH (ca
175 mL), and dil. to 1 L with FLO. To prep, buffered H 2
for dilns, dil. 1 .25 mL stock soln to 1 L with boiled and cooled
H 2 0. Autoclave 15 min at 121°.
(b) Buffered glucose broth (MR-VP medium). — For Me red-
Voges Proskauer (MR-VP) tests. Dissolve 7.0 g proteose pep-
tone, 5.0 g glucose, and 5.0 g K 2 HP0 4 in ca 800 mL FLO
with gentle heat and occasional stirring. Filter, cool to 20°,
and dil. to 1 L. Dispense 10 mL portions into test tubes and
autoclave 12-15 min at 121°. Max exposure to heat should be
<30 min. Final pH, 6.9 ± 0.2.
(c) Endo medium. — Suspend 3.5 g K 2 HP0 4 , 10.0 g pep-
tone, 20.0 g agar, and 10 g lactose in 1 L FLO. Boil to dis-
solve, add FLO to original vol., and clarify if necessary. Dis-
pense in 100 mL portions and autoclave 15 min at 121°. Final
pH, 7.4 ± 0.1. Before use, melt and add 0.25 g Na 2 S0 3 and
1.0 mL filtered 5% ale. soln basic fuchsin.
(d) Eosin methylene blue agar (Levine). — Dissolve 10.0 g
peptone, 2.0 g K 2 HP0 4 , and 15.0 g agar in 1 L H 2 0. Boil to
dissolve and add H 2 to original vol. Dispense in 100 or 200
mL portions and autoclave 15 min at 121°. Final pH, 7.1 ±
0.1. Before use, melt and to each 100 mL add 5 mL sterile
20% lactose soln, 2.0 mL 2% aq. Eosin Y soln, and 1.3 mL
0.5% aq. methylene blue soln.
(e) Koser's citrate broth. — Dissolve 1.5 g Na-
NH 4 HP0 4 .4H 2 0, 1 .0 g KH 2 P0 4 , 0.2 g MgS0 4 .7H 2 0, and 3.0
g Na citrate. 2H 2 in 1 L FLO. Dispense in 10 mL portions
into test tubes and autoclave 15 min at 121°. Final pH, 6.7 ±
0.1.
(f) Lactose broth. — Dissolve on H 2 bath, with stirring,
3.0 g beef ext and 5.0 g polypeptone or peptone in 1 L FLO.
Add 5.0 g lactose. Dispense into fermentation tubes and au-
toclave 15 min at 121°. Max. exposure to heat should be <30
min. Final pH, 6.7 ± 0.2.
(g) Plate count agar (tryptone glucose yeast agar). — Sus-
pend 5.0 g peptone-tryptone (pancreatic digest of casein), 2.5
g yeast ext, 1.0 g glucose, and 15.0 g agar in 1 L H 2 0. Heat
and boil until all ingredients are dissolved. Autoclave 15 min
at 121°. Final pH, 7.0 ± 0.1.
(h) Tryptophane broth. — Dissolve by heating, with stir-
ring, 10.0 g tryptone or trypticase in 1 L H 2 0. Dispense in 5
mL portions into test tubes and autoclave 15 min at 121°. Final
pH, 6.9 ± 0.2.
B. Other Media
(a) Malt agar. — Dissolve by boiling 30 g malt ext (Difco)
and 15.0 g agar in 1 L FLO. Autoclave 15 min at 121°. Just
before use, melt malt agar and acidify with 85% lactic acid to
pH 3.5. Do not reheat medium after addn of acid.
(b) Milk protein hydrolysate glucose agar. — BBL dehy-
drated, or prep, from 9.0 g milk protein hydrolysate, 1 g glu-
cose, 15 g agar, and 1 L H 2 0; adjust to pH 7.0. Autoclave 15
min at 121°, cool to room temp., and readjust pH to 7.0, if
necessary.
(c) Physiological salt soln. — Dissolve 8.5 g NaCl in 1 L
H 2 0. Autoclave 15 min at 121° and cool to room temp.
(d) Veal infusion agar. — Mix 500 g ground lean veal and
1 L H 2 0. Infuse overnight in refrigerator and strain thru
cheesecloth without pressure. Dil. to original vol. with H 2
428
Microbiological Methods
AOAC Official Methods of Analysis (1990)
and skim off any fat. Steam in Arnold sterilizer 30 min and
filter thru paper. Add 10.0 g peptone (Difco), 5.0 g NaCl, and
15.0 g agar.
Steam in Arnold sterilizer to dissolve ingredients. Adjust to
pH 7.6 and steam in Arnold sterilizer 15 min. Filter thru buch-
ner with paper pulp mat, with suction. (Use egg albumen for
clarification when necessary. Add fresh white of 1 egg pre-
viously beaten with 50 mL medium or its equiv. in desiccated
egg white (1.5 g) to each L of medium before adjusting pH
and after cooling to 50°. Shake thoroly to ensure soln of egg
white. Let stand 20 min. Heat in Arnold sterilizer 15 min to
coagulate egg white. Shake vigorously and reheat. Filter, ad-
just to pH 7.6, steam in Arnold sterilizer 15 min, and filter.)
Place 10 mL portions in test tubes or 80 mL portions into
bottles. Autoclave 20 min at 121°; final pH, 7.4.
For hemolytic tests, cool melted agar to 45° and add 5%
defibrinated horse, sheep, or rabbit blood prior to pouring plates
(0.5 mL blood/ 10 mL medium).
940.37 Technics for Eggs
and Egg Products
Microbiological Methods
Final Action
A. Preparation of Sample
(a) Liquid eggs, — Thoroly mix sample with sterile spoon
or sterile mech. stirrer and prep. .1:10 diln by aseptically
weighing 1 1 g egg material into sterile wide-mouth g-s or screw-
cap bottle; add 99 g sterile diln H 2 0, 940.36A(a), or sterile
physiological salt soln, 940.36B(c), and 1 tablespoonful sterile
glass shot, Thoroly agitate 1:10 diln to ensure complete soln
or distribution of egg material in diluent by shaking each con-
tainer rapidly 25 times, each shake being up-and-down move-
ment of ca 30 cm, time interval not exceeding 7 sec. Let bub-
bles escape. Transfer representative portion from 1:10 diln for
higher serial dilns as needed. Proceed as in 940.37B-F(a).
Pour all plates and inoculate other media within 15 min after
prepn of first diln to avoid growth or death of microorganisms.
(b) Frozen eggs. — Thaw frozen egg material as rapidly as
possible to avoid increase in number of microorganisms pres-
ent and at temp, low enough to prevent destruction of the mi-
croorganisms (<45° for <15 min). (Frequent rotary shaking
of sample container aids in thawing frozen material. Thawing
temp, may be maintained by use of H 2 bath or bacteriological
incubator.) Proceed as in (a).
(c) Dried eggs. — Thoroly mix sample with sterile spoon or
spatula. Prep. 1:10 diln as in (a). If material is relatively insol.
(stored samples), use 0.17V LiOH as diluent. Prep, serial dilns
as in (a) and proceed as in 940.37B~F(b).
B. Plate Counts
Inoculate one set of petri plates with 1 mL portion of each
suitable diln. Pour plates with tryptone glucose yeast agar or
milk protein hydrolysate glucose agar previously cooled to 42-
45°. Incubate inoculated plates 3 days at 32°. Count plates with
aid of Quebec colony counter, if available. Express final re-
sults as number of viable microorganisms /g egg material.
C. incidence of Conform Group
(a) Inoculate 1.0 mL portions from suitable dilns of egg
material into fermentation tubes of lactose broth. Incubate 24-
48 hr at 35°. Streak eosin methylene blue or Endo medium
plates from all lactose broth cultures showing gas production.
Incubate plates 24-48 hr at 35°. Examine plates of differential
media for colonies of microorganisms of coliform group. Re-
cord number of coliform bacteria/g egg material as reciprocal
of highest diln showing pos. confirmation on differential me-
dia.
(b) Biochemical reaction (optional). — Inoculate from col-
onies of coliform types of bacteria appearing on differential
agar plates to agar slants, 940.36A(g) or 940.36B(b). Incubate
24 hr at 35°. Purify cultures for further study. Obtain IMViC
biochem. reactions of purified cultures by following tests:
Kovacs test (indole production), 966.24(a);
Acid production in Me red indicator, 966.24(b);
Acetylmethylcarhinol production, 966.24(b);
Koser sodium citrate test (utilization of Na citrate as sole
source of C), 966.24(c).
Note: Follow methods for biochem. reactions recommended
in "Standard Methods for Examination of Water and Waste
Water," 16th ed., 1985, American Public Health Association,
American Water Works Association, and Water Pollution
Control Federation, 1015 15th St NW, Washington, DC 20005.
D. incidence of Hemolytic Staphylococci and Streptococci
— Procedure
Inoculate petri plates with 1 mL portions of suitable dilns
of sample. Pour plates with veal infusion agar contg 5% de-
fibrinated horse, sheep, or rabbit blood (0.5 mL blood/ 10 mL
medium). Cool agar to 45° and add blood just before pouring
plates. Incubate plates 24 hr at 35°. Confirm presence of coc-
cus types of microorganisms by microscopic examination of
smears taken from representative colonies and stained by Gram
method. Express final results as number/g.
E. Tests for Fungi — Procedure
Inoculate petri plates with 1 mL portions of suitable dilns
of sample. Pour inoculated plates with malt agar, 940.366(a),
previously cooled to 42-45°. Incubate plates 5 days at 20° or
at room temp., if 20° incubator is not available. Express final
results as number of fungi /g egg material. Confirm yeast col-
onies by microscopic examination of smears stained by Gram
method.
F. Direct Microscopic Counts
North aniline oil-methylene blue stain. — Mix 3.0 mL ani-
line oil with 10.0 mL alcohol, and slowly add 1.5 mL HC1
with const agitation. Add 30.0 mL satd ale. methylene blue
soln, dil. to 100.0 mL with H 2 0, and filter.
(a) Liquid and frozen eggs. — Place 0.01 mL undild egg
material on clean, dry microscopic slide and spread over area
of 2 sq cm (circular area with diam. of 1 .6 cm suggested). Let
film prepn dry on level surface at 35-40°. Immerse in xylene
<1 min; then immerse in alcohol <1 min. Stain >45 sec in
North aniline oil-methylene blue stain (10-20 min preferred;
exposure up to 2 hr does not overstain). Wash slide by re-
peated immersions in H 2 and dry thoroly before examination.
Observe subsequent operations and precaution as in "Standard
Methods for Examination of Dairy Products," 15th ed., 1985,
American Public Health Association. Express final result as
number of bacteria/g egg material (double microscopic factor,
since 2 sq cm area is used).
(b) Dried eggs.— Place 0.01 mL of 1:10 or 1:100 diln of
dried egg material on clean, dry microscopic slide and spread
over 2 sq cm.
Note: 0.17V LiOH may be used as diluent and is preferred
for samples that are relatively insol. Circular area with diam.
of 1.6 cm is preferable. Addn of drop of H 2 to each film
facilitates uniform spreading.
Proceed as in (a). Double microscopic factor, since area of
2 sq cm is used, and multiply count by 10 or 100, depending
on whether film was prepd from 1:10 or 1:100 diln.
Ref.: JAOAC 36, 91, 316(1953).
AOAC Official Methods of Analysis (1990)
Prepared Foods
429
CHILLED, FROZEN, PRECOOKED,
OR PREPARED FOODS, AND NUTMEATS
966.23 Microbiological Methods
First Action 1966
Final Action 1989
(For the detn of aerobic plate count, most probable number of
coliform bacteria and Escherichia coli, and Staphylococcus in
products such as frozen cooked meat, poultry, and vegetable
products; cooked and /or breaded seafood; bakery products;
salads; tree nut meats; and ingredients of food samples col-
lected during sanitation inspections of food producing estab-
lishments, unless specific directions are given for that prod-
uct.)
A. Media and Reagents
Ingredients and reagents used to prep, following media may
be product of any manufacturer if comparative tests show that
satisfactory results are obtained. Use pure carbohydrates suit-
able for biological use; ACS reagent grade inorg. chemicals;
and dyes certified by "Biological Stain Commission" for use
in media.
For convenience, dehydrated media of any brand equiv. to
formulation may be used. Test each lot of medium for sterility
and growth-promoting qualities of suitable organisms (e.g. , in-
oculate media contg lactose with coliform bacteria, Staphy-
lococcus media with Staphylococcus, etc.).
Det. pH before autoclaving with pH meter stdzd against std
buffers, 964.24. Adjust pH, when necessary, by adding IN
NaOH or iN HC1 so that stated final pH results after auto-
claving.
Use sterile glass or plastic, 100 x 15 mm, petri dishes.
(a) Plate count agar. — See 940.36A(g).
(b) Lauryl sulfate tryptose broth. — Dissolve 20.0 g trypti-
case or tryptose (pancreatic digest of casein), 5.0 g NaCl, 5.0
g lactose, 2.75 g K 2 HP0 4 , 2.75 g KH 2 P0 4 , and 0.1 g Na lau-
ryl sulfate in 1 L H 2 with gentle heat, if necessary. Dispense
10 mL portions into 20 X 150 mm test tubes contg inverted
fermentation tubes 10 x 75 mm. Autoclave 15 min at 121°.
Final pH, 6.8 ± 0.1.
(c) Brilliant green lactose bile (BGLB) broth. — Dissolve 10.0
g peptone and 10.0 g lactose in ca 500 mL H 2 0. Add soln
(pH 7.0-7.5) of 20 g dehydrated oxgall or oxbile in 200 mL
H 2 0. Dil. to 975 mL and adjust pH to 7.4. Add 13.3 mL 0. 1%
soln of brilliant green, and dil. to 1 L with H 2 0. Filter thru
cotton and dispense 10 mL portions into 20 x 150 mm test
tubes contg inverted 10 X 75 mm fermentation tubes. Auto-
clave 15 min at 121°. Final pH, 7.2 ±0.1.
(d) Eosin methylene blue agar (Levine). — See 940.36A(d).
(e) Baird-Parker medium (egg tellurite glycine pyruvate agar,
ETGPA). — (1) Basal medium. — Suspend 10.0 g tryptone, 5.0
g beef ext, 1 .0 g yeast ext, 10 g Na pyruvate, 12.0 g glycine,
5.0 g LiC1.6H 2 0, and 20.0 g agar in 950 mL H 2 0. Heat to
bp with frequent agitation to dissolve ingredients completely.
Dispense 95 mL portions into screw-capped bottles. Autoclave
15 min at 121°. Final pH, 7.0 ± 0.2 at 25°. Store <J month
at 4 ± 1°.
(2) Enrichment. — Bacto EY tellurite enrichment (Difco
Laboratories) or prep, as follows: Soak fresh eggs ca 1 min in
diln of satd HgCl 2 soln (1 + 1000). Aseptically crack eggs
and sep. yolks from whites. Blend yolk and physiological sa-
line soln, 940.36B(c), (3 + 7, v/v) in high-speed blender ca
5 sec. To 50 mL egg yolk emulsion add 10 mL filter sterilized
1% K tellurite soln. Mix and store at 4 ± 1°.
(J) Complete medium. — Add 5 mL warmed enrichment to
95 mL molten basal medium cooled to 45-50°. Mix well,
avoiding bubbles, and pour 15-18 mL into sterile 100 x 15
mm petri dishes. Store plates at room temp. (<25°) for <5
days before use. Medium should be densely opaque; do not
use nonopaque plates. Dry plates before use by 1 of following
methods: (a) in convection oven or incubator 30 min at 50°
with lids removed and agar surface downward; (b) in forced-
draft oven or incubator 2 hr at 50° with lids on and agar surface
upward; (c) in incubator 4 hr at 35° with lids on and agar sur-
face upward; or (d) on laboratory bench 16-18 hr at room
temp, with lids on and agar surface upward.
(4) Interpretation. —Colonies of S. aureus are typically cir-
cular, smooth, convex, moist, 2-3 mm in diam. on uncrowded
plates, gray-black to jet-black, frequently with light-colored
(off-white) margin, surrounded by opaque zone (ppt) and fre-
quently with outer clear zone; colonies have buttery to gummy
consistency when touched with inoculating needle. Occasional
non-lipolytic strains may be encountered which have same ap-
pearance, except that surrounding opaque and clear zones are
absent. Colonies isolated from frozen or desiccated foods which
have been stored for extended periods are frequently less black
than typical colonies and may have rough appearance and dry
texture.
(f) Trypticase (tryptic) soy broth with 10% sodium chlo-
ride. — Add 95 g NaCl to 1 L of soln of 17.0 g trypticase or
tryptose (pancreatic digest of casein), 3.0 g phytone (papaic
digest of soya meal), 5.0 g NaCl, 2.5 g K 2 HP0 4 , and 2.5 g
glucose. Heat gently if necessary. Dispense into 16-20 mm
diam. tubes to depth of 5-8 cm. Autoclave 15 min at 121°.
Final pH, 7.3 ± 0.2.
(g) EC broth. — Dissolve 20.0 g trypticase or tryptose (pan-
creatic digest of casein), 1.5 g Bacto bile salt No. 3 or bile
salt mixt., 5.0 g lactose, 4.0 g K 2 HP0 4 , 1.5 g KH 2 P0 4 , and
5.0 g NaCl in 1 L H 2 0. Dispense 8 mL into 16 x 150 mm
test tubes contg inverted 10 x 75 mm fermentation tube. Au-
toclave 15 min at 121°. Final pH, 6.9 ± 0.1.
(h) Brain-heart infusion. — See 967.25 A(r). Dispense into
bottles or tubes for storage and autoclave 15 min at 121°.
(i) Desiccated coagulase plasma (rabbit) with EDTA. — Re-
constitute according to manufacturer's directions. If not avail-
able, reconstitute desiccated coagulase plasma (rabbit) and add
Na 2 H 2 EDTA to final concn of 0.1% in reconstituted plasma.
(j) Tryptophane broth. — See 940.36A(h) but dispense in
10 mL portions,
(k) Buffered glucose broth (MR-VP medium). — See
940.36A(b). BBL Microbiological Systems, No. 11383, or
equiv.
(1) Koser's citrate broth. — See 940. 36 A (e).
(m) Butterfield's buffered phosphate diluent. — (1) Stock
soln. — Dissolve 34.0 g KH 2 P0 4 in 500 mL H 2 0, adjust to pH
7.2 with ca 175 mL IN NaOH, and dil. to 1 L. Store in re-
frigerator. (2) Diluent. —Dil. 1.25 mL stock soln to 1 L with
H 2 0. Prep, diln blanks with this soln, dispensing enough to
allow for losses during autoclaving. Autoclave 15 min at 121°.
B. Preparation of Sample
(Prep, all decimal dilns with 90 mL sterile diluent plus 10 mL
previous diln unless otherwise specified. Shake all dilns 25
times in 30 cm arc. Pipets must accurately deliver required
vol. Do not use to deliver <10% of their total vol. For ex-
ample, to deliver 1 mL, do not use pipet > 10 mL; to deliver
0.1 mL, do not use pipet >1 mL.)
(a) Frozen and /or prepared foods. — Use balance with ca-
pacity of >2 kg and sensitivity of 0.1 g to aseptically weigh
50 g unthawed (if frozen) sample into sterile high-speed blender
jar. Add 450 mL diluent, (m)(2), and blend 2 min. (If nec-
essary to temper frozen sample to remove 50 g portion, hold
430
Microbiological Methods
AOAC Official Methods of Analysis (1990)
<18 hr at 2-5°.) Not > 15 min should elapse from time sample
is blended until all dilns are in appropriate media.
If entire sample consists of <50 g, weigh portion equiv. to
1 /2 sample and add vol. of sterile diluent required to make 1:10
diln. Total vol, in blender jar must completely cover blades.
(b) Tree nut meat halves and larger pieces. — Aseptically
weigh 50 g sample into sterile jar. Add 50 mL diluent, (m)(2),
and shake vigorously (50 times thru 30 cm arc) to obtain 10°
diln. Let stand 3-5 min and shake just before making serial
dilns and inoculations.
(c) Nut meal. — Aseptically weigh 10 g sample into sterile
jar. Add 90 mL diluent, (m)(2), and shake vigorously (50 times
thru 30 cm arc) to obtain 10" I diln. Let stand 3-5 min and
shake to resuspend just before making serial dilns and inoc-
ulations.
C. Aerobic Plate Count
Seed duplicate petri dishes in dilns of 1:10, 1:100, 1:1000,
etc., using plate count agar, (a). Ordinarily 1:100 thru 1:10,000
are satisfactory. Place 1 mL appropriate diln in each plate, and
add molten agar (cooled to 42-45°) within 15 min from time
of original diln. Incubate 48 ± 2 hr at 35° and count duplicate
plates in suitable range (30-300 colonies). If plates do not
contain 30-300 colonies, record diln counted and note number
of colonies found. Average counts obtained and report as aero-
bic plate count/g.
966.24 Col'iform Group
and Escherichia coli in Tree Nut Meats
Microbiological Method
Final Action 1971
Seed 3-tube most probable number (MPN) series into lauryl
sulfate tryptose broth, (b), using I mL inocula of 1:10, 1:100,
and 1:1000 dilns, with triplicate tubes at each diln. (For nut
meats (halves and larger pieces), begin MPN detn with 10°
diln; for nut meal, begin with 10" l diln.) Incubate 48 ± 2 hr
at 35° for gas formation as evidenced by displacement of liq.
in insert tube or by vigorous effervescence when tubes are shaken
gently. Examine tubes for gas formation at 24 and 48 hr in-
tervals. Transfer, using 3 mm loop, from gassing tubes to
BGLB, (c) (omit this transfer for tree nuts), and EC broth,
(g), at time gas formation is noted.
Incubate BGLB broth 48 ± 2 hr at 35°. Using MPN Table
966.24, compute MPN on basis of number of tubes of BGLB
broth producing gas by end of incubation period. Report as
MPN of coliform bacteria/g.
Incubate EC broth 48 ± 2 hr at 45.5 ± 0.05° in covered
H 2 bath. Submerge broth tubes in bath so that H 2 level is
above highest level of medium. Examine tubes for gas for-
mation at 24 and 48 hr intervals. Streak gas-pos. tubes on Le-
vine's eosin methylene blue agar plates, (d), and incubate plates
24 ± 2 hr at 35°.
Pick 2 or more well, isolated typical colonies from Levine's
eosin methylene blue agar plates and transfer to agar slants
prepd from agar medium, (a). Incubate 18-24 hr at 35°. If
typical colonies are not present, pick 2 or more colonies most
likely to be E. coli. Pick >2 from every plate.
Transfer growth from plate count agar slants into following
broths for identification by biochem. tests:
(a) Tryptophane broth. — Incubate broth, (j), 24 ± 2 hr at
35° and test for indole by adding 0.2-0.3 mL Kovacs reagent,
967.25B(a), to 24 hr culture. Test is pos. if upper layer turns
red.
(b) MR-VP medium. — Incubate medium, (k), 48 ± 2 hr at
35°. Aseptically transfer 1 mL culture to 13 x 100 mm test
tube to test for acetylmethylcarbinol. Add 0.6 mL 5% ale. ct-
naphthol soln, 0.2 mL KOH soln (4 + 10), and few crystals
of creatine. Shake and let stand 2 hr. Test is pos. if eosin pink
develops. Alternatively, see 967.27D(c)(i).
Incubate remainder of MR-VP medium for addnl 48 hr and
test for Me red reaction by adding 5 drops Me red soln to
culture. Test is pos. if culture turns red; neg., if yellow. (Prep.
Me red soln by dissolving 0.1 g Me red in 300 mL 90% al-
cohol and dilg to 500 mL with H 2 0.)
(c) Koser citrate broth, (/). — Incubate 96 hr at 35° and rec-
ord growth as + or - .
(d) Lauryl sulfate tryptose broth, (b). — Incubate 48 ± 2 hr
at 35°. Examine tubes for gas formation.
(e) Gram stain. — Perform Gram stain on 18 hr agar slant
("Standard Methods for the Examination of Water and Waste
Water," 16th ed., 1985). Coliform organisms will stain red
(neg.); Gram-pos. organisms will stain blue-black.
(f ) Classification. — Classify biochem. types as follows:
Indole MR
+ +
+
+ +
VP
Citrate
+
+
Type
Typical E. coli
Atypical E. coli
Typical Intermediate
Atypical Intermediate
Typical Enterobacter
aerogenes
Atypical Enterobacter
aerogenes
Table 966.24
Most Probable Numbers (MPN) per
1 gof
Sample,
Using 3 Tubes with Each of 0.1,
0.01,
and 0.001
g Portions
Positive Tubes
Positive Tubes
Positive Tubes
Positive Tubes
0.1
0.01
0.001
MPN 0.1 0.01
0.001
MPN
0.1
0.01
0.001
MPN
0.1
0.01
0.001
MPN
<3 1
3.6
2
9.1
3
23
1
3 1
1
7.2
2
1
14
3
1
39
2
6 1
2
11
2
2
20
3
2
64
3
9 1
3
15
2
3
26
3
3
95
1
3 1 1
7.3
2
1
15
3
1
43
1
1
6.1 1 1
1
11
2
1
1
20
3
1
1
75
1
2
9.2 1 1
2
15
2
1
2
27
3
1
2
120
1
3
12. 1 1
3
19
2
1
3
34
3
1
3
160
2
6.2 1 2
11
2
2
21
3
2
93
2
1
9.3 1 2
1
15
2
2
1
28
3
2
1
150
2
2
12 1 2
2
20
2
2
2
35
3
2
2
210
2
3
16 1 2
3
24
2
2
3
42
3
2
3
290
3
9.4 1 3
16
2
3
29
3
3
240
3
1
13 1 3
1
20
2
3
1
36
3
3
1
460
3
2
16 1 3
2
24
2
3
2
44
3
3
2
1100
3
3
19 1 3
3
29
2
3
3
53
3
3
3
>1100
AOAC Official Methods of Analysis (1990)
Aerobic Plate Count
431
Other groupings may appear; in such cases cultures are usu-
ally mixed. Restreak to det. their purity.
Compute MPN of E. coll/ 'g, considering Gram neg., non-
spore-forming rods producing gas in lactose and producing
+ H or - + IMViC patterns as E. coli.
Refs.: JAOAC 49, 270, 276(1966); 51, 865, 867(1968); 58,
1154(1975).
977.27 Bacteria in Foods and Cosmetics
Spiral Plate Method
First Action 1977
Final Action 1981
A. Principle
Bacterial suspension from prepd sample of food or cosmetic
is deposited continuously on surface of rotating agar plate. Re-
sultant track on surface is in form of Archimedes spiral. Vol.
is decreased while dispensing stylus moves from center to edge
so that exponential relationship exists between vol. deposited
and radius of agar. On incubation, colonies develop along lines
where liq. was deposited. Counting grid is calibrated for sam-
ple vol. associated with different areas of agar. No. colonies
per known area is counted and calcd to bacterial concn.
B. Apparatus
Spiral plating machine. — For use with 150 X 15 mm (100
x 15 mm may be used) petri dishes and adjusted to deliver
total vol. of 0.035 mL/plate. Platform carrying plate is rotated
at ca 50 rpm and is connected mech. to lead screw driving
hollow syringe dispenser. Backflow syringe, 2- way valve, and
vac. trap control loading and dispensing of sample, disposal
of residual sample, and rinsing of system. Liq. is dispensed
from backflow syringe thru thin wall Teflon tubing thru stylus
to surface of agar plate. (Available com. from Spiral Systems
Marketing, 4853 Cordell Ave, Suite A10, Bethesda, MD
20014.)
C. Plates
Pour 40-45 mL portions plate count agar, 940.36A(g), into
150 x 15 mm (100 x 15 mm may be used) petri dishes; let
harden and dry to smooth, even surface.
D. Calibration of Spiral Counters
To det. vol. associated with different parts of counting grid,
prep. 11 bacterial suspensions by dilg 1:1 from 10 6 to 10 3
cells/mL (use nonspreaders). Plate all dilns in duplicate by
both 966. 23D and spiral plater, using same medium and in-
cubator. Count spiral plates as in 977. 27G and divide by av.
count/mL by 966. 23C to calc. vol. of counted grid area.
mL in counted area
No. spiral colonies on area
count/mL by 966.23C
E. Preparation of Samples
Weigh 50 g sample into sterile blender jar, add 450 mL diln
H 2 0, 940.36A(a), and blend 2 min. If necessary, let settle few
min before removing portion of supernate for spiral plating.
(Presence of particles may clog tubing.)
Liqs may be used directly or after dilg 1 + 9 with diln H 2 0.
F. Operation
Check stylus tip angle by letting vac. hold microscope cover
slip against face of stylus tip at I mm above platform. Cover
slip should be parallel to rotating platform in all directions.
Adjust angle if necessary. Check stylus at start position.
Clean stylus tip before use and between plating each sample
by rinsing 1 sec with com. 5.25% NaOCl soln and then 1 sec
with sterile H 2 0. Identify 3 disposable polyethylene sample
cups and fill with com. 5.25% NaOCl soln, sterile H 2 0, and
sample. Turn vac. filling valve to "on" and move sample holder
into position under stylus tip. Lower stylus into NaOCl soln
and lift out twice. Repeat with H 2 0. Lower stylus into sample
soln. Draw soln thru stylus until continuous column of liq. is
present in tube above vac. filling valve. With tip of stylus still
below surface of sample, close vac. valve. Raise stylus and
move sample holder out of way.
Identify lid of agar plate and remove lid. Place dish on turn-
table, and lower stylus until tip rests freely on agar surface.
Start app. and let rotate until stylus is lifted and app. stops
automatically. Remove dish and replace cover. Incubate 48 ±
3 hr at 35 ± 1°.
After all samples have been plated, flush app. with NaOCl
soln and H 2 0. When not in use, leave filled with H 2 0.
G. Counting Spiral Plates
Transparent viewing grid consists of 13.2 cm circle divided
into 5 areas by 4 concentric circles equidistant along diam.
(marked 1 (furthest) and 4 (nearest) to center) and into eight
45° wedges or octants, marked A thru H. Thus, each octant
is subdivided by 4 arcs linearly equidistant from each other.
Outer ring of 2 opposite octants (e.g., A and E) is further
subdivided in half by arc in middle (marked l / 2 ), and outer
ring thus formed is divided in half by line toward center. Addnl
lines are provided for use with 10 cm plates.
After incubation, center plate over grid. Choose any octant
sector and count colonies from outer edge toward center until
20 colonies have been counted. Continue counting remaining
colonies contained in segment in which 20th colony was ob-
served. Record this count together with No. segment that in-
cluded 20th colony (i.e., l /%, 1, 2, 3, or 4). Count opposite
similar segment and add together. If 20 colonies are not con-
tained in an octant, count all colonies on plate and designate
as T (total). If total No. colonies counted exceeds 75 in com-
pleting count in segment contg 20th colony, count will gen-
erally be low because of coincidence error associated with
crowding of colony. In this case, count circumferentially ad-
jacent annular segments starting with sector 1 until ^50 col-
onies are counted, and complete count of remaining colonies
in segment in which 50th colony was observed.
Divide No. colonies counted (or sum of 2 sector counts) by
corresponding vol. sectors counted in mL to obtain bacterial
count/mL. Use as vol. that calcd for that sector(s) from cal-
ibration, 977. 27D, based on std plate count.
Refs.: JAOAC 60, 807(1977); 64, 408(1981).
986.32 Aerobic Plate Count in Foods
Hydrophobic Grid Membrane Filter Method
First Action 1986
Final Action 1987
A. Principle
Hydrophobic grid membrane filter (HGMF) uses membrane
filter imprinted with hydrophobic material in grid pattern. Hy-
drophobic lines act as barriers to spread of colonies, thereby
dividing membrane filter surface into sep. compartments of
equal and known size. Number of squares occupied by colo-
nies is enumerated and converted to most probable number
value of organisms by using formula given below.
B. Apparatus, Culture Media, and Reagents
(a) Hydrophobic grid membrane filter (HGMF). — Mem-
brane filter has pore size of 0.45 |xm and is imprinted with
432
Microbiological Methods
AOAC Official Methods of Analysis (1990)
nontoxic hydrophobic material in grid pattern. ISO-GRID
(available from QA Laboratories, Ltd, 135 The West Mall,
Toronto, Ontario, Canada M9C 1C2) or equiv. meets these
specifications.
(b) Filtration units for HGMF. — Equipped with 5 fxm mesh
prefilter to remove food particles during filtration. One unit is
required for each sample. ISO-GRID (available from QA Lab-
oratories, Ltd) or equiv. meets these specifications.
(c) Pipets. — 1.0 mL serological with 0.1 mL graduations,
1.1 or 2.2 mL milk pipets are satisfactory. 5.0 mL serological
with 0.1 mL graduations.
(d) Blender. — Waring Blendor, or equiv. multispeed model,
with low-speed operation at 10 000-12 000 rpm, and 250 mL
glass or metal blender jars with covers. One jar is required for
each sample.
(e) Vacuum pump.— H 2 aspirator vac. source is satisfac-
tory.
(f ) Manifold or vacuum flask.
(g) Peptone /Tween 80 (PT) diluent. — Dissolve 1.0 g pep-
tone (Difco 0118) and 10.0 g Tween 80 in 1 L H 2 0. Dispense
enough vol. into diln bottles to give 90 ± 1 mL or 99 ± 1
mL after autoclaving 15 min at 121°.
(h) Tryptic soy-fast green agar (TSFA). — 15.0 g tryptone,
5.0 g phytone (or soy tone), 5.0 g NaCl, 0.25 g fast green FCF
(CI No. 42053), and 15.0 g agar dild to 1 L with H 2 0. Heat
to boiling. Autoclave 15 min at 121°. Temper to 50-55°.
Aseptically adjust pH to 7.3 ±0.1. Dispense ca 18 mL por-
tions into 100 x 15 mm petri dishes. Surface-dry plated me-
dium before use.
(i) Tris buffer. — 1.0M. Dissolve i21.1 g tris(hydroxy-
methyl)amino methane in ca 500 mL H 2 0. Adjust soln to de-
sired pH with coned HC1 and dil. to 1 L with H 2 0, Store at
either room temp, or 4-6°.
(j) Acetate buffer. — 1.0M. Dissolve 60 mL glacial acetic
acid in ca 500 mL H 2 0. Adjust soln to desired pH with 5M
NaOH and dil. to 1 L with H 2 0. Store at 4-6°.
(k) Amylase stock soln. — Dil. 10 g a-amylase (Sigma
Chemical Co., No. A 127 8 or equiv.) to 100 mL with tris buffer,
pH 7.0. Warm to 35° if necessary to aid soln. Filter thru What-
man No. 1 paper (or equiv.) to remove insoluble material; then
filter- sterilize using 0.45 |xm membrane filter. Store up to 1
week at 4-6° or up to 3 months at —18°.
(1) Cellulase stock soln. — Dil. 10 g cellulase (Sigma No.
C0901 or equiv.) to 100 mL with acetate buffer, pH 5.0. Warm
to 35° if necessary to aid soln. Filter thru Whatman No. 1
paper (or equiv.) to remove insoluble material; then filter-ster-
ilize using 0.45 jxm membrane filter. Store up to 1 week at
4-6° or up to 3 months at -18°.
(m) Diastase stock soln. — Dil. 10 g diastase (Sigma No.
A6880 or equiv.) to 100 mL with tris buffer, pH 7.0. Warm
to 35° if necessary to aid soln. Filter thru Whatman No. 1
paper (or equiv.) to remove insoluble material; then filter- ster-
ilize using 0.45 jxm membrane filter. Store up to 1 week at
4-6° or up to 3 months at -18°.
(n) Hemicellulase stock soln. — Dil. 10 g hemicellulase
(Sigma No. H2125 or equiv.) to 100 mL with acetate buffer,
pH 5.5. Warm to 35° if necessary to aid soln. Filter thru What-
man No. 1 paper (or equiv.) to remove insoluble material; then
filter- sterilize using 0.45 |xm membrane filter. Store up to 1
week at 4-6° or up to 3 months at -18°.
(o) Trypsin stock soln. — Dil. 10 g trypsin (Difco No. 0153
or equiv.) to 100 mL with tris buffer, pH 7.6. Warm to 35°
if necessary to aid soln. Filter thru Whatman No. 1 paper (or
equiv.) to remove insoluble material; then filter- sterilize using
0.45 )jLm membrane filter. Store up to 1 week at 4-6° or up
to 3 months at -18°.
(p) Lecithinase (phospholipase A 2 ) stock soln. — Dil. com.
enzyme soln (Sigma No. P9139 or equiv.) to 25 units /mL
with tris buffer, pH 8.0. Filter-sterilize using 0.45 [xm mem-
brane filter. Store up to 1 week at 4-6° or up to 3 months at
-18°.
(q) Pectinase stock soln. — Use com. enzyme soln of pec-
tinase from Aspergillus niger, contg 3-6 units /mg protein,
dissolved in 40% glycerol (Sigma No. P5146 or equiv.). Fil-
ter-sterilize using 0.45 p,m membrane filter. Store up to 1 week
at 4-6° or up to 3 months at -18°.
(r) Protease stock soln. — Use com. enzyme soln of pro-
tease from Bacillus subtilis, containing 7-15 units/mg protein
(Biuret) in aq. soln (Sigma No. P8775 or equiv.) Filter-ster-
ilize using 0.45 fxm membrane filter. Store up to 1 week at
4-6° or up to 3 months at -18°.
C, Sample Preparation
(a) Liquid egg. — Thoroly mix sample with sterile spoon or
spatula and prep. 1 : 10 diln by aseptically weighing 11 g egg
material into sterile wide-mouth g-s or screw -cap bottle; add
99 mL PT diluent, (g), and 1 tablespoon of sterile glass shot.
Thoroly agitate 1:10 diln to ensure complete soln or distri-
bution of egg material in diluent by shaking each container
rapidly 25 times, each shake being up-and-down movement of
ca 30 cm, time interval not exceeding 7 s. Let bubbles escape.
Transfer representative portion from 1 : 10 diln for higher serial
dilns as needed. If enzyme treatment is needed (see Table
986.32), combine 5 mL of 1 : 10 diln with 1 mL enzyme stock
soln. Incubate 20-30 min at 35-37° in H 2 bath. Correct for
addnl diln factor by filtering 1.2 mL of enzyme-treated sam-
ple.
(b) Other liquid samples. — Mix contents of sample con-
tainer thoroly. To prep. 1: 10 diln, aseptically transfer 10 mL
sample into 90 mL PT diluent, (g). Mix by shaking bottle 25
times thru 30 cm arc in 7 s. Transfer representative portions
Table 986.32 Enzyme Treatments for Foods 8
Food
Enzyme
Skim milk
none
Raw milk
none
Fluid dairy products other than skim milk
trypsin
Ice cream: without stabilizers
trypsin
contg gums
hemicellulase
contg cellulose derivatives
cellulase
Spray-dried milks
trypsin
Cheeses
trypsin
Spray-dried cheese powders
cellulase or protease 5
Sour cream
diastase
Yoghurt
trypsin
Butter
none
Margarine
none
Egg: liq. or powder
trypsin
Raw beef, pork, poultry
trypsin
Cooked meat or poultry
trypsin
Flour
none
Rice
none
Chocolate
amylase
Breakfast cereals
cellulase
Cake mixes
amylase
Fruit puree (e.g., fig paste)
pectinase
Raw vegetables
none
Lecithin
lecithinase
Food colorings
none
Gums
hemicellulase
Citrus juices
pectinase
Infant formula
trypsin
Sodium caseinate
protease
Nut meats
none
Shrimp
none
Oysters
trypsin
a Based on analysis of 1 mL of 1 :10 diln. Foods tested at dilns of 1:100
or higher do not usually need enzyme treatment.
b Varies, depending on individual product.
AOAC Official Methods of Analysis (1990)
Aerobic Plate Count 433
from 1:10 diln for higher serial dilns as needed. If enzyme
treatment is needed (see Table 986.32), combine 5 mL of 1 : 10
diln with 1 mL enzyme stock soln. Incubate 20-30 min at 35-
37° in H 2 bath. Correct for addnl diln factor by filtering 1.2
mL of enzyme-treated sample.
(c) Whole egg powder. — Thoroly mix sample with sterile
spoon or spatula and prep. 1 : 10 diln by aseptically weighing
11 g egg material into sterile wide-mouth g-s or screw-cap
bottle; add 99 mL PT diluent, (g), and 1 tablespoon of sterile
glass shot. Thoroly agitate 1:10 diln to ensure complete soln
or distribution of egg material in diluent by shaking each con-
tainer rapidly 25 times, each shake being up-and-down move-
ment of ca 30 cm, time interval not exceeding 7 s. Let bubbles
escape. Transfer representative portion from 1 : 10 diln for higher
serial dilns as needed. If testing 1: 10 diln is necessary, prep.
.1 : 100 diln and combine 10 mL of 1 : 100 diln with 1 mL tryp-
sin stock soln, (o). Incubate 20-30 min at 35-37° in H 2 bath.
Filter entire 11 mL vol. to test 1 :10 diln.
(d) Other foods. — To prep. 1 : 10 diln, aseptically weigh 10
g sample into sterile blender jar. Add 90 mL PT diluent, (g),
and blend 2 min at low speed (10 000-12 000 rpm). Transfer
representative portion from 1:10 diln for higher serial dilns as
needed. If enzyme treatment is needed (see Table 986.32),
combine 5 mL of 1:10 diln with 1 mL enzyme stock soln.
Incubate 20-30 min at 35-37° in H 2 bath. Correct for addnl
diln factor by filtering 1.2 mL of enzyme-treated sample.
D. Analysis
Select appropriate diln for analysis, depending on desired
counting range. Ordinarily, 1 MOO diln is satisfactory, pro-
ducing counting range of 100/g or mL to 500 000/g or mL.
Use 1 : 10 diln if very low counts are expected.
(See Figs 986.32A and 986.32B). Turn on vac. source. Place
sterile filtration unit on manifold or vac. flask. Open clamp
A. Rotate back funnel portion C. Aseptically place sterile HGMF
on surface of base D. Rotate funnel forward. Clamp shut by
sliding jaws L of stainless steel clamp over entire length of
flanges B extending from both sides of funnel C and base D,
and rotating moving arm K into horizontal (locked) position.
Aseptically add ca 15-20 mL sterile H 2 to funnel. Pipet
required vol. of appropriate diln into funnel. Apply free end
of vac. tubing E to suction hole F to draw liq. thru prefilter
mesh G. Aseptically add addnl 10—15 mL sterile H 2 to fun-
/v
TO
VACUUM
SOURCE
FIG. 986.32B— Filtration unit clamp
nel and draw thru mesh as before. Close clamp A to direct
vac. to base of filtration unit and draw liq. thru HGMF.
Open clamp A. Rotate moving arm K of stainless steel clamp
into unlocked (ca 45° angle) position and slide jaws L off of
flanges B. Rotate back funnel C. Aseptically remove HGMF
and place on surface of pre-dried TSFA (h) plate. Avoid trap-
ping air bubbles between filter and agar.
(a) Raw milk, pasteurized milks and creams, and egg pow-
ders. — Incubate 48 ± 3 h at 32°. Colonies will be various
shades of green. Count all squares contg one or more colonies
(pos. squares) except that if a single colony has clearly spread
to adjacent squares, count it as one pos. square. Convert pos.
square count to MPN with the formula, MPN = [N log e (N/
(N — x))] , where N = total number of squares and x — number
of pos. squares. Multiply by reciprocal of diln factor and re-
port as MPN of total bacteria/g or mL.
(b) Liquid egg.— Incubate 3 days (72 ± 3 h) at 32°. Pro-
ceed as in (a).
(c) All other foods. — Incubate 48 ± 3 h at 35°. Proceed as
in (a).
Ref.: J AOAC 69, 671(1986).
988.18
Aerobic Plate Count
Pectin Gel Method
First Action 1988
FIG. 986.32A— Filtration unit
A. Principle
Method uses pretreated petri plates contg thin "hardener"
layer, and liq. medium contg nutrients with pectin as only gel-
ling agent, Liq. medium, 12-15 mL, is poured into pretreated
petri plate and undild or dild sample is added. Plate is rotated
and rocked to mix sample and medium. Plates are then allowed
to stand on level surface 30-40 min until medium solidifies.
Total process is done at ambient temp. Plates are then incu-
bated and counted.
B. Materials
Note: Before pectin base medium formulated from individ-
ual ingredients is used, comparability to commercially avail-
able medium must be demonstrated.
Pectin gel tubes and plates. — Pectin gel is available as ster-
ile liq. in individual tubes contg sufficient gel to pour 1 plate.
Use tubes of Redigel and pretreated petri plates (RCR Scien-
tific, Inc., 206 W Lincoln Ave, Goshen, IN 46526), or equiv.
that meets specifications.
To prep, plate count pectin gel from individual ingredients,
suspend 5.0 g pancreatic digest of casein, 2.5 g yeast ext, and
1.0 g glucose, in 500 mL H 2 0. Suspend 15 g low methoxyl
pectin in 500 mL H 2 0. Heat individual mixts until all ingre-
434
Microbiological Methods
AOAC Official Methods of Analysis (1990)
dients are dissolved. Autoclave solns 15 min at 121°. Combine
nutrient and pectin solns and adjust pH to 7.0 ± 0.1. To prep.
pretreated petri plates, prep, hardener layer mixt. of 1% agar
with 0.02 CaCl 2 concn. Sterilize mixt. by autoclaving 15 min
at 121°. Aseptically dispense 5 mL portions of mixt. into ster-
ile petri plates.
C. Preparation of Samples
Prep, all decimal dilns with 90 mL sterile diluent (Butter-
field's phosphate buffer) plus 10 mL of previous diln unless
otherwise specified. Shake all dilns 25 times in 30 cm arc.
Pipets must accurately deliver required vol. Do not use to de-
liver <10% of their total vol. For example, to deliver 1 mL,
do not use pipet >10 mL; to deliver 0.1 mL, do not use pipet
>1 mL.
(a) Dairy products. — Measure (or weigh) 11 mL (or g)
sample and dil. in 99 mL Butterfield's diluent. For solid sam-
ples, blend 2 min at 10 000 to 12 000 rpm. Prep, addnl dilns
so that total colonies /plate is in 25-250 range. Incubate plates
48 ± 3 h at 32 ± 1°.
(b) Nondairy products. — Weigh 50 g sample into 450 mL
Butterfield's diluent and blend 2 min at 10 000 to 12 000 rpm.
Prep, further dilns by dispensing 10 mL sample into 90 mL
diluent so that total colonies/plate is in 30-300 range. Incu-
bate plates 48 ± 2 h at 35 ± 1°.
D. Determination
(1) Lift lid of pretreated petri plate and pour liq. pectin gel
from 1 tube (12-15 mL) into plate. Replace lid and swirl plate
to cover bottom with pectin gel. Prep, number of plates needed
for samples being run (duplicate plates for each diln). Plates
must be used within 5 min after liq . pectin gel is poured.
(2) Add 1 mL inoculum (sample) to liq. pectin gel in petri
plate. Touch pipet tip once to dry spot on inside wall of plate
(above level of liq. pectin gel) after dispensing sample to rest
point in pipet tip. Immediately rotate and rock plate to mix
sample thoroly with pectin gel. Do not spill pectin gel over
sides of plate. (Note: This step is primary difference in pro-
cedure between pectin gel and agar-based media. Do not add
inoculum (sample) to pretreated petri plate and pour pectin gel
over it. This would lock sample in one small area of plate
without sepn of individual colonies.)
(3) Let inoculated plates stand on level surface until pectin
gel is solid (ca 30-40 min), and then incubate 48 ± 2 h at 35
±1° for nondairy products and 48 ± 3 h at 32 ± 1° for dairy
products,
(4) Count duplicate plates in suitable range (30-300 colo-
nies for nondairy products, 25-250 colonies for dairy prod-
ucts). If plates do not contain proper range of colonies, record
diln counted and note number of colonies found. Average counts
obtained and report as aerobic plate count/g or mL.
Ref.: JAOAC 71, 343(1988).
COLIFORMS
989.11 Coliforms in Dairy Products
Pectin Gel Method
First Action 1989
A. Principle
Method uses pretreated plates contg thin "hardener" layer
and liq. medium contg nutrients with pectin as sole gelling
agent. Liq. medium (10-12 mL) is poured into pretreated plate
and undild or dild sample is added. Plate is rotated and rocked
to mix sample and medium. Plates are then allowed to rest on
level surface until medium solidifies. Then, 3-4 mL liq. me-
dium is poured as overlay and allowed to solidify. Total pro-
cess is done at ambient temp. Plates are then incubated and
counted as for agar-based prepns.
B. Materials
Note: Pectin base medium may be formulated from individ-
ual ingredients; suitability for analysis must be demonstrated.
Pectin gel and plates. — Violet red bile (VRB) pectin gel is
available as sterile liq. in individual units contg sufficient gel
to pour 1 plate or in units to pour 8 plates. VRB Redigel and
pretreated plates (RCR Scientific, Inc., 206 W Lincoln Ave,
Goshen, IN 46526), or equiv., meet specifications of method.
To prep. VRB pectin gel from individual ingredients, sus-
pend 7.0 g pancreatic digest of gelatin, 3.0 g yeast ext, 10.0
g lactose, 1.5 g bile salts No. 3, 5.0 g NaCl, 0.03 g neutral
red, and 0.002 g crystal violet in 500 mL H 2 0. Suspend 15 g
low methoxyl pectin in 500 mL H 2 0. Heat individual mixts
until all ingredients are dissolved. Autoclave solns 15 min at
121°. Combine nutrient and pectin solns and adjust pH to 7.4
± 0.2. To prep, pretreated petri plates, prep, hardener layer
mixt. of 1% agar with 0.02 CaCl 2 concn. Sterilize mixt. by
autoclaving 15 min at 121°. Aseptically dispense 5 mL por-
tions of mixt. into sterile petri plates.
C. Preparation of Samples
To prep, dilns, measure (or weigh) 11 mL (or g) sample and
dil. in 99 mL Butterfield's or 2% Na citrate diluent. For solid
samples, blend 2 min at 10 000 to 12 000 rpm. Prep, addnl
dilns so that total colonies/plate is in 25-250 range. Incubate
plates 48 ± 3 h at 32 ± 1°. Shake all dilns 25 times in 30 cm
arc. Pipets must accurately deliver required vol. Do not use to
deliver <10% of their total vol. For example, to deliver 1 mL,
do not use pipet > 10 mL; to deliver 0. 1 mL, do not use pipet
>1 mL.
D. Determination
(1) Lift lid of pretreated petri plate and pour ca 75% (10-
12 mL) of liq. medium from tube into plate. (Note: Remove
cap from each tube of liq. pectin gel medium as it is needed
to pour plate.) Prep, number of plates, in duplicate, needed
for samples being run. Replace lid and swirl plate to cover
bottom with liq. medium. Plates must be used within 5 min.
(2 ) Add inoculum (sample) to liq. pectin gel in petri plate.
Touch pipet tip once to dry spot on inside wall of plate (above
level of liq. medium) after dispensing sample to rest point in
pipet tip. Immediately rotate and rock plate to mix sample tho-
roly with pectin gel. Do not spill mixt. over sides of plate.
(Note: This step is primary difference in procedure between
pectin gel and agar-based media. Do not add inoculum (sam-
ple) to pretreated petri plate and pour pectin gel over it. This
would lock sample in one small area of plate without sepn of
individual colonies.)
(3) Let inoculated plates stand on level surface until pectin
gel is solid, and then pour remaining medium (3-4 mL) from
tube as overlay and let gel solidify. Incubate in same manner
as for agar-based plates (24 ± 2 h at 32 ± 1°).
(4) After 24 h incubation, count all red or pink colonies.
Report as coliforms /mL or g.
(5) Pick 5 colonies of each type present on each plate and
transfer to brilliant green lactose bile broth fermentation tubes,
966.23 A(c). Incubate 48 ± 3 h at 32 ± 1° and check for gas
production, which is considered pos. for coliforms.
(6) If any picks from step 5 are neg. for gas production,
adjust counts (step 4) accordingly.
Ref.: JAOAC 72, 298(1989).
AOAC Official Methods of Analysis (1 990)
Coliforms
435
986.33 Bacterial and Coliform Counts
in Milk
Dry Rehydratable Film Methods
First Action 1986
Final Action 1988
A. Principle
Method uses bacterial culture plates of dry medium and cold
H 2 0-sol. gel. Undild or dild samples are added directly to plates
at rate of 1.0 mL per plate. Pressure, when applied to plastic
spreader placed on overlay film, spreads sample over ca 20
sq. cm growth area. Gelling agent is allowed to solidify and
plates are incubated and then counted. Either pipet or plate
loop continuous pipetting syringe can be used for sample addn
for bacterial count analyses.
B. Apparatus
(a) Std method plates . — Plates contain std methods media
nutrients, 940.36A(g), cold H 2 0-sol. gelling agent coated onto
film base, overlay film coated with gelling agent, and 2,3,5-
triphenyltetrazolium chloride indicator. Circular growth area
of single plate contains ca twenty 1 cm squares outlined on
film base. Petri film Aerobic Count Plates® (available from
Medical-Surgical Division/3M, 225-5S 3M Center, St. Paul,
MN 55144) or equiv. meets these specifications.
(b) Violet red bile plates. — Plates contain violet red bile
nutrients conforming to APHA standards as given in Com-
pendium of Methods for the Microbiological Examination of
Foods, 2nd ed., 1984 (American Public Health Association,
1015 15th St, NW, Washington, DC 20005), cold H 2 sol.
gelling agent, and 2,3,5-triphenyltetrazolium chloride. Petri -
film VRB Plates® (available from Medical-Surgical Division/
3M), or equiv. meets these specifications.
(c) Plastic spreader. — Provided with Petrifilm plates, con-
sists of concave side and smooth flat side, designed to spread
milk sample evenly over plate growth area.
(d) Pipets. — Calibrated for bacteriological use of plate loop
continuous pipetting syringe to deliver 1.0 mL.
(e) Colony counter. — Std app., Quebec model preferred, or
one providing equiv. magnification and visibility.
C. Analysis
(a) Bacterial colony count. — Use Petrifilm Aerobic Count
Plates or equiv. plates. Place plate on flat surface. Lift top film
and inoculate 1 mL sample onto center of film base. Carefully
roll top film down onto inoculum. Distribute sample over pre-
scribed growth area with downward pressure on center of plas-
tic spreader device (recessed side down). Leave plate undis-
turbed 1 min to permit gel to solidify. Incubate plates 48 ± 3
h at 32° ± 1°.
In incubator, place plates in horizontal position, clear side
up, in stacks not exceeding 10 units. Count plates promptly
after incubation period. If impossible to count at once, store
plates after required incubation at 0-4.4° for not >24 h. This
should be avoided as a routine practice.
Use std colony counter for counting purposes. Magnifier-
illuminator may also be used to facilitate counting. Colonies
stain in various shades of red. Count all colonies in countable
range (30-300 colonies).
To compute bacterial count, multiply total number of col-
onies per plate (or av. number of colonies per plate if counting
duplicate plates of same diln) by reciprocal of diln used. When
counting colonies on duplicate plates of consecutive dilns,
compute mean number of colonies for each diln before detg
av. bacterial count. Estd counts can be made on plates with
>300 colonies and should be reported as estd counts. In mak-
ing such counts, circular growth area can be considered to con-
tain ca twenty 1 cm squares. To isolate colonies for further
identification, lift top film and pick colony from gel.
(b) Coliform count. — Use Petrifilm Coliform Count Plates
or equiv. plates. Proceed as in (a), but distribute sample over
plate by using plastic spreader, flat side down. Incubate plates
24 ± 2 h at 32° ± 1°. Count as in (a), but count only red
colonies that have one or more gas bubbles associated (within
1 colony diam.) with them. Count all colonies in countable
range (15-150 colonies). Red colonies without gas bubbles are
not counted as coliform organisms.
Ref.: JAOAC 69, 527(1986).
989.10 Bacterial and Coliform
Counts in Dairy Products
Dry Rehydratable Film Methods
First Action 1989
Method Performance:
AEROBIC COUNT
Chocolate milk:
s r - 0.102; s R = 0.177; RSD r = 4.3%; RSD R - 7.5%
Cheese:
s r = 0.113; s R - 0.117; RSD r = 3.6%; RSD R - 3.7%
Nonfat dry milk:
s r = 0.151; s R = 0.230; RSD r - 4.5%; RSD R = 6.9%
Evaporated milk:
s r = 0.193; s R = 0.198; RSD r - 8.3%; RSD R = 8.5%
Ice cream:
s r - 0.180; s R - 0.222; RSD r = 6.9%; RSD R - 8.5%
COLIFORM COUNT
Chocolate milk:
s r = 0.164; s R - 0.257; RSD r = 9.2%; RSD R = 14.4%
Cheese:
s r = 0.221; s R - 0.225; RSD r = 10.4%; RSD R = 10.6%
Nonfat dry milk:
s r = 0.197; s R = 0.151; RSD r = 8.5%; RSD R = 4.5%
Evaporated milk:
s r = 0.200; s R - 0.225; RSD r = 13.0%; RSD R = 13.0%
Ice cream:
s r - 0.081; s R = 0.131; RSD r = 4.1%; RSD R - 6.6%
A. Principle
Method uses bacterial culture plates of dry medium and cold
H 2 0-sol. gel. Undild or dild samples are added to plates at
rate of 1.0 mL per plate. Pressure, when applied to plastic
spreader placed on overlay film, spreads sample over ca 20
sq. cm growth area. Gelling agent is allowed to solidify and
plates are incubated and then counted. Pipet, plate loop con-
tinuous pipetting syringe, or automatic pipet can be used for
sample addn for bacterial count analyses.
B. Apparatus and Reagent
(a) Aerobic count plates. — Plates contain std methods me-
dia nutrients, 940.36A(g), cold H 2 0-sol. gelling agent coated
onto film base, overlay film coated with gelling agent, and
2,3,5-triphenyltetrazolium chloride indicator. Circular growth
area of single plate contains ca twenty 1 cm squares outlined
on film base. Petrifilm Aerobic Count Plates'® (available from
Medical-Surgical Division/3M, 225-5S 3M Center, St. Paul,
MN 55144) meet these specifications.
(b) Coliform count plates. — Plates contain violet red bile
436
Microbiological Methods
AOAC Official Methods of Analysis (1990)
nutrients conforming to APHA standards as given in Com-
pendium of Methods for the Microbiological Examination of
Foods, 2nd ed., 1984 (American Public Health Association,
1015 18th St, NW, Washington, DC 20005), cold H 2 0-sol.
gelling agent, and 2,3,5-triphenyltetrazolium chloride indica-
tor. Petrifilm Coliform Count Plates® (available from Medi-
cal-Surgical Division/3M) meet these specifications.
(c) Plastic spreader. — Provided with Petrifilm plates, con-
sists of recessed side and smooth flat side, designed to spread
sample evenly over plate growth area.
(d) Pipets. — Calibrated for bacteriological use, or plate loop
continuous pipetting syringe to deliver 1 .0 mL. Automatic pi-
pet to deliver 1.0 mL may be used.
(e) Colony counter. — Std app., Quebec model preferred, or
one providing equiv. magnification and visibility.
(f) Dilution water. —See 940.36A(a).
C. Sample Preparation
(a) For total plate counts: Aseptically prep. 1:10 diln (11 g/
99 mL diln H 2 0). Mix well and plate. Prep, addnl dilns as
required. Ordinarily, 1:10 and 1:100 dilns are sufficient.
(b) For coliform counts:
(1) Cream, half-and-half, condensed mild, egg nog, cottage
cheese, butter, margarine, and related products . — Make 1:5
diln (24.75 g/99 mL diln H 2 0). Mix well and plate 1 mL on
each of 2 plates. Multiply total of counts on 2 plates by 2.5
to obtain count/g.
(2) Sour cream, dips, and yogurt. — Proceed as in (7) except
after diln, adjust pH to 6.6-7.2 with 1.0N NaOH (ca 0.1 mL/
g sample).
(3) Buttermilk.— Make 1:10 diln (11 g/99 mL diln H 2 0).
Adjust p'H to 6.6-7.2 with 1.0N NaOH (ca 0.1 mL/g sam-
ples). Mix well and plate 1 mL on each of 2 plates. Multiply
total of counts on 2 plates by 5 to obtain count/g.
(4) Ice cream, sherbet, and mixes. — Hydrate dry-film plates
with 1 mL sterile diln H 2 and allow at least 1 h for gel to
solidify. Then, lift top film of prehydrated dry-film coliform
count plate (gel will adhere to top film) and dispense 0.5 mL
of 2:3 homogenate (10 g/5 mL diln H 2 0) onto bottom film of
each of 3 plates. Replace top film gently over sample. Add
counts on the 3 plates to obtain count/g. Alternatively, plate
1 plate and multiply result by 3 to obtain count/g.
(5) Cheese. — Proceed as in (7). Do not use citrate buffer to
homogenize sample.
(6) Chocolate milk. — Proceed as in (7).
D. Analysis
(a) Bacterial colony count. — Use dry-film aerobic count
plates. Place plate on flat surface. Lift top film and inoculate
1 mL sample onto center of film base. Carefully roll top film
down onto inoculum. Distribute sample over prescribed growth
area with downward pressure on center of plastic spreader de-
vice (recessed side down). Leave plate undisturbed 1 min to
permit gel to solidify. Incubate plates 48 ± 3 h at 32 ± 1°.
In incubator, place plates in horizontal position, clear side
up, in stacks not exceeding 20 units. Count plates promptly
after incubation period. If impossible to count at once after
required incubation, store plates at 0-4.4° for not >24 h. This
should be avoided as a routine practice.
Use std colony counter for counting purposes. Magnifier-
illuminator may also be used to facilitate counting. Colonies
stain in various shades of red. Count all colonies in countable
range (25-250 colonies).
To compute bacterial count, multiply total number of col-
onies per plate (or av. number of colonies per plate if counting
duplicate plates of same diln) by reciprocal of diln used. When
counting colonies on duplicate plates of consecutive dilns,
compute mean number of colonies for each diln before detg
av. bacterial count. Estd counts can be made on plates with
>250 colonies and should be reported as estd counts. In mak-
ing such counts, circular growth area can be considered to con-
tain ca twenty 1 cm squares. To isolate colonies for further
identification, lift top film and pick colony from gel.
(b) Coliform count. — Use dry -film coliform count plates.
Proceed as in (a), but distribute prepd sample over plate by
using plastic spreader, flat side down. Incubate plates 24 ± 2
h at 32 ± 1°. Count as in (a), but count only red colonies that
have one or more gas bubbles associated (within 1 colony diam.)
with them. Count all colonies in countable range (15-150 col-
onies). Red colonies without gas bubbles are not counted as
coliform organisms.
Ref.: J AOAC 72, 312(1989).
978.23 Fecal Coliforms
in Shellfish Growing Waters
Medium A-1 Method
First Action 1978
Final Action 1979
(Applicable to enumeration of feca] coliforms and also as pre-
sumptive test for Escherichia coli in shellfish growing waters)
A. Apparatus
(a) Pipets. — 1.0 mL serological with 1 mL graduations
and 10.0 mL with 0.1 mL graduations. Pipets conforming to
APHA stds as given in "Standard Methods for the Examina-
tion of Dairy Products," 15th ed., 1985, American Public Health
Association, 1015 15th St, NW, Washington, DC 20005, may
also be used.
(b) Incubator. — Air, 35 ± 0.5°.
(c) Water bath. — Covered, circulating, 44.5 ± 0,2°.
(d) Dilution bottles or tubes. — Borosilicate glass, with glass
or rubber stoppers or polyethylene screw caps equipped with
Teflon liners.
B. Media
Note: Because geographical differences may affect perfor-
mance of Medium A-1 method, det. comparability with LST-
EC tube method prior to using Medium A-1. Moreover, this
medium must be made from individual ingredients. Prefor-
mulated Medium A-1 is unacceptable.
(a) Butterfield's buffered phosphate diluent. — See
966.23A(m).
(b) Medium A- 1 broth. — Dissolve 5 g lactose, 20 g tryp-
tone, 5 g NaCl, and 0.5 g salicin in 1 L H 2 0. Heat to dissolve
ingredients, pipet in 1 mL Triton X-100 (Rohm & Haas Co.),
and adjust pH to 6.9 ±0.1. For 10 mL sample aliquots, prep,
and use double strength medium. To achieve approx. same
level of medium and inoculum in all tubes, dispense 10 mL
portions of single strength broth into 150 X 18 mm tubes contg
inverted fermentation vials; use 175 x 22 mm tubes contg in-
verted fermentation vials for double strength broth. Autoclave
10 min at 121°. Formation of flocculent ppt, particularly in
double strength medium, is common and does not impair per-
formance. Store in dark at room temp, and use within 7 days.
Store dehydrated ingredients and /or medium under conditions
that will prevent absorption of moisture.
C. Determination
Shake sample and each successive diln bottle vigorously us-
ing 25 complete up and down movements of ca 30 cm in 7
sec. Inoculate H 2 sample directly into tubes contg A-1 Me-
dium in suitable decimal dilns using 3 or 5 tubes/diln with
AOAC Official Methods of Analysis (1990)
Butterfield's buffered phosphate diluent. Place inoculated tubes
into air incubator and incubate 3 hr at 35 ± 0.5°. Transfer
tubes to H 2 bath and incubate 21 ± 2 hr at 44.5 ± 0.2°.
Maintain H 2 level in bath above level of liq. in inoculated
tubes. Presence of gas in inverted vial or of dissolved gas which
can be removed by slight agitation of tube constitutes pos. test.
Use std Most Probable Number (MPN) tables, Table 966.24
or Table 978.23, to det. MPN values. Report results as fecal
coliform MPN/ 100 ml sample.
Ref.: JAOAC61, 1317(1978).
983.25 Total Conforms, Fecal Coliforms,
and Escherichia coli in Foods
Hydrophobic Grid Membrane Filter Method
First Action 1983
Final Action 1985
A. Principle
Hydrophobic grid membrane filter (HGMF) uses membrane
filter imprinted with hydrophobic material in grid pattern. Hy-
drophobic lines act as barriers to spread of colonies, thereby
dividing membrane filter surface into sep. compartments of
equal and known size. Number of squares occupied by colo-
nies is enumerated and converted to most probable number
value of organisms by using formula given below.
Coliforms
437
B. Apparatus, Culture Media and Reagents
(a) Hydrophobic grid membrane filter (HGMF). — Mem-
brane filter has pore size of 0.45 (im and is imprinted with
nontoxic hydrophobic material in grid pattern. ISO-GRID
(available from QA Laboratories Ltd, 135 The West Mall, To-
ronto, Ontario, Canada M9C 1C2) or equiv. meets these spec-
ifications.
(b) Filtration units for HGMF. — Equipped with 5 |xm mesh
prefilter to remove food particles during filtration. One unit is
required for each sample. ISO -GRID (available from QA Lab-
oratories Ltd.) or equiv. meets these specifications.
(c) Pipets. — 1.0 mL serological with 0.1 mL graduations;
LI mL or 2.2 mL milk pipets are satisfactory. 5.0 mL ser-
ological with 0.1 mL graduations.
(d) Blender. — Waring Blendor, or equiv., multispeed model,
with low-speed operation at 10 000-12 000 rpm, and 250 mL
glass or metal blender jars with covers. One jar is required for
each sample.
(e) Vac. pump. — Water aspirator vac. source is satisfac-
tory.
(f ) Manifold or vac. flask.
(g) Filter paper. — Whatman No. I or No. 4, or equiv.
(h) Peptone /Tween 80 diluent. — Dissolve 1.0 g peptone
(Difco 01 18) and 10.0 g Tween 80 in 1 L H 2 0. Dispense enough
vol. into diln bottles to give 90 ± 1 mL after autoclaving 15
min at 121°.
Table 978.23 Most Probable Numbers per 100 mL of Sample, Planting 5 Portions in Each of 3 Dilutions in Geometric Series
Number of
Number of
Number of
Number of
Number of
Number of
Positive
Positive
Positive
Positive
Positive
Positive
Tubes
Tubes
Tubes
Tubes
Tubes
Tubes
10
1
0.1
10 1
0.1
10
1
0.1
10
1
0.1
10
1
0.1
10
1
0.1
mL
mL
mL
MPN mL mL
mL
MPN
mL
mL
mL
MPN
mL
mL
mL
MPN
mL
mL
mL
MPN
mL
mL
mL
MPN
2.0
2
4.5
3
7.8
4
13
5
23
1
1.8
1
4.0
2
1
6.8
3
1
11
4
1
17
5
1
31
o
2
3.6
2
6.0
2
2
9.1
3
2
13
4
2
21
5
2
43
o
3
5.4
3
8.0
2
3
12
3
3
16
4
3
25
5
3
58
o
4
7.2
1
4
10
2
4
14
3
4
20
4
4
30
5
4
76
5
9.0
5
12
2
5
16
3
5
23
4
5
36
5
5
95
o
1.8
4.0
2
6.8
3
11
4
17
5
33
1
3.6
1
6.1
2
1
9.2
3
1
14
4
1
21
5
1
46
o
2
5.5
2
8.1
2
2
12
3
2
17
4
2
26
5
2
64
3
7.3
3
10
2
3
14
3
3
20
4
3
31
5
3
84
o
4
9.1
4
12
2
4
17
3
4
23
4
4
36
5
4
110
5
11
5
14
2
5
19
3
5
27
4
5
42
5
5
130
o
2
3.7
1 2
6.1
2
2
9.3
3
2
14
4
2
22
5
2
49
o
2
1
5.5
2
1
8.2
2
2
1
12
3
2
1
17
4
2
1
26
5
2
1
70
o
2
2
7.4
1 2
2
10
2
2
2
14
3
2
2
20
4
2
2
32
5
2
2
95
o
2
3
9.2
1 2
3
12
2
2
3
17
3
2
3
24
4
2
3
38
5
2
3
120
o
2
4
11
1 2
4
15
2
2
4
19
3
2
4
27
4
2
4
44
5
2
4
150
o
2
5
13
1 2
5
17
2
2
5
22
3
2
5
31
4
2
5
50
5
2
5
180
o
3
5.6
1 3
8.3
2
3
12
3
3
17
4
3
27
5
3
79
o
3
1
7.4
3
1
10
2
3
1
14
3
3
1
21
4
3
1
33
5
3
1
110
o
3
2
9.3
1 3
2
13
2
3
2
17
3
3
2
24
4
3
2
39
5
3
2
140
o
3
3
11
1 3
3
15
2
3
3
20
3
3
3
28
4
3
3
45
5
3
3
180
o
3
4
13
1 3
4
17
2
3
4
22
3
3
4
31
4
3
4
52
5
3
4
210
3
5
15
1 3
5
19
2
3
5
25
3
3
5
35
4
3
5
59
5
3
5
250
4
7.5
1 4
11
2
4
15
3
4
21
4
4
34
5
4
130
4
1
9.4
1 4
1
13
2
4
1
17
3
4
1
24
4
4
1
40
5
4
1
170
o
4
2
11
1 4
2
15
2
4
2
20
3
4
2
28
4
4
2
47
5
4
2
220
o
4
3
13
1 4
3
17
2
4
3
23
3
4
3
32
4
4
3
54
5
4
3
280
o
4
4
15
1 4
4
19
2
4
4
25
3
4
4
36
4
4
4
62
5
4
4
350
4
5
17
1 4
5
22
2
4
5
28
3
4
5
40
4
4
5
69
5
4
5
430
o
5
9.4
1 5
13
2
5
17
3
5
25
4
5
41
5
5
240
5
1
11
5
1
15
2
5
1
20
3
5
1
29
4
5
1
48
5
5
1
350
o
5
2
13
1 5
2
17
2
5
2
23
3
5
2
32
4
5
2
56
5
5
2
540
o
5
3
15
1 5
3
19
2
5
3
26
3
5
3
37
4
5
3
64
5
5
3
920
5
4
17
1 5
4
22
2
5
4
29
3
5
4
41
4
5
4
72
5
5
4
1,600
5
5
19
5
5
24
2
5
5
32
3
5
5
45
4
5
5
81
438
Microbiological Methods
AOAC Official Methods of Analysis (1 990)
(i) M-FC agar. — 10.0 g tryptose, 5.0 g proteose peptone
No. 3, 3.0 g yeast ext, 5.0 g NaCl, 12.5 g lactose, 1.5 g bile
salts No. 3, 0.1 g aniline blue, and 15.0 g agar dild to 1 L
with H 2 (mFC Agar, Difco 0677, is satisfactory). Heat to
boiling. Temper to 50-55°. Adjust pH to 7.4 ±0.1. Dispense
ca 18 mL portions into 100 X 15 mm petri dishes. Surface-
dry plated medium before use.
(J) Tryptone bile agar (TBA). — 20.0 g tryptone, 1.5 g bile
salts No. 3, and 15.0 g agar dild to 1 L with H 2 (Tryptone
bile agar (Oxoid CM 595) is satisfactory). Heat to boiling.
Autoclave 15 min at 121°. Temper to 50-55°. Adjust pH to
7.2 ±0.1. Dispense ca 18 mL portions into 100 x 15 mm
petri dishes. Surface-dry plated medium before use.
(k) Tryptic soy-magnesium sulfate agar (TSAM). — 15.0 g
tryptone, 5.0 g phytone (or soytone), 5.0 g NaCl, 1.5 g
MgS0 4 .7H 2 0, and 15.0 g agar dild to 1 L with H 2 0. Heat to
boiling. Autoclave 15 min at 121°. Temper to 50-55°. Adjust
pH to 7.3 ±0.1. Dispense ca 18 mL portions into 100 x 15
mm petri dishes. Surface-dry plated medium before use.
(1) Indole reagent. — (I) Soln A: Dissolve 2.5 g p-dimeth-
yl ami no benzaldehyde and 10 mL HO in 90 mL alcohol. (2)
Soln B: Dissolve 2.0 g potassium persulfate in 200 mL H 2 0.
Mix equal vols of Soln A and Soln B just before use.
(m) Tris buffer. — l.QM, pH 7.6. Dissolve 121.1 g
tris(hydroxymethylamino)methane and dil. to 1 L with H 2 0.
Adjust pH to 7.6 with IN HC1.
(n) Trypsin stock soln. — Dil. 10 g trypsin to 100 mL with
tris buffer. Warm to 35° if necessary to aid soln. Filter thru
Whatman No. 1 paper (or equiv.) to remove insoluble mate-
rial, then filter-sterilize using 0.45 jxm membrane filter.
C. Sample Preparation
(a) Nut meat pieces. — Aseptically weigh 50 g sample into
sterile jar. Add 50 mL peptone/Tween 80 diluent (h) and shake
vigorously (50 times thru 30 cm arc). Let stand 3-5 min and
shake just before doing filtrations.
(b) Cheese. — Aseptically weigh 10 g sample into sterile
blender jar. Add 90 mL diluent (h) and blend 2 min at low
speed (10 000-12 000 rpm). Aseptically combine, in 16 X
150 mm tube, 3.5 mL of this 1:10 homogenate and 3.5 mL
trypsin soln (n). Incubate 20-30 min at 35 ± 0.5° in H 2 bath.
Vortex to remix suspension just before doing filtrations.
(c) Other foods needing digestion. — Aseptically weigh 10
g sample into sterile blender jar. Add 90 mL diluent (h) and
blend 2 min at low speed (10 000-12 000 rpm). Aseptically
combine in 1.6 x 150 mm tube 5.0 mL of this 1:10 homog-
enate and 1.0 mL trypsin soln (n). Incubate 20-30 min at 35
± 0.5° in H 2 bath. Vortex to remix suspension just before
doing filtrations.
(d) Other foods . — Aseptically weigh 10 g sample into ster-
ile blender jar. Add 90 mL diluent (h) and blend 2 min at low
speed (10 000-12 000 rpm).
D. Analysis
(See Figs. 986.32A and 986.32B). Turn on vac. source. Place
sterile filtration unit on manifold or vac. flask. Open clamp
A. Rotate back funnel portion C. Aseptically place sterile HGMF
on surface of base D. Rotate funnel forward. Clamp shut by
sliding jaws L of stainless steel clamp over entire length of
flanges B extending from both sides of funnel C and base D,
and rotating moving arm K into horizontal (locked) position.
Aseptically add ca 15-20 mL sterile H 2 to funnel. Pipet
required volume (see below) of sample suspension into funnel.
Apply free end of vac. tubing E to suction hole F to draw liq.
thru prefilter mesh G. Aseptically add addnl 10-15 mL H 2 6
to funnel and draw thru mesh as before. Close clamp A to
direct vac. to base of filtration unit and draw liq. thru HGMF.
Food
Filtering
Diln
Filtering Multiplication
Vol., mL Factor
Nut meat pieces 10° 0,5
Cheese 10~ 1 2.0 a
Other foods needing digestion 10" 1 1.2 a
Other foods 10" 1 1.0
2
10
10
10
a Filtering vol. of enzyme-digested suspension.
Open clamp A. Rotate moving arm K of stainless steel clamp
into unlocked (ca 45° angle) position and slide jaws L off of
flanges B. Rotate back funnel C. Aseptically remove HGMF
and place on surface of pre-dried agar plate (see below). Avoid
trapping air bubbles between filter and agar.
(a) Total coliform count. — Place HGMF on surface of pre-
dried M-FC agar (i). Incubate 24 ± 2 h at 35°. Count all squares
contg one or more blue colonies. Include any shade of blue.
Score each square as either pos. (blue) or neg. Convert pos.
square count to MPN with the formula
MPN = [N log e (N/(N - jc))]
where N = total number of squares and x - number of pos.
squares. Multiply by reciprocal of diln factor and report as
MPN of total coliform bacteria/g.
(b) Fecal coliform count. — Place HGMF on surface of pre-
dried TSAM (k). Incubate 4-5 h at 25° for dry foods and 4-
5 h at 35° for all other foods. Transfer HGMF to surface of
pre-dried M-FC agar (i) and incubate 24 ± 2 h at 44.5 ± 0.5°
in closed container. Proceed as in (a), and report as MPN of
fecal coliform bacteria/g.
(c) E. coli count. — Place HGMF on surface of pre-dried
TSAM (k). Incubate 4-5 h at 25° for dry foods and 4-5 h at
35° for all other foods. Transfer HGMF to surface of pre-dried
TBA (j) and incubate 24 ± 2 h at 44.5° ± 0.5° in closed
container. Prepare indole reagent (1) by combining equal vol.
of Soln A and Soln B. Place 9 cm filter paper disk in petri
dish lid and flood with 1-2 mL indole reagent (1). Transfer
HGMF to filter paper, ensuring that no air bubbles are trapped
between HGMF and paper. Let stand 10-15 min, then transfer
HGMF back to surface of TBA. Count all squares contg one
or more pink (indole pos.) colonies. Score each square as either
pos. (pink) or neg. Convert pos. square count to MPN with
formula above. Multiply by reciprocal of diln factor and report
as MPN of E. coli (biotype I)/g.
Refs.: JAOAC 66, 897(1983); 67, 812(1984).
ESCHERICHIA COLI
988.19 Escherichia coli in Chilled
or Frozen Foods
Fluorogenic Assay for Glucuronidase
First Action 1988
(Applicable only to chilled or frozen foods, except chilled or
frozen shellfish)
A. Principle
Lauryl sulfate tryptose broth with added 4-methyl-umbelli-
feryl-p-D-glucuronide (MUG) is used as medium in 3-tube MPN
method. Tubes are incubated 24 ± 2 h at 35°. Fluorescent-
pos. tubes are streaked onto eosin methylene blue agar (Le-
vine) plates, which are incubated 24 ± 2 h at 35°. Typical
colonies are picked and confirmed as E. coli.
B. Media and Reagents
See introductory par. to 966. 23A.
(a) Plate count agar. — See 940. 36 A (g).
(b) Eosin methylene blue agar (Levine).-See 940.36A(d).
AOAC Official Methods of Analysis (1990)
Escherichia Coli
439
(c) Tryptophane broth. — Dissolve by heating, with stirring,
10.0 g tryptone or trypticase in 1 L H 2 0. Dispense in 10 mL
portions into test tubes and autoclave 15 min at 121°. Final
pH, 6.9 ± 0.2.
(d) Buffered glucose broth (MR-VP medium). — For Me red-
Voges Proskauer (MR-VP) tests. Dissolve 7.0 g proteose pep-
tone, 5.0 g glucose, and 5.0 g K 2 HP0 4 in ca 800 mL H 2
with gentle heat and occasional stirring. Filter, cool to 20°,
and dil. to 1 L. Dispense 10 mL portions into test tubes and
autoclave 12-15 min at 3 21°. Max. exposure to heat should
be <30 min. Final pH, 6.9 ± 0.2. BBL, Division of Bioquest,
or Difco dehydrated medium may be used.
(e) Koser's citrate broth. — See 940.36A(e).
(f) Butterfield's buffered phosphate diluent. — See
966.23A(m).
(g) Lauryl sulfate tryptose broth with MUG. — Prep, lauryl
sulfate tryptose broth, 966.23A(b), and add 50 mg 4-methyl-
umbelliferyl-p-D-glucuronide (MUG). Dissolve with gentle heat,
if necessary. Dispense 10 mL portions into 20 X 150 mm test
tubes. Autoclave 15 min at 121°. Final pH, 6.8 ±0.1. Difco
dehydrated medium, or equiv., may be used.
(h) Peptone dilution water. — Dissolve 1.0 g peptone in 1
L H 2 0. Prep, diln blanks with this soln, dispensing enough to
allow for losses during autoclaving. Adjust pH to 7.0 ± 0.1.
Autoclave 15 min at 121°.
(i) Lactose broth. — Dissolve on H 2 bath, with stirring, 3.0
g beef ext and 5.0 g poly peptone or peptone in 1 L H 2 0. Add
5.0 g lactose. Dispense 450 mL portions into 750 mL flasks
and autoclave 15 min at 121°. Max. exposure to heat should
be <30 min. Final pH, 6.7 ± 0.2.
C. Preparation of Sample
Prep, all decimal dilns with 90 mL sterile diluent, peptone
diln H 2 (h) or 966.23A(m)(2), plus 10 mL previous diln un-
less otherwise specified. Shake all dilns 25 times in 30 cm arc.
Pipets must accurately deliver required vol. Do not use to de-
liver <10% of their total vol. For example, to deliver 1 mL,
do not use pipet >10 mL; to deliver 0. 1 mL, do not use pipet
>1 mL.
Frozen or chilled foods. — Use balance with capacity of ^2
kg and sensitivity of 0. 1 g to aseptically weigh 50 g unthawed
(if frozen) sample into sterile high-speed blender jar. Add 450
mL diluent, (i) or 966.23A(m)(2), and blend 2 min at 10 000-
12 000 rpm. (If necessary to temper frozen sample to remove
50 g portion, hold <18 h at 2-5°. ) Not >15 min should elapse
from time sample is blended until all dilns are made in ap-
propriate media.
If entire sample consists of <50 g, weigh portion equiv. to
V2 sample and add vol. of sterile diluent required to make 1 : 10
diln. Total vol. in blender jar must completely cover blades.
D. Determination
Notes: (J ) Test tubes used in MPN method should be checked
under UV light to be sure glass does not fluoresce. (2) To
avoid false-pos. fluoresence, longwave UV light used in method
should not exceed 6 watts (Blak-Ray, Model UVL-56 (avail-
able from UVP, Inc.), or equiv.).
Seed 3-tube most probable number (MPN) series into lauryl
sulfate tryptose broth contg MUG (g), using 1 mL inocula of
1 : 10, 1 : 100, and 1 : 1000 dilns, with triplicate tubes at each
diln. Incubate 24 ± 2 h at 35° and examine for fluorescence
of medium when tube is held under longwave UV light (366
nm).
Streak fluorescent-pos. tubes on eosin methylene blue agar
plates (b), and incubate plates 24 ± 2 h at 35°.
Pick 2 or more well isolated typical colonies from eosin
methylene blue agar plates and transfer to agar slants prepd
from agar medium (a). Incubate 18-24 h at 35°. If typical
colonies are not present, pick 2 or more colonies most likely
to be E. coli. Pick >2 from every plate.
Confirm E. coli as specified in 966.24.
Ref.: JAOAC71, 589(1988).
982.36 invasiveness by Escherichia coli
of Mammalian Cells
Microbiological Method
First Action 1982
Final Action 1987
A. Principle
Invasiveness is detected by intracellular growth on mono-
layer of HeLa cells on slides. To minimize extracellular bac-
terial multiplication, host-pathogen interaction is resolved into
2 phases, infective and intracellular, using appropriate sub-
strates and the following protocols: growth of monolayer in
chamber slides, using controlled inoculum and period of in-
cubation; detn of optimal pre-infection growth conditions for
pathogen; washing pathogen to remove toxic end products; in-
fection of host cell under controlled conditions of number and
multiplicity of infection, and medium and length of incuba-
tion; subsequent removal of unattached bacteria; use of post-
infection medium to permit only intracellular bacterial growth
for limited period.
B. Culture Media
(a) Minimal essential medium (MEM). — Eagle-type with
Earle's salts. Dissolve 126.4 mg L-arginine.HCl, 24 mg L-cys-
tine, 292 mg L-glutamine, 41.9 mg L-histidine.HCl.H 2 0, 52.5
mg L-isoleucine, 52.4 mg L-leucine, 73.1 mg L-lysine.HCl,
14.9 mg L-methionine, 33.0 mg L-phenylalanine, 47.6 mg l-
threonine, 10.2 mg L-tryptophan, 36.2 mg L-tryosine, 46.8 mg
L-valine, 1 mg D-calcium pantothenate, 1 mg choline chloride,
1 mg folic acid, 2 mg isoinositol, 1 mg pyridoxal HO, 1 mg
nicotinamide, 0.1 mg riboflavin, 1 mg thiamine. HC1, 1 g glu-
cose, 265 mg CaCl 2 .2H 2 0, 400 mg KC1, 200 mg MgS0 4 .7H 2 0,
6.8 g NaCl, 2.2 g NaHCO,, 140 mg NaH 2 P0 4 .H 2 0, and 10
mg phenol red in 1 L H 2 0. Sterilize by filtration. Final pH
should be 7.2 ± 0.2. Check sterility of all culture fluids before
use. Store at 4-8°.
(b) Fetal bovine serum (FBS). — Sterile, virus-screened,
my co-plasma-free, obtained aseptically during slaughter (Flow
Laboratories, Inc., 7655 Old Springhouse Rd, McLean, VA
22102). Store at 4-8°.
(c) Antibiotic concentrate (AC). — Dissolve 500 000 inter-
national units (IU) penicillin G and 500 mg streptomycin (Flow
Laboratories, Inc.) in 100 mL H 2 and sterilize by filtration.
Store at -10°.
(d) MEM-FBS-AC medium. — Routine medium for culti-
vation of HeLa mammalian cells. Mix 90 mL MEM (a), 10
mL FBS (b), and 1 mL AC (c). Store at 4-8°.
(e) MEM-FBS medium. — Medium for cultivation of HeLa
cells before infection. Mix 90 mL MEM (a) and 10 mL FBS
(b). Store at 4-8°.
(f) Earle's salts. — Prep, without phenol red as follows:
Dissolve 6.8 g NaCl, 400 mg KC1, 265 mg CaCl 2 , 200 mg
MgS0 4 .7H 2 0, 140 mg NaH 2 P0 4 .H 2 0, 1.0 g glucose, and 2.2
g NaHC0 3 in 1 L H 2 0. Sterilize by filtration. Final pH should
be 7.2 ± 0.2.
(g) Veal infusion broth. — Dissolve 500 g veal (infusion) and
10 g proteose peptone in 1 L H 2 with gentle heating. Dis-
pense 5 mL portions into 13 x 100 mm screw-cap tubes. Au-
toclave 15 min at 121°. Final pH should be 7.3 ± 0.2.
(h) Brain-heart infusion (BHI). — Dissolve 12.5 g BHI
440
Microbiological Methods
AOAC Official Methods of Analysis (1990)
(powder) in 1 L Earle's salts (f). Sterilize by filtration. Final
pH should be 7.2 ± 0.2.
(i) Bile salts No. 3. — Dissolve 5 g bile salts No. 3 for-
mulation in 1 L Earle's salts (f). Sterilize by filtration.
(j) Heat-inactivated HFBS.— Heat FBS (b) 2 h at 55 ± 1°.
Store at 4-8°.
(k) HFBS-BHI-BS medium. — Mix 20 mL heat-inactivated
FBS (j), 10 mL BH1 (h), 10 mL bile salts No. 3 (i), and 60
mL Earle's salts (f ). Store at 4-8°.
(I) Veal infusion agar slant. — For maintenance of cultures,
dissolve 500 g veal (infusion), 10 g proteose peptone No. 3,
5 g NaCl, and 15 g agar in 1 L H 2 with gentle heating. Dis-
pense 7 mL aliquots to 16 X 150 mm screw-cap tubes. Au-
toclave 15 min at 121°. Final pH should be 7.3 ± 0.2.
(m) Dulbecco's phosphate-buffered saline (PBS). — Dis-
solve 8.0 g NaCl, 200 mg KC1, 1.15 g Na 2 HP0 4 , 200 mg
KH 2 P0 4 , 100 mg CaCl 2 , and 100 mg MgCl 2 .6H 2 in 1 L H 2 0.
Sterilize by filtration. Final pH 7.2 ± 0.2.
(n) Calcium- and magnesium-free Dulbecco' s PBS. — Dis-
solve 8.0 g NaCl, 200 mg KC1, 1.15 g Na 2 HP0 4 , and 200 mg
KH2PO4 in 1 L FLO. Sterilize by filtration. Final pH 7.2 ±
0.2.
(o) Calcium magnesium, phenol red-free Hanks' PBS. —
Dissolve 8.0 g NaCl, 400 mg KC1, 90 mg Na 2 HP0 4 .7H 2 0,
60 mg KH 2 P0 4 , 1.0 g glucose, and 350 mg NaHC0 3 in 1 L
H 2 0. Sterilize by filtration. Final pH 7.2 ± 0.2.
(p) Trypsin stock soln. — 2.5%. Suspend 2.5 g 1 :250 tryp-
sin (Difco Laboratories) in 100 mL Ca- and Mg-free Hanks'
PBS (o) and let particles settle. Sterilize by filtration. Dil. 10
mL stock soln with 90 mL sterile Ca- and Mg-free Dulbecco's
PBS (n) to prep. 0.25% trypsin. Store at -10°.
(q) Gentamicin stock soln. — Dissolve 50 mg gentamicin
(Schering Corp., 2000 Galloping Hill Rd, Kenilworth, NJ
07033) in 100 rnL Dulbecco's PBS (m) to give soln contg 500
pg/mL. Dil 1 + 9 with Dulbecco's PBS to soln contg 50 \xg/
mL. Store at 4-8°.
(r) Lysozyme soln. — Weigh 0.3 g lysozyme, 3x crystal-
line, salt-free, ca 12 000 Shugar units/mg (Calbiochem Corp.),
into 100 mL Dulbecco's PBS and stir to dissolve. Store at 4-
8° not >2 weeks.
(s) Intracellular growth phase medium. — Mix 80 mL MEM-
FBS medium (e), 10 mL gentamicin soln (50 p,g/mL) (q),
and 10 mL lysozyme soln (r). Prep, immediately before use.
C. Diagnostic Reagents
(a) M ay -Grunwald stain. — Weigh 2.5 g stain (EM Science)
into 50 mL absolute MeOH, dissolve by grinding, and dil. to
1 L with MeOH. Stir 16 h at 37°. Hold stain J month at 22°
(room temp.). Filter for use.
(b) Giemsa stain. — Dissolve 1 g stain (EM Science, No.
GX0080) in 66 mL glycerol by heating 1.5-2.0 h at 55-60°.
Add 66 mL absolute MeOH. Store stain 2 weeks in tightly
stoppered bottle at 22°. Dil. stock soln (1 + 9) before use.
(c) Decolorizing and dehydrating reagents. — Acetone; ace-
tone-xylene (50 4- 50) and (33 + 67); xylene.
(d) Mounting medium. — Dil. mounting medium with xy-
lene to give easily dispensed colloidal suspension; 20 mL Per-
mount** (Fisher Scientific Co.) dild with 5 mL xylene is sat-
isfactory.
(e) Human cervical epithelial cell culture. — ATCC HeLa
culture. Other cultures, including Henle 407 human intestine
and human laryngeal carcinoma gave comparable data; how-
ever, HeLa cell culture was more suitable with regard to cul-
ture characteristics.
D. Apparatus
(a) Water baths. — Maintained at 35 ± 1° and 55 ± 1°.
(b) Microscopes. — Standard 900 X magnification; inverted
stage, 100 X magnification (Preiser Scientific, 94 Oliver St, St
Albans, WV 25177), or equiv.; microscope illuminator.
(c) Carbon dioxide incubator. — 95% air-5% C0 2 -moisture-
satd atmosphere, maintained at 36 ± 1° (Lab-Line Instru-
ments, Inc. Melrose Park, IL 60160, or equiv.).
(d) Tissue culture chamber slides. — Clean microscope slides
mounted with partitions on plastic gasket to facilitate multiple
testing. Lab-Tek units contg 4 chambers are satisfactory (Nunc,
Inc., 200 N. Aurora Rd, Naperville, IL 60566), or equiv.
(e) Culture containers. — Sterile 3 fluid oz (85 mL) glass
prescription bottles or plastic tissue culture flasks (Costar, 205
Broadway, Cambridge, MA 02139, or equiv.).
(f) Glass cover slips. — 1 x 2 in. (2.5 x 5.1 cm).
(g) Cell-counting chamber. — Spencer Bright Line, Fuchs-
Rosenthal (Preiser Scientific), or equiv.
(h) Refrigerated centrifuge with adapter. — To accommo-
date 13 x 100 mm tubes and covered centrf. cups to prevent
aerosolization of pathogens.
(i) Membrane fdters . — 0.45 |uunpore diam. (MilliporeCorp.,
or equiv.).
E. Preparation of HeLa Cell Culture
Using std cell culture technics, grow HeLa strain on inner
surface of 3 oz glass or plastic container, using 5 mL MEM-
FBS-AC medium, (d), for 7 days at 36° in C0 2 incubator.
Replace with fresh culture medium on fourth day to prevent
accumulation of toxic metabolites. In prepg cells in monolayer
for transfer to chamber slides, wash once with 5 mL Dulbec-
co's PBS (m) pre warmed at 36°. Add 5 mL pre warmed (36°)
0.25% trypsin and hold at room temp. 2 min. Aseptically re-
move ca 4.5 mL trypsin. Incubate flask at 36° with occasional
agitation. After monolayer has detached and cells are fairly
uniformly distributed in residual trypsin, add 25 mL pre-
warmed (36°) MEM-FBS medium, (e). Est. cell density, using
counting chamber. Add MEM-FBS medium, if necessary, to
dil. suspension to density of 1 X 10 5 cells/mL. With occa-
sional agitation, rapidly transfer 1 mL aliquots to chambers of
slide. Incubate 20-24 h at 36° in C0 2 incubator. Aseptically
remove spent medium before infection. Wash each monolayer
once with 1 mL pre warmed (36°) Earle's salts, (f ), and 1 mL
prewarmed (36°) uninoculated infection medium, (k) (see be-
low).
F. Preparation of Bacteria
Inoculate, with needle, 5 mL veal infusion broth, (g), using
growth from veal infusion agar slant (I) incubated at 22°. In-
cubate presumptive E. coli broth cultures 18-24 h at 36°. Centrf.
suspension 20 min at 1200 X g at 18°. Resuspend cells in
equal vol. of Earle's salts, (f). Recentrifuge 20 min at 1200
x g. Resuspend cells in 5 mL Earle's salts. Dil. latter sus-
pension with prewarmed (36°) HFBS-BHI-BS medium, (k), to
final density of 5 X 10 7 cells /mL. Add 0.2 mL of each sus-
pension to prepd chamber (above). Use 0.2 mL HFBS-BHI-
BS for uninoculated neg. control.
G. Infection Stage
Incubate chambers 2.5 h at 36° in C0 2 incubator. Time fac-
tor is critical; shorter period results in min. number of infected
host cells and longer period may result in cytotoxic effect aris-
ing from medium and possibly bacterial metabolites.
H. Intracellular Growth Stage
Remove infection medium from chamber with Pasteur pipet.
To prevent contamination, use sep. pipet for each chamber.
Wash each chamber twice with 1 mL aliquots of prewarmed
(36°) Earle's salts. Subsequently wash with I mL aliquot of
prewarmed intracellular growth phase medium (s) prepd im-
mediately before use. Add 0.8 mL prewarmed intracellular
growth phase medium to each chamber. Incubate 5 h at 36° in
AOAC Official Methods of Analysis (1990)
Escherichia Coli
441
C0 2 incubator. Control of extracellular growth is critical at
this stage; sensitivity of culture to gentamicin and other anti-
biotics should be examined by std procedures before patho-
genicity testing. Problem is critical in meats and dairy products
where antibiotics may have been used in therapy or in feeds.
/. Staining
Remove fluid contents of chambers. Wash monolayer 3 times
with 1 mL Dulbecco's PBS (n). Add 1 mL absolute MeOH
fixative per chamber. Hold at room temp. 5 min. Remove
MeOH and side walls of chamber slide. Insert single-edge ra-
zor blade between gasket and slide, and gently pry gasket from
slide. If necessary, cautiously remove remnants of gasket from
slide with razor blade. Do not let specimen dry while slide is
prepd for staining. Immerse slides in May-Grunwald stain (a)
10 min. Withdraw slides, remove excess stain, and immerse
in Giemsa stain (b) 20 min. Withdraw slides, remove excess
stain, and immerse in H 2 10-20 s. Briefly rinse twice in
acetone. Briefly immerse slides in following sequence of solvs;
acetone-xylene (50 + 50), acetone-xylene (33 + 67), and xy-
lene. Evenly distribute 4 drops of mounting medium, (d) to
slide. Place large cover slip on prepn. Remove excess mount-
ing medium and xylene by gently blotting. Gently apply pres-
sure to remove air bubbles from prepn.
J. Detection and Criteria of Invasiveness
Examine specimens with 900 x magnification. Criterion for
intracellular location of bacteria is parfocality of cytoplasmic
ground substance and bacteria. If invasive, E. coli occurs within
cytoplasm. Frequently, they may be located along nuclear
membrane. In addition, they may be elongated. Finally, bac-
teria may occur within a membrane (phagolysosome) individ-
ually or in groups, indicative of intracellular growth. Examine,
at random, 10 fields contg 15-25 HeLa cells. Count bacteria
in each cell. Criterion for infection is ^5 bacteria per cell.
Criterion for invasiveness of bacterial culture is 5:1.0% in-
fected HeLa cells.
HeLa cell results with E. coli strains must be confirmed by
Sereny keratoconjunctivitis test.
Refs.: Acta Microbiol. Acad, Sci. Hung. 2, 292(1955); 4,
367(1957). J . Hyg. Epidemiol. Microbiol. Immunol. 3,
292(1959). JAOAC 65, 602(1982).
984.34 Detection of Escherichia coli
Producing Heat-Labile Enterotoxin
DNA Colony Hybridization Method
First Action 1984
Final Action 1987
(Caution: This procedure uses radioactive and mutagenic com-
pounds. Personnel must receive adequate training and moni-
toring and have proper facilities available for handling these
substances.)
A. Method Performance
Results
Percent
95% Confidence
Range (approx.)
Correct
False positive
False negative
96.9
2.1
4.6
95-99
1-5
1-11
Of 13 laboratories, 8 (62%) correctly identified all unknown
samples (25/25); 11 laboratories (85%) identified >96% of
the samples.
B. Principle
Isolated and purified genes (DNA) that code for determi-
nants of bacterial virulence can be used to detect pathogenic
strains. Specific fragments of DNA are isolated by cleaving
plasmid DNA with appropriate restriction endonucleases and
sepg resulting pieces by gel electrophoresis. Purified frag-
ments are radioactively labeled in vitro. Bacterial cultures to
be tested are spotted on nitrocellulose filters on agar medium:
and incubated until colonies are visible. Cells are lysed in situ,
DNA is fixed to filter, and radioactive virulence gene DNA
fragments are added. Colonies which contain same gene as
radioactive DNA will bind this DNA and become radioactive.
These colonies are detected by autoradiography.
C. Reagents
(Prep, all media according to manufacturer's instructions.)
(a) 10X M9 Salts.— Dissolve 10 g NH 4 C1, 60 g Na 2 HP0 4 ,
30 g KH 2 P0 4 , and 5 g NaCl in final vol. of 1 L H 2 0. Dispense
into 100 mL aliquots and autoclave 15 min at 121°.
(b) Amplification medium. — Sterilize all components sep.
Aseptically combine 100 mL 10X M9 salts (a), 835 mL H 2 0,
10 mL 0.1M MgS0 4 , 10 mL 0.01M CaCl 2 , 25 mL 20% (w/
v) casamino acids, 20 mL 20% (w/v) glucose, and 0.2 mL
thiamine (10 mg/mL).
(c) TE Buffer.— Combine 10 mL 1.0M Tris-HCl (tris-hy-
droxy methyl aminomethane HC1) and 2 mL 0.5M Na?EDTA.
Adjust to pH 8.0 with ION NaOH. Add H 2 to final" vol. of
1 L.
(d) TES Buffer.— Combine 30 mL 1.0M Tris, 10 mL 0.5M
Na 2 EDTA, and 10 mL 5.0M NaCl. Add H 2 to final vol. of
1 L.
(e) CsCl saturated isopropanol. — Add ca 50 mL TE buffer
(c) to ca 350 mL isopropanol. Add solid CsCl (reagent or op-
tical grade) until bottom layer is satd.
(f ) Triton lytic mix.— Add 0. 1 mL Triton X-100, 5 mL 1 .0M
Tris, pH 8,0, and 12.5 mL 0.5M Na 2 EDTA, pH 8.0, to H 2
(final vol. 100 mL).
(g) J OX TBE electrophoresis buffer. — Dissolve 108 g Tris,
9.3 g Na 2 EDTA, and 55 g boric acid in ca 800 mL H 2 0. Ad-
just pH to 8.2 with coned HC1 and bring final vol. to 1 L with
H 2 0.
(h) 10X Hindlll reaction buffer. — Combine 50 mL 1.0M
Tris, pH 8.0, 10 mL 1.0M MgCl 2 , 10 mL 5.0JV1 NaCl, and
10 mL 100 mM dithiothreitol in final vol. of 1 L H 2 0.
(i) Stop soln. — Combine 1.0 mL 10% (w/v) sodium do-
decyl sulfate, 10 mg bromophenol blue, 2 mL 0.5M Na 2 EDTA,
pH 8.0,5 g glycerol in a final vol. of 10 mL of H 2 0.
(j) I OX nick translation buffer. — Combine 500 u-L 1.0M
Tris, pH 7.8, 50 |xL 1.0M MgCl 2 , 7 [xL 2-mercaptoethanol,
and 500 \xL nuclease-free bovine serum albumin (1 mg/mL).
(Commercially available nick translation kits contain similar
reagents. Follow supplier's instructions.)
(k) Hybridization mixture. — Combine 22 mL distd form-
amide, 12.5 mL 20X SSC (I), 0.5 mL 10% (w/v) sodium
dodecyl sulfate, 5.0 mL 10X Denhardt's soln (m), 0.1 mL
0.5M Na 2 EDTA, pH 8.0, and 9.9 mL H 2 0.
(1) 20X Standard saline citrate soln (.SSC).— Add 175.4 g
NaCl and 88.2 g Na citrate to final vol. of 1 L H 2 0. 5X and
2X SSC may be prepd by dilg 20X SSC with H 2 0.
(m) I0X Denhardt's soln.— Combine 2.0 g Ficoll (400,000
mol. wt), 2.0 g polyvinyl pyrrolidone (360,000 mol. wt), and
2.0 g nuclease-free bovine serum albumin in 1 L H 2 0. Store
5 mL aliquots at -20°.
(n) Calf thymus DNA. — Dissolve 1 g purified calf thymus
DNA in 100 mL H 2 by stirring for several hours. Sonicate
until av. mol. wt is 300,000-500,000 which may be detd by
442
Microbiological Methods
AOAC Official Methods of Analysis (1990)
electrophoresis with appropriate stds. Store in 1 mL portions
at -20°.
(o) Brain heart infusion broth. — Prep, and sterilize accord-
ing to supplier's instructions.
(p) DE 52 column chromatography medium. — Prep, ac-
cording to manufacturer's instructions in loading buffer, (q).
(q) DE 52 loading buffer.— Combine 30 mL 5.0M NaCl,
0.2 mL 0.5M Na 2 EDTA," and 10 mL 1.0M Tris (pH 8,0) in
final vol. of 1 L H 2 0.
(r) DE 52 elating buffer.— Combine 200 mL 5.0M NaCl,
0.2 mL 0.5M Na.EDTA, and 10 mL 1.0M Tris (pH 8.0) in
final vol. of 1 L H z O.
(s) Sephadex G-50 column chromatography medium. — Prep.
according to manufacturer's instructions in TE buffer, (c).
D. Apparatus and Materials
(a) Preparative ultracentrifuge and fixed angle rotor. —
100,000 x g and 13 mL tubes.
(b) Shaker.— In 37 ± 1° H 2 bath with clips for holding 1
L erlenmeyers.
(c) Longwave ultraviolet lamp. — 302 nm transilluminator
preferred. Camera for photographing gels is useful.
(d) Refrigerated super speed centrifuge and fixed angle ro-
tor. — 37,000 X g and -20°, capable of holding 50 mL tubes
and adapters for 15 or 30 mL tubes.
(e) Siliconized glass tubes. — 15 or 30 mL capable of with-
standing 10,000 X g.
(f ) Spectrophotometer or colorimeter and sample holder. —
Measure bacterial cell growth at 550 or 600 nm.
(g) Escherichia call strain C600(pEWD299)(ATCC
37218). — Contains cloned heat-labile enterotoxin gene. Pos.
and neg. strains such as E. coll H 10407 (ATCC 35401) and
pi asm id pBR313 (ATCC 37018) are needed as controls during
hybridization.
(h) Alpha- 32 ? deoxycytosine triphosphate. — dCTP, 2000-
3000 Ci/mmole, aq, stabilized (ICN Biomedicals, Inc., ICN
Plaza, 3300 Hyland Ave, Costa Mesa, CA 92626; New Eng-
land Nuclear, 549 Albany St, Boston, MA 02118; Amersham
Corp., Div. of Amersham International, 2636 S. Clearbrook
Dr, Arlington Heights, IL 60005-4692).
(i) Ultralow temperature freezer . — Capable of —70° is pre-
ferred; however, -20° (not frost-free) may be substituted.
(j) Vacuum desiccator. — Large enough to contain 15 or 30
mL tubes.
(k) Polycarbonate tubes. — 50 mL.
(1) Variable volume microplpettors and tips. — To cover range
of 1-1000 uX.
(m) Electrophoresis apparatus. — Horizontal and vertical
units with bed dimensions ca 12 x 12 cm and appropriate power
supplies (to 125 mA; 200 V).
(n) Incubator. — H 2 bath or dry heating block capable of
maintaining 37 ± 1°.
(o) Plastic conical centrifuge tubes. — 500 and 1500 jxL sizes
able to withstand 15,000 X g with appropriate racks.
(p) Centrifuge. — For spinning tubes (o) at greater than
10,000 x g.'
(q) Dialysis tubing. — 1 / 4 in. diam., 10-12,000 molecular
weight cut-off. Boil 3 min before use.
(r) Glass wool. — Boiled or siliconized.
(s) Disposable plastic syringes. — 1 mL.
(t) Vacuum side arm flask. — 250 mL for degassing.
(u) Cooling block or refrigerated H 2 bath. — 15 ± 1°.
(v) Plastic column. — Disposable, ca 4 X 0.9 cm.
(w) Scintillation counter. — Or Geiger-Mueller counter if
calibrated in cpm.
(x) Nitrocellulose filters. — 0.45 |xm pore size, 82 mm diam.
(y) Absorbent paper filters. — 82 mm diam.; similar in
characteristics to Whatman No. 1 .
(z) Petri dishes. — 100 X 15 or 20 mm, plastic.
(aa) Vacuum oven. — Maintain 80 ± 3°.
(bb) X-ray film. — 8 x 10 in. is convenient size.
(cc) X-ray film holder cassette .—With intensifying screens
(Kodak regular or Dupont Cronex lightening plus).
E. Isolation of Plasmid DNA
Inoculate 25 mL brain heart infusion broth (o) contg 10 |xg
ampicillin (filter-sterilized) /mL with frozen stock of strain C600
(pEWD299). Incubate overnight at 37° with shaking. Read A
at 550 nm, using 25-fold diln. Inoculate 1.5 L amplification
medium (b) to A 550 = 0.02. [Note: This procedure can be scaled
up to 10 L.] Shake or aerate well at 37°. When A 550 = 0.4,
add solid chloramphenicol to 100 |xg/mL. Reduce shaking to
75 rpm or aeration to 2 Lpm. Incubate overnight. Harvest cells
by centrifugation at 4°, resuspend pellets in TES buffer (d),
and centrf. again. Resuspend cells in 8 mL 25% sucrose (w/
v, nuclease-free) in TE buffer (c) in 50 mL polycarbonate centrf.
tube. Add 1 mL 1% lysozyme (egg white, grade 1), mix gently,
and let sit on ice 5 min. Add 13 mL Triton lytic mix (f), stir
briefly to mix, and incubate on ice 30 min. Centrf. 30 min at
27,000 x g. Decant supernate thru gauze. If pellet is very soft,
centrf. again at 37,000 X g for 30 min and combine this su-
pernate with first one. Measure vol. of supernate (to 0.1 mL)
and add 0.97 g solid CsCl for each mL. Add so In to ultra-
centrf . tubes and layer on surface 0. 1 mL ethidium bromide,
10 mg/mL ? for each mL supernate (before addn of CsCl) on
liq. surface. [Caution: Ethidium bromide is mutagenic. Handle
with care.] Fill tubes with light mineral oil, balance to 50 mg,
and cap or seal. Centrf. 40 h in fixed angle rotor at 100,000
x g or 18 h in vertical rotor at 180,000 x g at room temp.
(23°).
Observe ultracentrifuge tube in subdued room light, without
fluorescent lights. Locate lower, orange band with longwave
UV light and remove band with needle and syringe. Place band
into polystyrene or siliconized glass tube. Ext and discard
ethidium bromide with isopropanol satd with TE buffer and
CsCl (e). Repeat until pink color is gone and then ext twice
more. Measure remaining sample vol. and add 3 vols H 2 and
25 |ag yeast transfer RNA (2.5 mg/mL, stored at -20°). After
addition of water, add one-ninth total vol. of 3.0M Na acetate-
lOmM MgCl 2 . Add 2.5 vols -20° alcohol and hold at -20°
1 h. Centrf. 10 min. at 9,000 x g at 0°. Discard supernate
and let pellets drain until alcohol odor is gone. Tubes may be
dried 15 min in vac. desiccator but do not over-dry. Gently
resuspend pellet in 1 mL TE buffer (c). Est. DNA concn by
electrophoresis against known stds. If A 258 is measured, DNA
concn will be over-estd because of presence of RNA. [For pure
DNA, A 2 58 =1.0 corresponds to 50 jxg/mL and ratio A l5% /
A no ~ 1.8]. Store DNA in plastic or siliconized glass tubes
at 4°.
Enterotoxin Gene DNA Isolation
F. Enzyme Titration
Titr. restriction endonuclease against plasmid (pEWD299),
using estd DNA concn to det. correct amt of enzyme. Usually,
one unit of enzyme will digest about 1 |xg DNA. However,
this can vary by several fold, depending on plasmid, enzyme,
or impurities. Generally, it is best to follow methods suggested
by supplier.
If Hindlll is used to cleave pEWD299, an 850 base-pair
fragment will be generated which contains nucleotide sequence
for entire B subunit and about one-third of the A subunit of
AOAC Official Methods of Analysis (1990)
Escherichia Coli 443
the heat-labile enterotoxin. Dispense ca 1 |xg DNA into four
500 |ulL conical plastic tubes. Add 2.5 |xL 10X - HindlU re-
action buffer (h). Add 0, 2, 5, or 25 units of enzyme to each
tube. Add 2.5 jxL bovine serum albumin (1 mg/mL, nuclease-
free). Add H 2 to bring vol. to 25 |JiL. Incubate 1 h at 37°.
Add 5 |ulL stop soln (i) and electrophorese 25 jxL of each mix-
ture for 3 h at 100 V in 0.7% agarose in IX TBE dild from
(g). As control, run 30-50 ng linear bacteriophage lambda DNA.
Stain gel with ethidium bromide (2 ixg/mL) until lambda DNA
band is visible under longwave UV light. If record is desired,
rinse gel briefly with H 2 0, and photograph with 302 nm tran-
silluminator and camera with Wratten No. 23 A or 9 filter.
G. Preparative Digest
Scale up titrn digest using lowest amt of enzyme that achieves
complete digestion. After 1 h of incubation at 37°, add one-
tenth vol. of stop soln (i). Prep. 10% poly acrylamide gel. For
50 mL gel, combine 32.5 mL H 2 0, 5.0 mL 10X TBE (g),
12.5 mL 40% acrylamide (w/v; caution: acrylamide monomer
is a neurotoxin). Degas 15-30 min in sideartn flask under vac.
Add 0.5 mL freshly prepd 10% (w/v) ammonium persulfate
soln. Add 50 uX TEMED (MA^A^N'-tetramethylethylenedi-
amine) but mix gently so as not to aerate degassed soln. Pour
vertical gel which should harden in 10-20 min. Layer digest
on gel and electrophorese for 3 h at 100 V in IX TBE (diluted
from g). Stain with ethidium bromide (2 jxg/mL) until bands
are visible with longwave UV. Slice 850 base-pair band (near-
est bottom) from gel and place into dialysis tubing with 1-2
mL IX TBE. Place bag in horizontal electrophoresis unit and
cover with IX TBE. Electroelute band from gel at 50 V for
16 h. Reverse polarity of electrodes and turn on power for 15
s at 150 V. Remove buffer contg DNA from dialysis bag with
plastic pipet. Add one-tenth vol. of stop soln (i). Repeat elec-
trophoresis and electroelution as described above.
H. DE52 Chromatography
Prep. DE52 according to manufacturer's instructions, using
loading buffer (0.15M NaCl, ImM Na 2 EDTA, pH 8.0, 0.01M
Tris, pH 8.0). Construct 0.3 to 0.4 mL DE52 column in 1 mL
plastic syringe plugged with boiled or siliconized glass wool.
Wash column with 2 mL loading buffer. Apply 1-2 mL DNA
to top of column bed. Wash column with 3-4 mL loading
buffer. Elute DNA with 10 column vols of eluting buffer (1.0M
NaCl, ImM Na 2 EDTA, pH 8.0, 10mM Tris, pH 8.0) in 0.3
mL aliquots. Collect 0.3 mL fractions in 500 |xL plastic tubes.
Most of DNA should be in first 2 or 3 fractions. Spot 2 |xL
of each fraction onto 1 % agarose with 2 jxg/mL ethidium bro-
mide and illuminate with UV light. Fractions contg DNA will
fluoresce; pool these fractions and alcohol-ppt by measuring
total vol. and adding 10 jjlL transfer-RNA (2.5 mg/mL) and
one-ninth soln vol. of 3.0M NaOAC-lOmM MgCl 2 . Add 2.5
vols of -20° alcohol and hold at -20° >1 h. Centrf. at 10,000
x g for 10 min. Discard supernate and gently rinse pellet (which
may not be visible) with 0.5 mL —20° alcohol. Drain well until
alcohol odor is gone but do not dry completely. Gently resus-
pend DNA pellet in 200-300 \xL TE buffer (c).
/. In Vitro DNA Labeling
Kits are available commercially for nick translation reaction.
Following procedure uses 50 ng DNA rather than 1 p,g often
suggested by suppliers. To 500 |ulL conical plastic centrf. tube,
add 50 ng DNA (as prepd above in max. vol. of 5 |xL). Add
3 jxL 10X reaction buffer. Add 1.5 (jlL of soln 333 jxM in
each of deoxyadenosine triphosphate, deoxyguanosine tri-
phosphate, and thymidine triphosphate. Add 16 jxL alpha- 32 P
deoxycytosine triphosphate (dCTP) (h). Add H 2 to final vol.
of 26 (xL. Add 2 (xL DNase I (100 ng/mL, dild immediately
before use). Incubate 10 min at 15°. Add 2 jxL DNA poly-
merase I (1 IU/jxL). Incubate 1 h at 15°. Add 2 uX 0.5M
Na 2 EDTA, pH 8.0. Prep. 2 mL column of Sephadex G-50
(prepd according to manufacturer's instructions) using TE buffer
(c). Load reaction mix onto column and elute by adding 100
|ulL portions of TE buffer (c). Collect twenty 2-drop fractions
into 500 iulL tubes. Spot 2 fiL of each fraction onto 2 X 2 cm
paper squares (e.g., Whatman 3 MM), dry, add scintillation
fluid (e.g., 5 g 2,5-diphenyloxazole/L toluene), and count.
Geiger-counter may suffice to assay fractions. Labeled DNA
is eluted from column usually between fractions 6 and 12. Un-
incorporated dCTP elutes as larger peak, starting between frac-
tions 12 and 15. Pool fractions from earliest peak and count
2 (xL as previously described. Using 3000 Ci/mmol of dCT 32 P,
specific activities of 2-8 X 10 8 cpm/|xg usually result.
J. Colony Hybridization Filter Preparation
When received, transfer sample cultures to 5 mL rich broth
and incubate at 37° for 18-24 h. Aseptically add 2 mL culture
to 0.5 mL sterile 50% (v/v) glycerol. Store at -70° if pos-
sible. [Note: Frost-free freezers will decrease culture viability.
If cultures must be stored at —20°, use non-frost-free unit. This
caveat holds for all frozen material in this procedure.]
Boil nitrocellulose filters (0.45 |xm, 82 mm diam.) for 2-3
min in ca 2 L H 2 0. While still wet, flatten filters (to minimize
wrinkles) between paper filters (such as Whatman No. 1 or
Schleicher and Schuell No. 597), using forceps to avoid touch-
ing filters. Loosely wrap filters in Al foil and sterilize at 121°,
15 lb, for 10-20 min on liq. (slow exhaust) cycle.
Store at room temp. Aseptically inoculate ca 5 mL rich broth
with portion of frozen bacterial culture. [It is not necessary to
completely thaw culture and it may be re-used several times.]
Incubate cultures 18-24 h at 37°. Aseptically, place sterile ni-
trocellulose filter on dry MacConkey agar plate. Ensure that
no bubbles are trapped under filter and that it wets completely.
Discard filters that do not lie flat. Label filters, using soft lead
pencil or by perforating filter in distinctive pattern with needle.
This may be more easily done after baking at 80° (see below).
Filters marked with 5 mm square grid are useful for arranging
cultures in orderly array. Inoculate filters with sterile micro-
biological needle, using 1: 100-fold diln, in sterile normal sa-
line, of overnight culture. Always inoculate each filter with
known pos. and neg. control cultures. Record location of each
culture; 30-50 cultures should fit on each filter. It is vital that
filters and the resulting autoradiogram can be oriented un-
ambiguously. Make duplicate filters, since procedure may have
to be repeated. Incubate filters 18-24 h at 37°. Mark cultures
which have failed to grow, or a false-neg. result may be re-
ported .
Lyse colonies by transferring filters for 10 min onto paper
filters (in 100 x 15 mm plastic petri plates) wetted with 1.5
mL 0.5M NaOH. Ensure that no bubbles are trapped under
filters. Transfer nitrocellulose filters for >1 min each, to series
of 3 paper filters each wetted with 1.5 mL 1 .0M ammonium
acetate-0.02N NaOH. Shift nitrocellulose filter to fourth am-
monium acetate-NaOH filter for 10 min. Keep filters horizon-
tal during transfers so that lysed colonies will not run together.
Air dry nitrocellulose filter on absorbent paper >30 min. Bake
in vac. oven 2 h at 80°. Cool filters to room temp, and label
with H 2 0-proof ink or pencil. Store between paper filters un-
der vac.
K. Colony Hybridization
Freshly prep. 50 mL hybridization mixt. (k). Boil 0.5 mL
sonicated calf thymus DNA (n) 10 min and add to hybridiza-
444
Microbiological Methods
AOAC Official Methods of Analysis (1990)
tion mixt. (k). Pre-incubate each nitrocellulose filter 3 h at 37°
in 100 X 15 mm plastic petri dish contg 5 mL hybridization
mixt. with boiled calf thymus DNA. After 3 h, alkali-denature
radioactive toxin gene DNA. Det. vol. of DNA required to
contain 1 x 10 6 cpm. Correct for 14.2-day half-life of 32 P.
Add 1 X 10 6 cpm DNA to 500 |ulL plastic conical tube and
bring total vol, to 300 >xL with H 2 0. Add 6 jjlL ION NaOH
and mix briefly with pipet tip. After 10 min, neutze with 6 |xL
ION HO Ac. Boil 0.5 mL sonicated calf thymus DNA for 10
min and add 50 mL hybridization mixt. (k). Place nitrocel-
lulose filter into 5.0 mL fresh hybridization mixt. and 1 x 10 6
cpm alkali denatured and neutzd probe DNA. Incubate 18-24
hat 37°.
Rinse filters 5-10 s in 10-15 mL 5X SSC (dild from soln
1)-0.I% (w/v) sodium dodecyl sulfate (SDS). Place filter into
clean petri dish and cover with 10-15 mL 5X SSC-0.1% SDS
and incubate 1 h at 70°. Place filter in fresh 5X SSC-0.1%
SDS and incubate addnl 1 h. Rinse filter 5-10 s in 2X SSC
(dild from soln 1). Air-dry 15-30 min. Mount filter with small
pieces of tape onto paper and cover with plastic sheet (such
as document holder).
L. Autoradiography
In dark room, place film on plastic-covered filters in cassette
film holder with intensifying screens. Enclose film holder in
plastic bag and expose film preferably at —70° but at least
-20°. Exposure length is dictated by amt of radioactive DNA
bound to filter. If increase of 2-3 cps is observed when Geiger-
Mueller counter is held over filter, it is likely that pos. reaction
will be visible after 1 d exposure. After exposure, let cassette
reach room temp. Develop following manufacturer's instruc-
tions. If spots are too faint for analysis, expose new film for
longer period.
M. Interpretation of Results
DNA of cells having gene for heat-labile enterotoxin of
Escherichia coli bind radioactive toxin gene DNA. Film will
be exposed and dark spots will appear after development. Since,
colony size and hybridization efficiency can vary, this test is
best used qual. and not quant. If there are dark areas on film
where no colonies should be, unhybridized radioactive DNA
has probably not been completely washed away. Rewash filter
twice in 5X SSC-0. 1 % SDS at 70° for 1 h. Let dry and expose
film. After film development, make pos. or neg. detn of each
unknown culture by comparing intensity of spot with pos. and
neg. control cultures. Neg. control should show no darkening
of film or, at most, very faint darkening. Pos. control should
show distinct darkening of film clearly discernible above back-
ground.
H. Troubleshooting
If autoradiograms are unsatisfactory, a number of factors
might be responsible. False-neg. results could be due to spon-
taneous loss of virulence determinants, insufficient growth of
colonies on filters, failure to bake filters to fix DNA, or in-
sufficient radioactivity during hybridization. False-pos. results
may result from insufficient filter washing after hybridization,
failure to add Denhardt's soln or sonicated calf thymus DNA,
or use of probe DNA fragment which was not purified ade-
quately. Possible remedies include use of new bacterial cul-
tures, prepg new filters with lysed colonies, reviewing pro-
cedures and reagent composition, rewashing filters, or checking
darkroom methods.
ReL: JAOAC 67, 801(1984).
986.34 Enterotoxigenic Escherichia coli
DNA Colony Hybridization Method Using Synthetic
Oligodeoxyribonucleotides and Paper Filters
First Action 1986
Final Action 1987
(Caution: This procedure uses radioactive compd. Personnel
must receive adequate training and monitoring and have proper
facilities available for handling this substance.)
A. Principle
Chemically synthesized pieces of DNA (oligodeoxyribonu-
cleotides) that code for regions of genes detg bacterial viru-
lence can be used to identify pathogenic strains of bacteria.
These oligomers are radioactively labeled in vitro and hybrid-
ized with colonies of bacterial cells that have been lysed and
fixed to paper filters. Colonies contg same region of a gene
will bind labeled DNA and become radioactive. Such colonies
can be detected by autoradiography.
B. Reagents
(Prep, all media according to manufacturer's instructions and
use analytical grade materials whenever possible. Note: DNA
often adheres to unsiliconized glass. When working with solns
contg DNA, use siliconized glassware or disposable plas-
tic ware unless otherwise specified.)
(a) Lysis mixture A.— Combine 50 mL ION NaOH, (s), 300
mL 5.0M NaCl, (u), and 650 mL H 2 0.
(b) Lysis mixture B. — Combine 50 mL 2.0M Tris, pH 7.0,
(v), 400 mL 5.0M NaCl, (u), and 550 mL H 2 0.
(c) Hybridization mixture. — Combine in plastic tube or
beaker: 28.9 mL H 2 0, 15.0 mL 20X SSC, (d), 5.0 mL 50X
Denhardt's soln, (e), and 0.1 mL 0.5M EDTA soln, pH 8.0,
(f ). Final vol. is 49 mL. Use immediately.
(d) 20X std saline citrate soln (SSC). — Dissolve 175.4 g
NaCl and 88.2 g Na citrate in final vol. of 1 L H 2 0.
(e) 5 OX Denhardt's soln. — Dissolve 2.0 g Ficoll (av. mo-
lecular wt 400 000), 2.0 g polyvinyl pyrrolidone (av. molec-
ular wt 360 000), and 2.0 g bovine serum albumin in 200 mL
H 2 0. Store at -20° in 5.0 mL aliquots.
(f ) 0.5M Disodium ethylenediamine tetraacetate soln, pH
8.O.— Dissolve 186. 12 g Na 2 EDTA in 800-900 mL H 2 0. Ad-
just to pH 8.0 with ION NaOH, (s). DiL to 1 L with H 2 0.
(g) Sonicated calf thymus DNA. — Dissolve 1 g purified calf
thymus DNA in 100 mL H 2 by stirring 3-4 h. Sonicate until
av. molecular wt is 300 000-500 000, which may be detd by
electrophoresis with appropriate stds such as 123-base ladder
(Bethesda Research Laboratories (BRL), Div. Life Technol-
ogies, Inc., 8717 Grovemont Circle, Gaithersburg, MD 20877).
Store in 1 mL portions in 13 X 100 mm screw-cap tubes. Glass
may be used in this instance only.
(h) 6X SSC soln.— Combine 300 mL 20X SSC, (d), with
700 mL H 2 0.
(i) 2X SSC soln.— Combine 100 mL 20X SSC, (d), with
900 mL H 2 0.
(j) Synthetic DNA stock soln. — Approx, 150-350 [xg/mL.
(A 260 = 5-10 units.) Soln of 2 2- base, single stranded DNA
molecules [STH (human) and STP (porcine) oligodeoxyribo-
nucleotide probes for enterotoxin genes] will have concn ca
20-50fjLM. Store at -20°,
(k) Synthetic DNA working soln.~D\\. stock soln, (j), in
H 2 to IOjjlM. Store at -20°.
(1) 2.0M Tris soln, pH 7.6.— Dissolve 242.28 g Tris in ca
800 mL H 2 0. Adjust to pH 7.6 with coned HC1. Dil. to 1 L
with H 2 Q.
AOAC Official Methods of Analysis (1990)
Escherichia Coli 445
(m) 1.0M MgCl 2 so/n.— Dissolve 9.52 g MgCL, in final vol.
of 100 mL H 2 0.
(n) 0.5M Dithiothreitol soln. — Weigh 0.77 g dithiothreitol
and combine with H 2 to final vol. of 10.0 mL. Store at 4°.
(o) I OmM Spermidine soln. — Dissolve 14.5 mg spermidine
in final vol. of 10.0 mL H 2 0. Store at -20°.
(p) 10X Kinase buffer .—Combine 2.5 mL 2.0M Tris, pH
7.6, (I), 1.0 mL 1.0M MgCl 2 , (m), 1.0 mL 0.5M dithiothrei-
tol, (n), 1.0 mL lOmM spermidine, (o), 20 |xL 0.5M EDTA,
(f ), and 4.5 mL H 2 0. Store at 4°.
(q) (y- 32 P)ATP. — Aq. soln of adenosine triphosphate, spe-
cific activity 3000-7000 Ci/mmole. ("Crude" prepn from ICN
Biomedicals, Inc. , ICN Plaza, 3300 Hyland Ave, Costa Mesa,
CA 92626, or equiv.). Store at -70° if possible.
(r) Bacteriophage T4 polynucleotide kinase. — 20 units/ixL
(BRL or equiv.).
(s) ION NaOH soln.— Dissolve 400 g NaOH in final vol..
of 1 L H 2 0.
(t) 2.0M Tris soln, pH 8.0. — Follow instructions for (1) but
adjust pH to 8.0.
(u) 5.0M NaCl soln.— Dissolve 292.2 g NaCl in final vol.
of 1 L H 2 0.
(v) 2 .OM Tris soln, pH 7.0. — Follow instructions for (1) but
adjust pH to 7.0.
(w) Glycerol freezing soln. — Combine 50.0 mL glycerol and
50.0 mL H?0. Dispense 0.5 mL aliquots into 1 dram vials.
Sterilize by autoclaving 15 min at 121°.
(x) NACS PREPAC column loading buffer. — Dissolve 19.3
g ammonium acetate in final vol. of 1 L H 2 0.
(y) NACS PREPAC column eluting buffer.— Dissolve 308.4
g ammonium acetate in final vol. of 1 L H 2 0.
(z) Brain heart infusion or trypticase soy broth and agar. —
For microbial growth.
(aa) Scintillation fluid. — Dissolve 5.0 g 2,5-diphenyloxa-
zole in 1 L toluene.
(bb) ST probe soln. — Combine equal vols of STH and STP
working soln, (k).
(cc) Phosphor amidite soln. — 0.5 g (Applied Biosy stems,
Inc., 850 Lincoln Centre Dr, Foster City, CA 94404; Amer-
ican Bionetics, Inc., 21377 Cabot Blvd, Hayward, CA 94545,
or equiv.), reagent grade (2:95%), made up to 0.1M using
anhyd. CH 3 CN, (nn), and glass syringe transfer procedures
with protection from atm. H 2 0. Vortex mix until dissolved.
(dd) Thiophenol soln. — Mix 80 m'L /?-dioxane (<0.01%
H 2 0), 80 mL triethylamine (99 + %), and 40 mL thiophenol
(99 + %) ("Gold Label," Aldrich Chemical Co., or equiv.).
(ee) lH-Tetrazole soln. — Add 300 mL anhyd. CH 3 CN, (nn),
to 10 g resublimed tetrazole, (oo), with protection from atm.
H 2 0, and sonicate until dissolved. Warm (30—40°), if neces-
sary.
(ff) Ammonium hydroxide soln. — 28-30% NH 3 , as sup-
plied.
(gg) Acetic anhydride soln. — Combine 160 mL tetrahydro-
furan (<0.01% H 2 0), 20 mL 2,6-lutidine ("Spectro Grade,"
Eastman Kodak Co., or equiv), and 20 mL acetic anhydride
(99 + %).
(hh) 4-Dimethylaminopyridine soln. — Dissolve 13 g re-
cry std 4-dimethylaminopyridine, (pp), in 200 mL tetrahydro-
furan (<0.01% H 2 0).
(ii) Trichloroacetic acid soln. — Weigh 125 g trichloroace-
tic acid (Aldrich Chemical Co., Inc., No. 25,139, or equiv.,
99 + %) in beaker with min. exposure to atm. moisture and
transfer to storage container using 4 L CH 2 C1 2 (<0.006% H 2 0).
( jj) Iodine soln. — Combine 320 mL tetrahydrofuran, 80 mL
2,6-lutidine, and 10.2 g I crystals. Sonicate until dissolved.
Add 8.0 mL H 2 0, dropwise, with stirring.
(kk) Dimethoxytrityl (DMT) assay soln. — Dissolve 19 g p-
toluenesulfonic acid monohydrate in 1 L LC grade CH 3 CN
(0.1M).
(11) Triethylammonium acetate (TEAA) buffer. — With const
stirring, add 28 mL triethylamine, (qq), to 1.8 L H 2 fol-
lowed by 10 mL glacial acetic acid. Titr. slowly with more
acid to pH 7.0 and then vac. filter thru type HA 0.45 (xm filter
(Millipore Corp. or equiv.).
(mm) Detritylation soln. — Add 3 mL glacial acetic acid to
97 mL H 2 0.
(nn) Anhydrous acetonitrile. — Store 1 L LC grade CH 3 CN
(<0.007% H 2 0, Burdick & Jackson Laboratories, Inc., or
equiv.) over type 4 A molecular sieves >24 h.
(00) Resublimed IH-tetrazole. — Sublime 20 g 1 //-tetrazole
(99 + %, Aldrich "Gold Label" or equiv.) in std sublimation
app. at <0.25 torr and 130-140°. (Yields ca 15 g sublimate.)
(pp) Recrystallized 4-dimethylaminopyridine . — Dissolve 200
g 4-dimethylaminopyridine in ca 1 L hot (50-60°) tetrahydro-
furan contg 20 g decolorizing charcoal. Filter while still hot
thru glass fiber paper (Grade 934- AH, "Reeve Angel," What-
man, Inc., or equiv).
(qq) Triethylamine. — 99 + % (Aldrich "Gold Label" or equiv.
LC grade).
C. Apparatus and Materials
(a) Labware. — 100 X 15 mm glass petri plates; plastic
beakers and tubes to contain up to 100 mL; 100 x 15 or 20
mm plastic petri plates; plastic conical tubes to contain up to
500 |xL; plastic pipets to cover range 1-10 mL; variable vol.
micropipettors and tips to cover range 1—1000 \lL.
(b) Incubators. — (I) Capable of maintaining 37 ± 1°; (2)
capable of maintaining 40 ± 1°; (3) capable of maintaining
50 ± 1°; (4) H 2 bath or dry block capable of maintaining
37 ± 1°.
(c) UV spectrophotometer. — To measure DNA concn at 260
nm. (1 A 260 unit is 50 u.g/mL for double stranded DNA and
33 jig/mL for single stranded DNA.)
(d) Ultralow temperature freezer . — Capable of maintaining
-70° is preferred, but freezer (not frost- free) at -20° may be
substituted .
(e) Freezer. — Capable of maintaining —20° (not frost-free).
(f) Cellulose filters . — No. 541 (Whatman), 82-85 mm diam.
(g) Absorbent filters. — Whatman No. 1 or similar, ca 85
mm diam.
(h) NACS PREPAC column.^DNA binding resin (BRL or
equiv.).
(a) Scintillation counter. — Or Geiger-Mueller counter if
calibrated in cpm,
(j) X-ray film and developing chemicals. — 8 X 10 in. is
convenient size. Kodak XAR X-ray film or equiv.
(k) Darkroom. — Facilities for X-ray film development with
appropriate safelight.
(1) X-ray film holder cassette. — With intensifying screens
(Kodak regular, Eastman Kodak Co.; Dupont Cronex Light-
ening Plus, E.I. Dupont de Nemours & Co.; or equiv.).
(m) Centrifuge. — Capable of spinning 500 fiL conical plas-
tic tubes (Eppendorf Model 5412, Brinkmann Instruments, Inc.,
or equiv.).
(n) Vacuum desiccator. — Needed only if prepd colony hy-
bridization filters must be stored 1 week.
(o) DNA synthesizer. — Manual or automated synthesis sys-
tem (i.e., Applied Biosystems synthesizer Model 380A; other
synthesis systems providing equiv. results are also acceptable).
(p) Synthesis ("reaction") columns. — 1 jxmol long chain
alkylamine-functionalized controlled pore glass, either pre-
packed or handpacked (Applied Biosystems or equiv.).
446
Microbiological Methods
AOAC Official Methods of Analysis (1 990)
(q) Fraction collector, — To collect fractions from auto-
mated synthesis system. Should have auxiliary signal input.
(r) Liquid chromatographic system. — App. with gradient
elution capability, UV detection at 254 or 260 nm, and jxBon-
dapak® C I8 , 7.8 mm x 30 cm column (Waters Associates,
Inc., or equiv.).
(s) Rotary vacuum centrifuge. — To cone. LC-purified oli-
godeoxyribonucleotides (SpeedVac concentrator/dryer, Sa-
vant Instruments, Inc., 110-103 Bi-County Blvd, Farming-
dale, NY 11735, or equiv.)-
(t) Glass syringes. — Capacity up to 10 mL for transfer of
anhyd. CH 3 CN with protection from atm. moisture.
(u) Type HV, 0.45 pm filters. — To remove LC column par-
ticulates (Millipore or equiv.).
D. Colony Hybridization Filter Preparation
Transfer candidate cultures to 5 mL brain heart infusion or
trypticase soy broth and incubate 18-24 h at 37°. If culture
must be stored before analysis can be performed, aseptically
add 2.0 mL culture to 0.5 mL freezing soln, (w). Store at -70°
if possible. (Note: Frost-free freezers will decrease culture vi-
ability and may result in loss of virulence determinants. If cul-
tures must be stored at -20°, use non-frost-free unit. This pre-
caution holds for all frozen material in this procedure.)
Aseptically inoculate 5 mL rich broth with portion of frozen
bacterial culture. Sterile cotton swabs are well suited for this
purpose. Always include known pos. and neg. control cultures
on every fiber (see below). (If culture is not thawed, it may
be reused innumerable times.) Incubate culture 18-24 h at 37°.
At same time, aseptically prepare 100 x 15 mm petri plates
contg either brain heart infusion or trypticase soy agar and dry
inverted 18-24 h at 37°. After inoculating cultures in orderly
array and ensuring that resulting colonies will not ultimately
merge while growing, inoculate agar plates with test cultures,
using sterile microbiological needle, toothpick, cotton swab,
or replicator; 9-10 mm is convenient distance between cul-
tures. Record location of each culture; it is vital that culture
patterns and resulting autoradiogram(s) can be oriented un-
ambiguously. Prep, multiple plates and concomitant filters be-
cause hybridization procedure may have to be repeated and
number of steps to be repeated is thereby lessened. Incubate
plates inverted 18-24 h at 37°. Mark cultures failing to grow;
otherwise, false-neg. results may be reported.
Label Whatman No. 541 cellulose filters, (f), 82-85 mm
diam., using soft lead pencil, and also mark filter so it can be
oriented unambiguously after replication. (Note; Other man-
ufacturers make filters with physical properties equiv. to
Whatman No. 541. However, DNA binding abilities of such
filters are not always suitable for use in DNA hybridization.)
Apply filter so that side with pencil markings faces colony
array on agar surface of plate contg colonies. Wetting initial
edge of filter paper and rolling to opposite edge usually elim-
inates formation of air pockets. If air bubbles are entrapped
between filter and agar plate, remove by applying gentle pres-
sure with glass spreader. This maneuver also ensures more ef-
ficient attachment of cultures to filter paper, but care must be
taken to avoid spreading colonies because of excessive pres-
sure. Filters may be peeled from plate immediately, but more
definitive reactions are usually obtained if filter remains sit-
uated 1-2 h. (Note: Colony array on filter is now mirror image
of array originally applied to agar plate.)
Lyse colonies replicated onto filters by transferring filters
with colony side up onto absorbent cellulose filters, ca 85 mm
diam. (such as Whatman No. 1 or Schleicher & Schuell No.
597) contained in glass 100 x 15 mm petri plates and previ-
ously wetted with 1 .5-2.0 mL lysis mixt. A, (a). Be sure that
no air is entrapped between filters. Heat filters in glass plate
for 3-5 min in steam. Transfer steamed filters to glass petri
plates contg absorbent cellulose filters previously wetted with
1 .5-2.0 mL lysis mixt. B, (b). Again, be sure that no air pockets
result. Maintain filters in horizontal position when transferring
so that lysed colonies (DNA) will not become confluent. Let
filters become completely neutralized by remaining situated 5~
10 min.
If filters are not to be used immediately, air-dry on absorb-
ent paper at room temp, and store under vac. between filter
papers. Such filters have been kept ca 1 year without notice-
able change in results.
E Oligodeoxyribonucleotide Synthesis
(Note 1: A number of companies will custom-synthesize oli-
godeoxyribonucleotides. Also, several oligodeoxyribonucleo-
tide synthesis systems are com. available, both automated and
manual. Results are generally satisfactory if manufacturer's in-
structions are followed. This method uses one of com. avail-
able, automated synthesizers and procedure described below
is meant to serve only as example.)
(Note 2: All solns for prepn and isolation of synthetic oli-
godeoxyribonucleotides should be prepd in deionized H 2
passed thru 0.2 jxm filter ("Versacap Filter Unit," Gelman Sci-
ences, Inc., or equiv.).)
According to manufacturer's instructions, use Applied Bio-
systems, "fast" cycle but with following modifications of step
times: trichloroacetic acid to column detritylation step, 75 s
(retained in fraction collector); CH 3 CN to column post-detri-
tylation step, 50 s (also retained and pooled with above in frac-
tion collector); CH 3 CN to column, pre-coupling step, 120 s;
coupling step, 180 s; capping step, 120 s. Synthesis is ended
with dimethoxytrityl (DMT) group retained at 5' terminus. Au-
tomated cleavage from support is achieved with coned NH 4 OH
at room temp, for 1 h. DiL delivered NH 4 OH soln with 1 mL
coned NH 4 OH, heat 10 h at 60° in 3.7 mL vial with Teflon-
lined screw cap (Supelco, or equiv.). Let cool to room temp.
Add 50 jxL triethylamine, (qq). Evap. NH 3 with N stream to
ca 2 mL.
F. Quantitation of Coupling Yield
To det. isolated product yield (see below) and ensure sat-
isfactory coupling at each addn, theoretical yields of product
must be calcd. Dil. each collected fraction (from detritylation
and post-detritylation steps above) to 5 mL with DMT-assay
soln (kk). Mix each fraction well and read A at 530 nm. Use
assay soln (kk) as reference std. Compare A with that of pre-
vious fraction to det. coupling efficiency of each step (gen-
erally 97-99%). To det. overall theoretical yield, multiply all
individual step-yields.
G. Oligodeoxyribonucleotide Purification and Isolation
To det. chromatgc properties of prepn, perform anal. run.
Set detector for 0.1 AUFS. Inject 10 |ulL soln evapd to 2.0
mL. In ambient temp, column, start 20-30% gradient (at 1%/
min) of CH 3 CN in triethylammonium acetate buffer, (II). Con-
tinue at 30% CH 3 CN after 10 min. Generally, major DMT-
product elutes at 10 ± 3 min. After elution time is detd, repeat
chromatgy on preparative scale (inject 100 |ulL crude soln, 1.0
AUFS), Collect center position of major peak.
H. Oligodeoxyribonucleotide Processing
Before synthetic oligonucleotide can be used as substrate for
polynucleotide kinase, LC solvs and DMT group must be re-
moved. Cone, collected LC fraction using N ca 10-20 min to
remove most CH 3 CN. Cone, sample to dryness using concen-
trator/dryer, (s). Add 1 mL 3% (v/v) acetic acid to remove
DMT protecting group. Vortex-mix to dissolve. After 5—10
min at room temp., freeze in crushed dry-ice and cone, using
AOAC Official Methods of Analysis (1990)
Escherichia Coli
447
concentrator /dryer, (s). Dissolve residue in 1 mL H 2 0. Add
1 mL anal . grade ethyl acetate to ext org . impurities and vor-
tex-mix thoroiy. Let org. layer sep. from aq. layer contg DNA
and possible LC column particulates (centrf. if necessary). Re-
move org. layer with Pasteur pipet and discard. If insoluble
LC column particulates are present, syringe-filter DNA soln
thru type HV, 0.45 [xm filter (u). Let DNA soln gravity-filter
and collect residual soln by rapidly depressing syringe plunger.
Remove 50 uX aliquot from 1 mL filtered DNA soln for A
measurement. Cone, both remaining sample and A aliquot to
dryness. Dissolve aliquot in 1 mL H 2 and measure A at 260
nm. Since V20 of sample has been removed, multiply reading
by 19 to obtain A units in total purified sample. Discard A
aliquot. Multiply A in total purified sample by 10 (because
only 10% of total synthesis reaction was purified) to obtain A
units of entire isolable product. Compare this yield with calcd
value (1 |xmole x theoretical yield [see above] x molar A of
oligonucleotide synthesized x 10~ 3 ) to det. yield of isolable
product. Molar A is calcd by adding number of purines (dA
plus dG) times 14 000 plus number of pyrimidines (dC plus
T) times 7000. These factors are molar extinction coefficients
and 10~ 3 is used to convert molar A to (jimoles/mL which is
a millimolar concn.
/. End-Labeling of Synthetic DNA
Synthetic oligodeoxyribonucleotides are rehydrated to ca 5—
10 A 260 units (ca 150-350 (xg/mL) to serve as stock soln (j).
One A 260 unit corresponds to ca 33 (Xg/mL single-stranded DNA.
Molecular wt of 22- base, single-stranded DNA molecule is ca
7260. Prep. 10|xM working soln for each DNA probe (10
pmoles/uX, 72.6 |xg/mL). If desired, STH and STP synthetic
DNA probes can be combined into single soln, 5(jlM in each
probe, (bb).
Mix 5 (xL DNA probe soln, (bb), 2.5 uX 10X kinase buffer,
(p), 15 (jlL H z O, 1.5 |xL Cy- 32 P) ATP, (q), and 1 pX T4 ki-
nase, (r), in 500 (xL plastic conical centrf. tube, (a), on ice.
Add kinase, (r), last and return enzyme immediately to -20°
because it is quite heat-labile. Centrf., (m), 2-3 s to ade-
quately mix reagents. Incubate at 37° in H 2 bath or dry block
heater, (b), 1 h. Add 2 uX 0.5M EDTA, (f), to terminate
reaction. Add 1.6 julL 4.0M ammonium acetate soln, (y), to
bring ammonium acetate concn to 0.25M before applying sam-
ple to NACS PREPAC column.
Unincorporated 32 P is removed by binding DNA to NACS
PREPAC column, (h). Equilibrate column with 0.25M am-
monium acetate, (x), 2 h. Load reaction mixt. onto column
and wash, using gravity or very gentle pressure, with ca 4 mL
loading buffer, (x), to remove free ATP. Elute bound DNA
with 200 |uX aliquots of eluting buffer, (y). Do not force liq.
thru column rapidly. Collect three 200 uX fractions in 500 uX
plastic tubes, (a). Spot 2 uX of each fraction onto ca 2 x 2
cm paper (e.g., Whatman 3MM), dry, add ca 5 mL scintil-
lation fluid, (aa), and assay radioactivity by scintillation
counting. Geiger-Mueller counter, (i), may suffice if properly
calibrated and used. Most labeled DNA is eluted from column
in fractions 1 and 2. Pool fractions and count triplicate 2 uX
portions as described above. Est. total vol. of prepn by care-
fully drawing into plastic 1 mL pipet. Calc. total amt of ra-
dioactivity recovered in prepn. Usually, 1-2 x 10 8 cpm is
obtained if specific activity of ATP, (q), is 3000-7000 Ci/
mmol. Store at -20°.
/. Colony Hybridization
Freshly prep. 50 mL hybridization mixt., (c). Boil 1.0 mL
sonicated calf thymus DNA, (g), 5 min in H 2 bath and add
to hybridization mixt., (c). Dispense 10 mL sonicated calf thy-
mus DNA-hybridization mixt. into 100 x 15 or 20 mm plastic
petri dish and insert cellulose filter contg lysed colony array.
To use std amt of probe for each hybridization, det. vol. of
probe DNA soln required to contain 1 x 10 6 cpm after cor-
recting for 14.2 day half-life of 32 P. Add 1 X 10 6 cpm probe
DNA to soln contg filter. Mix briefly and incubate plate over-
night at 40°.
Wash hybridized filters free of 32 P-labeled DNA not specif-
ically bound to DNA from colonies on filter by removing filter
from hybridization mixt. and rinsing 5-10 s in plastic petri
dish contg 10 mL 6X SSC, (h). Drain and recover filter with
6X SSC. Incubate 1 h at 50°. Again, drain plate, recover with
6X SSC, and incubate 1 h at 50°. Finally, rinse filter 5-10 s
at room temp, in 2X SSC, (i). Air-dry on absorbent paper at
room temp, to prevent curling. Mount filter to 8 x 10 in. stiff
paper (e.g., Whatman 3MM) using small pieces of tape. Cover
with plastic or glassine sheet (such as document or neg. holder)
to prevent contamination of intensifying screens in X-ray film
holders.
K. Autoradiography
Exposure time is dictated by amt of radioactive DNA bound
to filter. If increase above background exceeds 10 cps when
Geiger-Mueller counter is held over filter, it is likely that pos.
reaction will be visible after 4 h exposure at room temp. How-
ever, if increase of 2-3 cps is observed, enclose loaded film
cassette in sealed plastic bag and expose film overnight, pref-
erably at -70° or at least -20°. If -70° is not available, cas-
sette can be sandwiched between slabs of dry ice to reduce
exposure time.
In darkroom, place X-ray film onto plastic-covered filter in
cassette film holder with intensifying screens. Expose film for
appropriate length of time as detd above. After exposure, let
cassette equilibrate at room temp, (to prevent moisture accu-
mulation) before removing plastic bag. Develop X-ray film by
following manufacturer's instructions. If spots are too faint or
too intense for analysis, expose new film for appropriate length
of time.
L. Reporting of Results
Lysed colonies of E. coli strains contg DNA coding for heat-
stable enterotoxins will bind radioactively labeled oligonu-
cleotide probe for ST. These radioactive lysed colonies will
expose X-ray film, and dark spots will be evident after de-
velopment. Det. if each unknown culture is pos. or neg. by
comparing spot intensity to that of pos. and neg. culture con-
trols. However, many factors can influence quality of these
results: size of colonies, amt of cellular debris, amt of DNA
per lysed colony, hybridization and washing temps, hybrid-
ization time, specific activity of probe, and length of autora-
diogram exposure. Well documented pos. and neg. controls
must be present on every filter to ensure that the procedure
has been performed correctly and that compensation for non-
specific binding of labeled probe DNA (neg. colonies that may
be seen as faint spots) has been made.
If neg. control cultures exhibit faint spots, and pos. culture
spots are intense, re-wash filter(s) in 6X-SSC, (h), at 52-55°
twice for 1 h each time. Dry filters and re-expose autoradio-
gram. Take care because thermal stability of oligonucleotide
hybrids is much less than that of longer DNA molecules.
M. Troubleshooting
Unsatisfactory autoradiograms can result from several fac-
tors, some of which have been listed in the previous section.
False-neg. results can be due to spontaneous loss of plasmids,
especially when strains are cultivated excessively under non-
selective laboratory conditions (i.e., re-isolation or further
subculture). Also, hybridization and/or washes at excessively
high temps can result in decreased DNA probe binding which
448
Microbiological Methods
AOAC Official Methods of Analysis (1990)
in turn can lead to neg. observation. Occasionally, very large
colonies do not become affixed to filters and cellular material
is lost from hybridization filters. False-pos. results can be ob-
served if either hybridization or washing temp, is too low.
Nonspecific DNA probe binding will occur. Autoradiogram
exposures of excessive time can result in overemphasis of lim-
ited, nonspecific binding of probe to neg. cultures; this may
be falsely reported as pos. results. Other possible sources of
error and their remedies have been discussed (984.34N; J AOAC
67, 801(1984)).
Finally, it is essential to note that resulting autoradiogram
spot arrays are mirror images of plate inoculation patterns. This
is not the case with 984.34. Results are accurately read if au-
toradiograms are reversed (left to right) before interpretation.
Films must be marked so that they can be unambiguously ori-
ented with recorded location of each test culture.
Ref.: JAOAC 69, 531, 151A(1986).
984.35 Escherichia coli Enterotoxins
Mouse Adrenal Cell and Suckling Mouse Assays
First Action 1984
Final Action 1987
A. Principle
When exposed to cholera toxin or heat-labile enterotoxin of
Escherichia coli, mouse adrenal cell line, designated Yl, re-
sponds by change in morphology from flat to round. Response
is mediated by adenyl cyclase and is irreversible. Intragastric
administration of heat-stable enterotoxin of E. coli to suckling
mouse causes fluid accumulation in intestinal lumen. This
measurable response is mediated by guanyl cyclase.
B. Media and Reagents
(a) Casamino acids -yeast extract {CAYE) broth. — So In a:
Casamino acids, 20 g; yeast ext, 6 g; NaCl, 2.5 g; K 2 HP0 4 ,
8.71 g; adjust to pH 8.5 with 0.1N NaOH, and to final vol.
of 1 L. Soln b: MgS0 4 , 50 g; MnCl 2 , 5 g; FeCl 2 , 5 g; dissolve
in min. amt of 0.01N H 2 S0 4 , and adjust to final vol. of 1 L
with H 2 0.
Add 1 mL soln b to soln a before sterilizing; autoclave 15
min at 121 °C after dispensing.
(b) Trypticase soy -yeast extract (TSYE) broth. — Com.
trypticase soy broth rehydrated as directed with 0.6% yeast ext
added.
(c) Tissue culture media. — (7) Growth medium: Ham's F-
10 with glutamine and NaHC0 3 (Flow Laboratories), 100 mL;
newborn calf serum, 10 mL; antibiotic cone. (5000 IU peni-
cillin G, and 5000 \xg streptomycin /mL), 1 mL. (2) Mainte-
nance medium: Same as (1) except serum level is 1%.
(d) Dulbeccos PBS, pH 7.5.— NaCl, 8 g; KC1, 200 mg;
Na 2 HP0 4 .7H 2 0, 2.16 g; KH 2 P0 4 , 200 mg; make up to 1 L
with H 2 and autoclave 15 min at 121°.
(e) Trypsin.— 0.25% in Dulbecco's PBS.
(f ) Cholera enterotoxin. — 1 mg/mL when reconstituted as
directed (Schwartz /Mann).
(g) Mice. — Outbred white Swiss mice, 3-5 days old.
(h) Evans blue.~2% soln.
C. Equipment and Materials
(a) Serological pipets. — 1 and 5 mL, small tip.
(b) Pipets.— 25 )ulL.
(c) Swinnex filters. — 25 mm, 0.45 u,m membrane.
(d) Disposable syringes .—5 mL, accommodating Swinnex
filters.
(e) Tissue culture flasks . — Plastic, 75 sq. cm.
(f) Vertical laminar flow hood. — Biological containment,
equipped with HEPA filters.
(g) Incubator. — C0 2 , set at 35° and 5% C0 2 .
(h) Microliter tissue culture plates. — 96 wells, with lids,
sterile.
(i) Syringe. — 1 mL, disposable.
(j) Animal feeding needle. — 24 gage, 1 in., straight.
(k) Needle. — 27 gage. Not needed if per os procedure is
followed.
Labile Toxin (LT)
D. Day 1
(a) Inoculate control cultures and cultures to be assayed into
TSYE broth in 16 X 125 mm screw-cap tubes. Incubate in
shaker incubator overnight at 37°. Both known enterotoxin-
pos. and enterotoxin-neg. E. coli cultures should be used as
controls, in addition to cholera toxin-pos. control.
(b) Remove growth medium from confluent layer of Yl cells
in 75 sq. cm flask. (One flask will provide enough cells for 2
microtiter assay plates.) Wash cell layer with PBS. Remove
PBS wash and add 5 mL trypsin. After 1 min exposure, re-
move 4.5 mL trypsin and place flask in 35° incubator. Observe
at 5 min intervals for cell detachment. When cell sheet has
detached, add 5 mL growth medium and pipet repeatedly to
break up cell clumps.
(c) Add cells from Day 1 (b) to 35 mL growth medium
(total vol. is 40 mL) in small beaker. Stir this suspension while
pipeting 0.2 mL into each well of two 96- well microtiter plates,
using macro! iter pipet. Cover finished plates and incubate ca
48 h at 35° in C0 2 incubator.
E. Day 2
(d) Add 2 drops of previously prepd starter culture, Day 1
(a), to 10 mL CAYE broth in 50 mL erlenmeyer and incubate
24 h at 37° in shaker incubator at 250 rpm.
F. Day 3
(e) Centrf. 24 h culture from Day 2 (d). (Twenty min at
2500 rpm will clarify most cultures of E. coli.). Filter super-
nate thru 0.45 jim membrane in Swinnex syringe-end filter
holder.
(f ) Divide filtrate into 2 portions. Heat one portion 30 min
at 80°; leave other portion unheated. Both heated and unheated
portions are assayed. Heated portion serves as neg. control.
Store both at 4°.
(g) Prep, cholera toxin soln of 1 ng CT/mL in PBS. Soln
is used as pos. control for cell reactivity. Note: CT is unstable
at this concn, even at 4°; prep, daily from stock soln.
(h) Remove microtiter plates prepd in Day 1 (c) and replace
growth medium with maintenance medium, 0.2 mL/well.
(i) Add 0.025 mL assay and control solns to one or more
wells (4/test substance recommended) of microtiter plate, us-
ing microtiter syringe. Incubate microtiter plates 30 min at 35°
in C0 2 incubator.
Replace maintenance medium, 0.2 mL/well. Incubate mi-
crotiter plates overnight at 35° in C0 2 incubator.
G. Day 4
( j) Examine microtiter plates for degree of rounding, start-
ing with controls. Score rounding as follows:
= no rounding
1 = ca 25% rounding
2 = ca 50% rounding
3 = ca 75% rounding
4 = 100% rounding
AOAC Official Methods of Analysis (1990)
Staphylococcus 449
Score >2 is reported as pos. for LT. Score <2 is recorded as
neg. Neg. controls should show <10% rounding.
Stable Toxin (ST)
H. Day 1
(a) Inoculate starter culture. Procedure is identical to Day
1 (a), and need not be repeated when both assays (LT and ST)
are done concurrently.
/. Day 2
(b) Inoculate CAYE assay culture. Procedure is identical to
Day 2 (d), and need not be repeated when both assays (LT
and ST) are done concurrently.
J. Day 3
(c) Prep, cultures for assay. Procedure is same as Day 3 (e)
and (f ), and need not be repeated if both assays (LT and ST)
are done concurrently. Heated portion only is used in ST assay
(suckling mouse). Material for ST assay may be stored at 4°
for several days without noticeable loss of activity.
K. Day 4
(d) Add 2 drops of sterile Evans blue to 1 mL filtrate to be
assayed.
(e) Inject suckling mice precutaneously with 0. 1 mL filtrate
into milk-filled stomach. Use tuberculin syringe and 27 gage
needle. Inject min. of 4 mice for each filtrate. Discard all in-
jections in which blue filtrate is not confined to stomach (im-
mediate visual inspection) or
(f ) Inject per os 0. 1 mL filtrate into stomach of each mouse,
using tuberculin syringe equipped with 24 gage feeding needle.
This procedure may be used instead of precutaneous injection
described in Day 4 (e). Either method works and preference
is based on analyst's familiarity. Both methods yield equiv.
results.
(g) Hold mice 3 h at room temp. Sacrifice mice by C0 2
inhalation. Open each abdomen and remove intestinal tract with
exception of stomach and liver. Pool intestines treated with
same filtrate in tared weighing vessel. Pool remainder of car-
casses in another tared weighing vessel. Weigh both vessels
on balance accurate to 0.01 g. Compute ratio of intestine wt/
carcass wt.
L. Interpretation
Report ratio >0.083 as pos. for ST. Report ratio <0.074 as
neg. for ST. Ratio 0.075-0.082 calls for re-examination of
filtrate involved.
Ref.: J AOAC 67, 946(1984).
STAPHYLOCOCCUS
980.37 Staphylococcus aureus in Foods
Microbiological Method
Final Action 1984
Repealed First Action 1987
(Applicable to detection and enumeration of small numbers of
S. aureus in raw food ingredients and non-processed foods ex-
pected to contain large population of competing species.)
Inoculate 3 tubes of trypticase soy broth with 10% NaCl,
966.23A(f ), at each test diln with 1 mL aliquots of decimal
dilns of sample. Max. diln of sample must be high enough to
yield neg. end point. Incubate 48 hr at 35-37°.
Using 3 mm loop, transfer 1 loopful from each growth-pos.
tube to dried Baird-Parker medium plates, 966.23A(e)(3). Streak
so as to obtain isolated colonies. Incubate 45-48 hr at 35-37°.
From each plate showing growth, pick >1 colony suspected
to be S. aureus, 966.23 A(e)(4). Transfer colonies to tubes contg
0.2 mL brain heart infusion (BHI) broth, 967.25A(r), and
emulsify thoroly. Withdraw I loopful of resulting culture sus-
pension and transfer to agar slant contg any suitable mainte-
nance medium, e.g. , trypticase soy agar: Suspend 40 g powder
in 1 L H 2 0. Let stand 5 min and mix thoroly. Heat gently with
occasional agitation and boil ca 1 min or until soln is com-
plete. Autoclave 15 min at 121°. Incubate BHI culture sus-
pensions and slants 18-24 hr at 35-37°. Retain slant cultures
at room temp, for ancillary or repeat tests, in case coagulase
test results are questionable.
To BHI cultures add 0.5 mL reconstituted coagulase plasma
with EDTA, 966.23A(i), and mix thoroly. Incubate at 35-37°
and examine periodically over 6 hr interval for clot formation.
Any degree of clot formation is considered pos. reaction. Small
or poorly organized clots may be observed by gently tipping
tube so that liq. portion of reaction mixt. approaches lip of
tube; clots will protrude above liq. surface. Coagulase-pos.
cultures are considered to be S. aureus. Test pos. and neg.
controls simultaneously with cultures of unknown coagulase
reactivity. Recheck doubtful coagulase test results on BHI cul-
tures which have been incubated at 35-37° for >18 but <48
hr.
Report most probable number (MPN) of S. aureus /g from
tables of MPN values, Table 966.24.
987.09 Staphylococcus aureus in Foods
Most Probable Number Method for Isolation
and Enumeration
First Action 1987
(Applicable to detection and enumeration of small numbers of
S. aureus in food ingredients and food expected to contain
large population of competing species)
A. Apparatus
(a) Pipets. — 1.0 mL with 0.1 mL graduations; 5.0 mL and
10.0 mL with 0.5 and 1.0 mL graduations.
(b) Blender. — Waring Blendor, or equiv., 2-speed model,
with high-speed operation at 16 000-18 000 rpm, and 1. L
glass or metal blender jars with covers. One jar is required for
each analytical unit.
(c) Mixer. — Vortex Genie, or equiv.
(d) Water bath.— Maintained at 35-37°.
(e) Incubator. — Maintained at 35°.
B. Media and Reagents
(a) Trypticase (tryptic) soy broth with 10% sodium chloride
and 1% sodium pyruvate . — Add 95 g NaCl to 1 L soln of 17.0
g trypticase or tryptose (pancreatic digest of casein), 3.0 g
phytone (papaic digest of soya meal), 5.0 g NaCl, 2.5 g
K 2 HP0 4 , 2.5 g dextrose (dehydrated trypticase or tryptic soy
broth is satisfactory), and 10 g sodium pyruvate. Adjust to pH
7.3. Heat gently if necessary. Dispense 10 mL into 16 X 150
mm tubes. Autoclave 15 min at 121°. Final pH, 7.3 ± 0.2.
Store <1 month at 4 ± 1°.
(b) Physiological salt soln. — Dissolve 8.5 g NaCl in 1 L
H 2 0. Autoclave 15 min at 121° and cool to room temp.
(c) Baird-Parker medium (egg tellurite glycine pyruvate agar,
ETGPA). — (1) Basal medium. — Suspend 10.0 g tryptone, 5.0
g beef ext, 1.0 g yeast ext, 10.0 g Na pyruvate, 12.0 g glycine,
5.0 g LiC1.6H 2 0, and 20.0 g agar in 950 mL H 2 0. Heat to
bp with frequent agitation to dissolve ingredients completely.
450
Microbiological Methods
AOAC Official Methods of Analysis (1990)
Dispense 95 mL portions into screw-cap bottles. Autoclave 15
min at 121°. Final pH, 7.0 ± 0.2 at 25°. Store <1 month at
4 ± 1°.
(2) Enrichment. — Bacto EY tellurite enrichment (Difco
Laboratories) or prep, as follows: Soak fresh eggs ca 1 min in
diln of satd HgCl 2 soln (1 + 1000). Aseptically crack eggs
and sep. yolks from whites. Blend yolk and physiological sa-
line soln, (fo), (3 + 7, v/v) in high-speed blender ca 5 s. To
50 mL egg yolk emulsion add 10 mL filter-sterilized 1% K
tellurite soln. Mix and store at 4 ± 1°.
(3) Complete medium. — Add 5 mL warmed enrichment to
95 mL molten basal medium cooled to 45-50°. Mix well,
avoiding bubbles, and pour 15-18 mL into sterile 100 x 15
mm petri dishes. Store plates at room temp. (<25°) for <5
days before use. Medium should be densely opaque; do not
use nonopaque plates. Dry plates before use by 1 of following
methods: (a) in convection oven or incubator 30 min at 50°
with lids removed and agar surface downward; (b) in forced-
draft oven or incubator 2 h at 50° with lids on and agar surface
upward; (c) in incubator 4 h at 35° with lids on and agar sur-
face upward; or (d) on laboratory bench 16-18 h at room temp,
with lids on and agar surface upward.
(d) Brain-heart infusion (BHl) broth. — Dissolve infusion
from 200 g calf brain and from 250 g beef heart, 10.0 g pro-
teose peptone or gelysate, 5.0 g NaCl, 2.5 g Na 2 HP0 4 .12H 2 0,
and 2.0 g glucose in 1 L H 2 0, heating gently if necessary.
Dispense 5 mL portions into 16 x 150 mm test tubes and au-
toclave 15 min at 121°. Final pH, 7.4 ± 0.2.
(e) Desiccated coagulase plasma (rabbit) with EDTA. —
Reconstitute according to manufacturer's directions. If not
available, reconstitute desiccated coagulase plasma (rabbit)
and add Na 2 H 2 EDTA to final concn of 0.1% in reconstituted
plasma.
(f) Butterfield' $ buffered phosphate diluent. — (J) Stock
soln. —Dissolve 34.0 g KH 2 P0 4 in 500 mL H 2 0, adjust to pH
7.2 with ca 175 mL IN NaOH, and dil. to 1 L. Store in re-
frigerator. (2) Diluent. — Dil. 1.25 mL stock soln to 1 L with
H 2 0. Prep, diln blanks with this soln, dispensing enough to
allow for losses during autoclaving. Autoclave 15 min at 121°.
C. Preparation of Food Homogenate
Aseptically weigh 50 g u nth awed food sample into sterile
blender jar. Add 450 mL phosphate-buffered diln H 2 and ho-
mogenize 2 min at high speed (16 000-18 000 rpm). Use this
1:10 diln to prep, serial dilns from 10~ 2 to 10~ 6 by transferring
10 mL of 1 : 10 diln to 90 mL diln blank, mixing well with
vigorous shaking, and continuing until 10~ 6 is reached.
D. Most Probable Number Technique
Inoculate 3 tubes of trypticase soy broth with 10% NaCl and
1% sodium pyruvate, (a), at each test diln with 1 mL aliquots
of decimal dilns of sample. Max. diln of sample must be high
enough to yield neg. end point. Incubate 48 h at 35°.
Using 3 mm loop, transfer 1 loopful from each growth-pos.
tube to dried Baird-Parker medium plates, (c)(3). Vortex-mix
tubes before streaking if growth is visible only on bottom or
sides of tubes. Streak so as to obtain isolated colonies. Incu-
bate 48 h at 35-37°.
E. interpretation
Colonies of S. aureus are typically circular, smooth, con-
vex, moist, 2-3 mm in diam. on uncrowded plates, gray-black
to jet-black, frequently with light-colored (off-white) margin,
surrounded by opaque zone (ppt), and frequently with outer
clear zone; colonies have buttery to gummy consistency when
touched with inoculating needle. Occasional non-lipolytic strains
may be encountered which have same appearance, except that
surrounding opaque and clear zones are absent. Colonies iso-
lated from frozen or desiccated foods which have been stored
for extended periods are frequently less black than typical col-
onies and may have rough appearance and dry texture.
F. Confirmation Technique
For each plate showing growth, pick >1 colony suspected
to be S. aureus. With sterile needle transfer colonies to tubes
contg 0.2 mL BHI broth, (d), and to agar slants contg any
suitable maintenance medium, e.g., trypticase soy agar, std
plate count agar, etc. Incubate BHI culture suspensions and
slants 18-24 h at 35°. Retain slant cultures at room temp, for
ancillary or repeat tests, in case coagulase test results are ques-
tionable.
To BHI cultures add 0.5 mL reconstituted coagulase plasma
with EDTA, (e), and mix thoroly. Incubate at 35-37° and ex-
amine periodically over 6 h interval for clot formation. Any
degree of clot formation is considered pos. reaction. Small or
poorly organized clots may be observed by gently tipping tube
so that liq. portion of reaction rnixt. approaches lip of tube;
clots will protrude above liq, surface. Coagulase-pos. cultures
are considered to be S. aureus. Test pos. and neg. controls
simultaneously with cultures of unknown coagulase reactivity.
Recheck doubtful coagulase test results on BHI cultures which
have been incubated at 35-37° for >18 but <48 h.
Report most probable number (MPN) of S. aureus /g from
tables of MPN values, Table 966.24.
Ref.: JAOAC 70, 35(1987).
975.55 Staphylococcus aureus in Foods
Surface Plating Method for Isolation and Enumeration
First Action 1975
Final Action 1976
(Applicable for general purpose use in testing foods expected
to contain >10 cells of 5. aureus / f g. For small numbers, see
987.09.)
A. Apparatus
Sterile, bent glass streaking rods. — Hockey stick or hoe-
shape, with fire-polished ends, 3-4 mm diam., 15-20 cm long,
with angled spreading surface 45-55 mm long.
B. Determination
At each diln plated, aseptically transfer 1 mL of sample sus-
pension, 987. 09C, to triplicate plates of Baird-Parker medium,
987.09B(c)(5) ? and equitably distribute the 1 mL inoculum over
the triplicate plates (e.g., 0.4 mL-0.3 mL-0.3 mL). Spread
inoculum over surface of agar using sterile, bent glass streak-
ing rods. Avoid extreme edges of plate. Retain plates in up-
right position until inoculum is absorbed by medium (ca 10
min on properly dried plates). If inoculum is not readily ab-
sorbed, plates may be placed in incubator in upright position
ca 1 hr before inverting. Invert plates and incubate 45-48 hr
at 35-37°. Select plates contg 20-200 colonies, unless only
plates at lower dilns (>200 colonies) have colonies with typ-
ical appearance of S. aureus, 987. 09E. If several types of col-
onies are observed which appear to be S. aureus, count num-
ber of colonies of each type and record counts sep. When plates
at lowest diln plated contain <20 colonies, these may be used.
If plates contg >200 colonies have colonies with typical ap-
pearance of S. aureus and typical colonies do not appear at
higher dilns, use these plates for enumeration of S. aureus,
but do not count non-typical colonies. Select >1 colony of
each type counted and test for coagulase production, 987. 09F.
Add number of colonies on triplicate plates represented by col-
onies giving pos. coagulase test and multiply by sample diln
AOAC Official Methods of Analysis (1990)
Staphylococcus
451
factor. Report this number as number of S. aureus /g of food
tested.
Ref.: JAOAC58, 1154(1975).
976.31 Staphylococcal Enterotoxin
in Foods
Microslide Gel Double Diffusion Test
First Action 1976
Final Action 1977
(Detects 0.1-0.01 |mg enterotoxin/mL and is applicable to de-
tection of enterotoxin in culture fluids and coned food exts)
A. Principle
Pptn line occurs when serological type of enterotoxin dif-
fuses thru gel and reacts with its specific antibody. Coales-
cence with ref. pptn line which results from serological reac-
tivity of enterotoxin serotype and specific antibody confirms
identity.
B. Apparatus
(a) Debubblers. — Fine glass rods. Prep, by pulling glass
tubing very fine, as in making capillary pipets. Break into ca
6 cm lengths and seal ends in flame.
(b) Electrical tape. — Insulating tape, 0.25 X 19.1 mm
(Temflex 1700, 3M Co., Electrical Products Div., Bldg 225-
4N-05, 3M Center, St Paul, MN 55144-1000, or equiv.)
(c) Microscope slides. — Plain glass, pre-cleaned, 7.62 x
2.54 cm (3 x 1"), 0.96-1.06 mm thick.
(d) Pasteur pipets. — Prep, by drawing out ca 7 mm od glass
tubing or use disposable 30 or 40 |xL pipets (Kensington Sci-
entific Corp., 1399 64th St, Emeryville, CA 94608, or equiv.).
(e) Petri dishes. — 20 x 150 mm and 15 x 100 mm.
(f) Plastic templates. — See Fig. 976.31 A. (Available from
Toxin Technology, 845 E. Johnson St, Madison, WI 53703.)
(g) Silicone lubricant. — High vac. grease (Dow Corning
Corp., or equiv.).
(h) Staining jars. — Coplin or Wheaton jars.
(i) Sterile bent glass spreaders. — Bend glass rods like hockey
sticks and fire polish.
(j) Water- saturated synthetic sponge strips. — Approx. 1.5
x 1.5 x 6.5 cm H 2 0-satd absorbent cotton is also satisfactory.
C. Media and Reagents
(a) Agar soln for coating slides. — 0.2%. Add 2 g bacteri-
ological grade agar to 1 L boiling H 2 and heat until agar
dissolves. Pour 20-30 mL portions agar into 180 mL (6 oz)
prescription bottles or equiv. containers and store at room temp.
Remelt when needed for coating slides.
(b) Brain-heart infusion (BHI) agar. — 0.7% (w/v). Adjust
BHI broth to pH 5.3; add bacteriological grade agar to prep.
0.7% concn and dissolve by boiling gently. Distribute in 25
mL portions into 25 X 200 mm test tubes, and autoclave 10
min at 121°. Immediately before use, aseptic ally empty tubes
of sterile medium into 15 x 100 mm petri dishes.
(c) Enterotoxin antisera. — Dil. lyophilized sera (Toxin
Technology) with normal physiological saline according to
specific instructions of supplier. Store liq. stocks (highly coned)
and working dilns of antisera at 4°; for long term storage, freeze -
drying or freezing is recommended.
(d) Enterotoxin references. — Rehydrate lyophilized enter-
otoxin prepns, (c), according to specific instructions of sup-
plier.
(e) Gel diffusion agar. — Add 1.2% purified agar (Noble
special agar, Difco Laboratories) to boiling fluid base (0.85%
NaCl-0.80% Na barbital with final concn of 1:10,000 mer-
thiolate (Eli Lilly and Co., Pharmaceutical Div., Lilly Cor-
porate Center, Indianapolis, IN 46285, or equiv.) adjusted to
pH 7.4). Filter hot agar thru 2 layers of anal, grade paper and
store in 15-25 mL portions in screw-cap bottles.
(f) Staining soln. — 0.1% Thiazine Red R stain (Fluka
Chemical Corp., or equiv.) in 1% HOAc.
(g) Sterile distilled water. — Dispense 5 mL distd H 2 into
tubes and autoclave 15 min at 121°. Normal physiological sa-
line may be substituted for H 2 0.
(h) Turbidity std.—\% BaCl 2 -l% H 2 S0 4 (1 + 99) (No. 1
of McFarland nephelometer scale) .
D. Preparation of Sample
Select ^4 isolated staphylococcal colonies from enumera-
tion and recovery media, and streak nutrient media agar slants,
or equiv. Incubate slants 18-24 hr at 35-37°. Add loopful of
growth from agar slants to 5.0 mL sterile distd H 2 or saline
and prep. aq. suspension of organisms from each slant which
is equiv. to turbidity of No. 1 tube of McFarland nephelometer
scale (ca 3 x 10 8 organisms/mL). Inoculate surface of semi-
solid BHI agar with 4 drops aq. suspension of organisms de-
Dimensions of Well
Template
0.3175 cm
0.45 cm
FIG. 976.31 A— Plastic template schematic for microslide assembly
452
Microbiological Methods
AOAC Official Methods of Analysis (1990)
(1) Bivalent
1. Combination Antisera
(e.g., Anti A and B)
2. Prepn under test
3. Ref. enterotoxin (e.g.,
4. Prepn under test
5. Ref. enterotoxin (e.g.,
Type A)
Type B)
(2) Monovalent
1. Antiserum (e.g., Anti A)
2. Dilns of prepn under test
3. Ref. enterotoxin (e.g.. Type A)
4. Dilns of prepn under test
5. Dilns of prepn under test
FIG. 976.31 B — Arrangement of antisera and homologous reference enterotoxins (1) when assaying preparation(s) under test for
presence of 2 staphylococcal enterotoxins simultaneously (bivalent detection system) or (2) when assaying dilutions of preparation
under test with apparent enterotoxin excess (monovalent detection system)
livered from sterile 1 .0 mL pipet. Spread drops of aq. culture
suspension over entire surface of semisolid agar with sterile
glass rod and incubate plates upright 48 hr at 35-37°. Transfer
contents of petri dish to 50 mL centrf . tube with aid of wood
applicator stick and centrf. 10 min at 32,800 g to remove agar
and organisms. Examine culture fluid for presence of serolog-
ically identifiable enterotoxins.
E. Preparation of Slides
Wrap double layer of elec. tape around pre-cleaned micro-
scope slide, leaving 2.0 cm space in center, as follows: Start
piece of tape ca 9.5-10 cm long ca 0.5 cm from edge of bot-
tom surface of slide and wrap tightly around slide twice. Wipe
area between tapes with cheesecloth soaked with alcohol, and
dry with dry cheesecloth. Coat upper surface area between tapes
with 0.2% bacteriological grade agar as follows: Melt 0.2%
agar, and maintain at ^55° in screw-cap bottle. Hold slide
over beaker on hot plate adjusted to 65-85° and pour or brush
0.2% agar over slide between 2 pieces of tape. Let excess agar
drain off, wipe bottom surface of slide, and collect agar in
beaker for reuse. Place slide on tray and dry in dust-free atm.
(e.g., incubator). If slides are not clean, agar will not coat
slides uniformly.
F. Preparation of Slide Assemblies
Prep, plastic templates according to specifications in Fig.
976. 31 A. Spread thin film of silicone grease on side of tem-
plate that will be placed next to agar (i.e., side with smaller
holes). Place ca 0.4 mL melted and cooled (55-60°) 1.2% gel
diffusion agar between tapes. Immediately lay silicone-coated
template on melted agar and edges of bordering tapes. Place
1 edge of template on 1 piece of tape, and bring opposite edge
to rest gently on other piece. Sat. strips of synthetic sponge
(ca 1.5 x 1.5 x 6.5 cm) with H 2 0, and place 2 strips on
periphery of each 20 x 150 mm petri dish. Place slide in prepd
petri dish (2-4 slide assemblies /dish) soon after agar hardens,
and label slide.
G. Slide Gel Diffusion Test
To prep, record of assay, draw hole pattern of template on
record sheet and indicate number (same as that used for slide)
and contents of each well. Place suitable diln of antiserum or
sera in central well, homologous ref. enterotoxin in peripheral
well(s), and material under examination in well adjacent to
that contg ref. enterotoxin. See Fig. 976.31 B(7) for reagent
arrangement for simultaneous detection of 2 enterotoxin types
(bivalent detection system). Prep, control slide with only ref.
toxin and antienterotoxin serum to det. proper reactivity of re-
agents. Fill wells to convexity with reagents, using Pasteur or
disposable 30 or 40 u,L pipet. Partially fill capillary pipet with
soln and remove excess liq. by touching pipet to edge of sam-
ple tube. Slowly lower pipet into well until it touches agar
surface, and fill to convexity. Remove trapped air bubbles from
all wells by probing with debubbler, (a), against dark back-
ground. Let slides incubate 48-72 hr at room temp, in covered
petri dishes contg moist sponge strips (24 hr slide incubation
at 35° is generally sufficient for testing of culture fluids). Care-
fully remove template by sliding it to 1 side. If necessary,
clean slide by dipping in H 2 and wiping bottom of slide.
Enhance lines of pptn by immersing slide in staining soln, (f),
5-10 min. To preserve slide as permanent record, rinse any
reactant liq. remaining on slide by dipping in H 2 and then
immerse slide in each of following baths 10 min: staining soln,
1% HO Ac, 1% HO Ac, and 1% HOAc contg 1% glycerol. Drain
excess fluid from slide and dry in 35° incubator. After pro-
longed storage, lines of pptn may not be visible until slide is
immersed in H9O.
H. Interpretation
Examine slide for lines of pptn by holding at oblique angle
to light source against dark background. Coalescence of test
sample lines of pptn with ref. line(s) of pptn indicates pos.
reaction. Fig. 976.3 1C shows microslide gel diffusion test as
bivalent detection system: Antisera to enterotoxins A and B
are in well 1; known ref. enterotoxins A and B are in wells 3
and 5, resp., to produce ref. lines of A and B; prepns under
test are in wells 2 and 4. Interpret 4 reactions as follows: (1)
No line development between test prepns — absence of enter-
otoxins A and B; (2) coalescence of test prepn line from well
4 with enterotoxin A ref. line (intersection of test prepn line
with enterotoxin B ref. line) — absence of enterotoxins A and
B in well 2, presence of enterotoxin A and absence of enter-
otoxin B in well 4; (3) presence of enterotoxin A and absence
of enterotoxin B in both test prepns; and (4) absence of en-
terotoxins A and B in test prepn in well 2, presence of enter-
otoxins A and B in well 4. Operator can simplify assay by
AOAC Official Methods of Analysis (1990)
Staphylococcus 453
A Enterotoxin Ref.
o
est rrepn ..^wr"" Ami A and B I est rrepr
o o
B Enterotoxin Ref.
o
o
o
o o\ o
o
o
e» o
FIG. 976.31 C— Examples of 4 possible reactions in bivalent detection system. See 976.31 H for explanation of reactions
testing only 1 prepn for presence of 2 different enterotoxin s
on same set of slides.
If concn of enterotoxin in test material is excessive, for-
mation of ref. line will be inhibited because of fast migration
of toxin thru gel, thus localizing antibody in its well. Fig.
976.31D(A) shows this inhibition of ref. line formation when
10 and 5 |xg enterotoxin/mL, resp., are used. Figs. 976.31D(#)-
(F) show ppt patterns when successively less enterotoxin is
used. If test prepn inhibits formation of ref. line as in Fig.
976.31D(A), dil. test material, utilizing monovalent system
shown in Fig. 976.31E. Reactant arrangement for assaying dilns
of prepn under test is shown in Fig. 976.31B(2). Figure 976.31E
shows microslide gel diffusion test as monovalent system in
which antiserum is placed in well I; ref. enterotoxin in well
3; and dilns of test prepn in wells 2, 4, and 5. Do not make
starting diln of culture fluid (test material) so high as to dil.
beyond reactive concn of enterotoxin.
Occasionally, atypical ppt patterns form which may be dif-
ficult for inexperienced analysts to interpret. One of most com-
mon atypical reactions is formation of lines not related to toxin,
but caused by other antigens in test material. Examples of such
patterns are given in Fig. 976. 31F, which shows microslide
*3
3
3
3
3
3
O
O
O
O
O
O
20 O
1
O 4
20 04
1
20 (61 04
1
20
\0 } 04
20
o
1
04
20 O
1
O 4
o
5
O
5
O
5
O
5
o
5
O
5
(1) Antiserum
(2) 10 /jg/ml
Enterotoxin
(3) Enterotoxin Ref.
(4) 5 jug/ml
Enterotoxin
B
(1) Antiserum
(2) 4 jjg/ml
Enterotoxin
(3) Enterotoxin Ref.
(4)3 uq/m{
Enterotoxin
(1) Antiserum
(2) 2 jug/ml
Enterotoxin
(1) Antiserum
(2) 0.5 pg/ml
Enterotoxin
(1) Antiserum
(2) 0.125 /j g/ml
Enterotoxin
<3) Enterotoxin Ref. (3) Enterotoxin Ref. (3) Enterotoxin Ref.
{1} Antiserum
(3) Enterotoxin Rel.
(4) 1 jug/ml
Enterotoxin
(4) 0.25 ;ug/ml
Enterotoxin
(4)0.0625jLjg/ml
Enterotoxin.
FIG. 976.31 D— Effect of amount of enterotoxin in test preparation on development of reference line of precipitation. See 976.31 H
for explanation of reactions
454 Microbiological Methods
AOAC Official Methods of Analysis (1990)
Enterotoxin Ref. A
***%*.
t '■ lu
! O
FIG. 976.31 E— Appearance of microslide gel diffusion test as
monovalent system
geJ diffusion test as bivalent detection system. (See reactant
arrangement in Fig. 976.315(7).) In ppt pattern 976.31F<7),
test prepn in well 4 produced atypical reaction indicated by
nonspecific line of pptn (lines of nonidentity with enterotoxin
refs A and B), which intersects enterotoxin ref. lines. In ppt
pattern 976.31F(2), both test prepns (wells 2 and 4) are neg.
for enterotoxins A and B but produce nonspecific lines of pptn
which intersect enterotoxin A and B ref. lines of pptn.
/. Slide and Template Recovery
To recover slides for reuse, clean without removing tape.
Rinse slides with tap H 2 to remove agar gel, boil 3-5 min
in tap H 2 contg mild detergent, rinse in tap H 2 and then in
distd H 2 0, immerse momentarily in alcohol, and wipe dry with
cheesecloth. Wash templates with hot (not boiling) H 2 contg
moderately strong detergent, using cheesecloth to remove sil-
icone film. Rinse templates with tap H 2 0, distd H 2 0, and al-
cohol; dry with cheesecloth, and tap alcohol out of wells. In
cleaning plastic templates, avoid exposure to excessive heat or
plastic-dissolving sol vs. Templates and especially wells must
be dry before reuse.
Ref.: J AOAC 59, 594(1976).
980.32 Staphylococcal Enterotoxin
in Foods
Extraction and Separation Methods
First Action 1980
Final Action 1981
A. Apparatus
(a) Centrifuge. — High-speed, preferably refrigerated, with
285 mL stainless steel bottles, or equiv.
(b) Dialysis sac. — 1.25 in. (32 mm) flat width tubing, av.
pore diam. 4.8 u>m. Cut piece of tubing long enough to ac-
commodate vol. of food to be extd. Soak tubing in 2 changes
of H 2 to remove glycerol coating. Tie 1 end with 2 knots
close together. Test for leaks by filling sac with H 2 and
squeezing, while untied end is held tightly with fingers. Empty
sac and place in H 2 until ready for use.
(c) Chromatographic columns. — 400 X 20 (id) mm, with
stopcock (or use rubber tube attachment with finger clamp),
packed with carboxymethyl cellulose (CMC), Whatman CM
22, 0.6 meq/g, or equiv. Pack as follows: Suspend 1 g CMC
in 100 mL 0.005 M Na phosphate buffer, pH 5.7, in 250 mL
beaker, and adjust to pH 5.7 with 0.005 Af H 3 P0 4 . Stir inter-
mittently 15 min, recheck pH, and adjust, if necessary. Pour
suspension into tube containing plug of glass wool, and let
settle. Withdraw liq. from column to within ca 25 mm of sur-
face of settled CMC in column. Place loosely packed plug of
glass wool on column. Pass 0.005MNa phosphate buffer, pH
5.7, thru column until washing is clear and pH is 5.7 (150-
200 mL). Leave enough buffer in column to cover glass wool
to prevent column from drying out.
(d) Reservoir. — Attach ca 60 cm latex tubing to stem of
separator of appropriate size and attach other end of tube to
piece of glass tubing inserted thru No. 3 rubber stopper to fit
chromatgc column. Suspend separator from ring stand above
chromatgc tube.
B. Reagents
(a) Polyethylene glycol (PEG) soln. — 30%. See 974.38B(f).
(b) Sodium phosphate buffer solns. — (J) pH 5.7, 0.2M. —
Add 0.2 M NaH 2 P0 4 (27.60 g 1H 2 0/L) to 0.2M Na 2 HP0 4
(53.61 g 7H 2 0/L) to pH 5.7. (2) pH 5.7, 0.005M.— Dil. 0.2A/,
pH 5.7, buffer with H 2 (1 + 39). Adjust to pH 5.7 with
0.005 M H3PO4. (3) pH 6.4, 0.2M.— Add 0.2 M Na 2 HP0 4 to
0.2 M NaH 2 P0 4 to pH 6.4. (4) pH 6.5, 0.05M Na phosphate-
NaCL— Add NaCl (11.69 g/L) to pH 6.4, 0.2M soln to give
0.2M NaCl (pH is ca 6.3). Dil. with H 2 (1 + 3) and adjust
to pH 6.5 with 0.05 M H3PO4 or 0.05 M Na 2 HP0 4 .
Enterotoxin Ref. A
I0T0
o
Enterotoxin Ref. B
Enterotoxin Ref. A
1 O |" O
Enterotoxin Ref. B
FIG. 976.31 F— Precipitate patterns in microslide gel diffusion test demonstrating nonspecific (atypical) lines of precipitation
AOAC Official Methods of Analysis (1990)
Sterility
455
C. Extraction of Toxin
Homogenize 100 g sample in 500 mL (or 20 g with 100
mL) 0.2 M NaCl 3 min in high-speed blender to very fine con-
sistency. Adjust to pH 7.5 with IN NaOH or HC1 if food is
highly buffered or with 0. IN if weakly buffered. Let stand 10-
15 min, recheck pH, and readjust to pH 7.5, if necessary.
Transfer homogenate to two 285 mL stainless steel bottles and
centrf. 20-30 min at 27,300 x g at 5° in refrigerated centrf.
(Lower speeds for longer times may be used.) If refrigerated
centrf. is not available, centrf. at room temp., but chill su-
pernate 1 h at 4° before filtering.
Decant supernate into beaker thru fine mesh screen (or other
filtering material (e.g., miracloth) placed in funnel). Re-ho-
mogenize solids left in centrf. bottles with 125 mL (for 100 g
sample; 25 mL for 20 g) 0.2M NaCl as above. Centrf., filter
and combine filtrate with original supernate.
D. Purification of Toxin
Place combined exts in dialysis sac, immerse sac in 30%
PEG soln and let cone, at 5° to <15 mL. Remove sac from
soln and wash outside thoroly with tap H 2 0. Soak sac in distd
H 2 1—2 min and let stand in 0.2M NaCl few min. Pour con-
tents of sac into 50 mL beaker. Rinse inside of sac with 2-3
mL portions 0.2M NaCl by running fingers up and down out-
side of sac to remove material adhering to insides. Add rins-
ings to beaker. Repeat rinsings until clear, keeping vol. at min.
Quant, transfer ext to separator, add V4-V2 vol. CHC1 3 , and
shake vigorously 10 times thru arc of 90°. Centrf. 10 min at
32,800 X g at 5°. Return mixt. to separator. Slowly drain lower
CHCI3 layer and discard. Repeat extn at least once (twice with
high protein foods). After final extn, measure vol. of aq. phase,
and dil. with 40 vols pH 5.7, 0.005M Na phosphate buffer.
Adjust pH to 5.7 with 0.005M H 3 P0 4 or Na 2 HP0 4 . Place ad-
justed soln in separator large enough to accommodate vol. for
percolation thru CMC column.
Place stopper (attached thru tubing to separator) loosely into
top of chromatgc column and slowly fill tube nearly to top
with di Id ext from separator. Tighten stopper in tube and open
stopcock of separator. Let liq. percolate thru column at 5° at
1-2 mL/min by adjusting flow rate with stopcock at bottom
of tube. Stop flow when liq. reaches top of glass wool layer.
(If liq. has passed, rehydrate column with 25 mL H 2 0.)
Wash column with 100 mL pH 5.7 0.005M Na phosphate
buffer at same flow rate, stopping flow when liq. level reaches
top of glass wool. Discard wash.
Elute enterotoxin from CMC column with 200 mL pH 6.5
0.05M Na phosphate-NaCl buffer at rate of 1-2 mL/min at
room temp. Force last of liq. from column by applying air
pressure to top of tube.
E. Concentration of Toxin
Place eluate in dialysis sac. Place sac in 30% PEG at 5° and
cone, to almost dryness. Remove sac and wash thoroly with
tap H 2 0. Soak sac in pH 6.5, 0.05M phosphate-NaCl buffer,
and remove cone, from sac by rinsing with five 2-3 mL por-
tions pH 6.5, 0.05M phosphate-NaCl buffer.
Transfer soln to separator, add V4-V2 vol. CHC1 3 , and shake
vigorously 10 times thru arc of 90°. Centrf. 10 min at 32,800
x g at 5°. Return mixt. to separator. Slowly drain lower CHC1 3
layer and discard.
Place ext in short dialysis sac (ca 16 cm). Place sac in 30%
PEG and let stand until all liq. has been removed from inside
of sac. Remove sac from soln and wash outside thoroly with
tap H 2 0. Place sac in distd H 2 1-2 min. Remove contents
of sac by rinsing inside with 1 mL portions distd H 2 until
rinse is clear, keeping vol. to min. Place rinsings in 18 X 100
mm test tube or other container (e.g., 2-3 dram vial), and
freeze-dry. Dissolve freeze-dried sample in as small vol. saline
soln. 974.38B(e) as possible (0.15-0.1 mL).
Det presence of enterotoxin as in 976.31.
Ref.: JAOAC63, 1205(1980).
STERILITY (COMMERCIAL) OF FOODS
(CANNED, LOW ACID)
972.44
Microbiological Method
First Action 1972
Final Action 1978
(Personnel with beards, mustaches, or sideburns below ear lobe
should not perform sterility examination unless these are com-
pletely covered with sterile caps and masks. Wear clean
laboratory coat for examination.)
A. Principie
"Low acid foods" means any food with finished equilibrium
pH value >4.6. Method applies only to containers which show
no distention of either end. Incubate containers >10 days at
21-35° before examination.
Com. sterility is defined as that condition achieved by ap-
plication of heat which renders food free of viable forms of
microorganisms having public health significance, as well as
microorganisms not of health significance capable of repro-
ducing in the food under normal non-refrigerated conditions
of storage and distribution.
B. Media and Reagents
See also 966.23A.
(a) Tryptone broth. — (Aerobic medium.) Dissolve 10.0 g
tryptone or trypticase, 5.0 g glucose, 1.25 g K 2 HP0 4 , 1.0 g
yeast ext, and 2.0 mL 2% ale. soln of bromocresol purple in
1 L H 2 with gentle heat, if necessary. Dispense 10 mL por-
tions into 20 x 150 mm screw-cap test tubes and autoclave
20 min at 121°. Do not exhaust before using.
(b) Modified PE-2 medium. — (Anaerobic medium.) Dis-
solve 20.0 g peptone, 3.0 g yeast ext, and 2.0 mL 2% ale.
soln of bromocresol purple in 1 L H 2 with gentle heat, if
necessary. Dispense 19 mL portions into 20 x 150 mm screw-
cap test tubes contg 8-10 untreated Alaska seed peas (Rogers
Brothers Co., Seed Div., PO Box 2188, Idaho Falls, ID 83401,
No. 423; or hardware store). Autoclave 30 min at 121°. If not
freshly prepd, heat to 100° and cool to 55° before using.
(c) Glucose starch agar. — (Aerobic medium.) Dissolve 15.0
g proteose peptone No. 3, 2.0 g glucose, 10.0 g sol. starch,
5.0 g NaCl, 3.0 g Na 2 HP0 4 , 20.0 g gelatin, and 10.0 g agar
in 1 L H 2 0, heat to bp, and autoclave 15 min at 121° in er-
lenmeyer. Aseptically pour into sterile petri dishes and allow
to solidify.
(d) Nutrient agar. — (Aerobic medium for spore produc-
tion; Difco dehydrated, or equiv.) Dissolve 3.0 g beef ext, 5.0
g peptone, and 15.0 g agar in 1 L H 2 0, heat to bp, and au-
toclave 30 min at 121°.
(e) Detergent sanitizer soln. — pHisoHex (3% hexachloro-
phene), or equiv.
C. Apparatus
(a) Can opener. — Bacti-Disc Cutter (Wilkens-Anderson Co.,
4525 W Division St, Chicago, 1L 60651, No. 10810-01), bac-
teriological can opener (Marmora Machine Co., 1956 N
Latrobe Ave, Chicago, IL 60639), or equiv.
(b) Caps. — Disposable, operating room-type (Baxter Hos-
456
Microbiological Methods
AOAC Official Methods of Analysis (1990)
pital Supply Div. , 1450 Waukegan Rd, McGaw Park, IL 60085,
or equiv.).
(c) Pipets,— Straight wall, 200-250 mm long x 7 mm id,
9 mm od (Scientific Products, Inc., No. G6100-9, cut and
fire polished, or equiv.).
D. Sampling
Conduct test in clean room. (If necessary, open room may
be used but outside windows must be closed and direct drafts
across work area must be eliminated.) If available, use laminar
flow cabinet. Strip labels from cans, examine cans for external
defects, and record descriptions. Wash cans with soap (or de-
tergent sanitizer soln) and H 2 0, and dry with clean paper towels.
Wipe counter top with 100 ppm CI soln (e.g., Clorox or dild
NaOCl soln) immediately before placing washed and dried can
on it. Place code end of can in down position and number cans
in ink or with CuS0 4 marking soln to right of side seam.
Wash hands and face with soap, and re wash hands and face
with detergent sanitizer soln. Completely cover hair with clean
disposable operating room cap.
Hold noncoded end of can over large Meker burner, just
above blue portion of flame. Heat this end of can until all
condensation is evapd; then return can to table in former po-
sition. Clean handle and blade of special can opener, (a), with
paper towel moistened with 70% alcohol, flame metal portion
enough to destroy all microorganisms, and use it to make 4
cm (1.5") diam. hole in noncoded, heated end of can. Im-
mediately and without moving can, use straight- wall sterile glass
pipet, (c), to transfer ca 2 g food to sep. tubes, 2 each of
aerobic and 2 of anaerobic media (4 total). (No other trans-
ferring tool may be substituted.) Preloosen screw cap and hold
it between little and ring fingers while transfer is being made.
Flame lips of media tubes both before and after addn of food.
When transferring food to anaerobic tubes, food must be in-
oculated into lower portion of medium. Tighten screw caps
after inoculation, incubate tubes 72 hr at 35°, and observe daily.
Record results for each tube sep.
Remove addnl >10 g food sample from each container with
sterile pipet and place in sterile 25 x 200 mm screw-cap test
tube. Use pipet-like spatula, if necessary, for this operation
(thermophilic contamination unlikely). Number tube to cor-
respond to can and refrigerate for later testing, if necessary.
E. Contamination Control
Use sterile loop or glass rod to streak plate of glucose starch
agar, (c). On table, open plate of glucose starch agar for time
equal to longest duration that any medium tube or plate is ex-
posed. Incubate plates 72 hr at 35°, and observe daily.
F. Microscopic Examination
With pair of metal cutting shears, enlarge hole in can and
record odor. Microscopically (oil immersion) examine heat-
fixed thin smear of food, stained 10 sec with 1% gentian (or
crystal) violet and washed in running tap H 2 0, or, alterna-
tively, examine wet mounts with phase contrast microscope.
If food contains appreciable fat, xylol should be dripped across
food smear while it is still hot from heat fixing. Compare stained
smear with one made from normal product, if possible.
G. pH Determination
Det. pH with pH meter, using ref. buffer near normal pH
of food. Record both ref. buffer pH and sample pH. Compare
to normal can of food, if available.
H. Confirmation of Results
If there is any abnormal odor, abnormal appearance, ab-
normal pH, numbers of bacteria on microscopic examination,
and/or growth in media from any can of food, subculture cor-
responding refrigerated tube as follows: Flame lip of tube and,
with straightwall sterile glass pipet, (c), transfer ca 2 g food
to 2 tubes each of aerobic and anaerobic media (4 total). Flame
lips of media tubes both before and after addn of food. Tighten
caps after inoculation, incubate tubes 72 hr at 55°, and observe
daily. Record results for each tube sep.
Any organisms isolated from normal cans having obvious
vac. which produce gas in anaerobic medium at 35° should
immediately be suspected as being from laboratory contami-
nation. Aseptically inoculate growing organism into another
normal can, close hole with solder, and incubate 14 days at
35°. Any swelling of container indicates that organism was not
in original sample. Record as laboratory contamination and re-
view results of addnl cans to verify finding of contamination.
Growth in aerobic medium at 35° from normal cans indi-
cates either non-com. sterility or laboratory contamination.
Unless there is abnormal odor, abnormal appearance, abnor-
mal pH, and/or numbers of bacteria on microscopic exami-
nation from product in original can, record results as labora-
tory contamination and review results of addnl cans to verify
finding of contamination. Otherwise, observe subculture re-
suits at 55°. Growth at 35° and absence of growth at 55° con-
firm nonsterility of original container. Check growth under
aerobic conditions on nutrient agar plates, (d), at 55° and con-
firm for spores after 72 hr. Confirmation indicates nonsterility
due to flat sour spoilage. Record growth at 55° under anaerobic
conditions with gas production as com. sterile. Growth is caused
by dormant spores incapable of growth at normal temps of
storage and distribution.
If only one of duplicate tubes is pos. after incubation and
streaked glucose starch agar is also neg., record as laboratory
contamination. Growth on air control plate of glucose starch
agar also indicates potential laboratory contamination.
Ref.: JAOAC 55, 613(1972).
984.36 Microleak Detection
in Low-Acid Canned Food Containers
Helium Leak Test
First Action 1984
Final Action 1987
A. Principle
(This test does not detect bacterial contamination.)
He is inert gas with small MW that can be forced through
micron size openings and be easily detected by gas chromatgc
analysis. After can is pierced aseptically, and sample is taken
for microbiological analysis, can is sealed with rubber disc and
subjected to He at 45 psi for 30 min. Headspace sample is
then taken and analyzed for He.
B. Apparatus
(a) Gas chromato graph. — Instrument capable of sepg He
from N, O, H, and C0 2 as described, or equiv., with strip
chart recorder, gas partitioner (Model 1200, Fisher Scientific)
with dual thermal conductivity ceils and dual in-line columns.
Column 1: 6 l / 2 ft x Vs in. Al packed with 80-100 mesh Col-
umpak® PQ. Column 2: 11 ft X 3 / 16 in. Al packed with 60-
80 mesh Molecular Sieve 13 x.
Operating conditions: column temperature 75°; attenuation
128; Ar carrier gas inlet pressure 40 psi, flow rate 26 mL/min
thru gas partitioner; bridge current 125 mA; column mode 1
& 2; temperature mode, column; injector temp., off.
(b) Puncturing press (Fig. 98436). — Made from drill press
for electric hand drill with internal spring reversed to push head
AOAC Official Methods of Analysis (1990)
Sterility
457
FIG. 984.36— Puncturing press
down. Metal valve, 3- way (stopcock No. 3161 Becton-Dick-
inson & Co., Stanley St, Rutherford, NJ 07070); vac. pressure
gage, 30 in. Hg/0-60 psi, 2V2 in. face (Ametek, US Gauge
Div., PO Box 152, Sellerville, PA 18960). Stainless steel piercer
IV2 in. X V2 in. (machined in local machine shop) with No.
2 taper in piercer top, beveled Vs in. x 3 / 16 in. at bottom.
V4 in. X l / 2 in. silicone rubber gasket around beveled Vs in.
piercer to maintain seal.
(c) Helium exposure tank. — ASME paint tank, 10 gal., tested
to 100 psi, equipped with inlet and outlet microcontrol valves
(Harrison Rubber and Supply Co., Court and Race Sts, Cin-
cinnati, OH 45202).
(d) Pressurized helium tank. — With 2 stage regulator.
(e) Timer and solenoid. — To automate release of He from
exposure tank.
(f) Helium gas standards. — Scott Specialty Gasses, 2330
Hamilton Blvd, South Plainfield, NJ 07090.
(g) Cyanoacrylate glue. — SuperGlue (3M, AC&S Div., 3M
Center, St Paul, MN 55144-1000, or equiv.).
(h) Can opener. — Bacteriological, 972.44C(a).
(i) Rubber discs. — 2 3 /s in. x Vs in. and 70 durometer
(Netherland Rubber Co., 629 Burbank, Cincinnati, OH 45206).
C. Calibration Test Procedure
For gas chromatographs equipped with side port loop (0.5
mL), inject 5.0 mL calibrated He stds (suggested range of 5,
15, 25, 50, and 75% He). For instruments not equipped with
side port loop, inject appropriate vol. of stds. Use same vol.
for analysis of headspace gas samples. Plot percent He vs He
peak ht at attenuation used. Depending on qual. of instrument,
plot should approximate a straight line.
Check gage on can piercer against known pressure and vac.
Test resealing procedure, 984. 36E, on control cans.
D. Helium Exposure Tank
Control introduction rate of He into exposure tank, and time
cans are exposed to He pressure at 45 ± 2 psi. Timer, sole-
noid, and microvalves with vernier scales can facilitate pro-
cedure. Connect He source to exposure tank. Turn timer on to
close outlet solenoid valve. Approx. 15-20 min are needed to
reach 45 psi in tank. Make minor adjustments if necessary.
Adjust timer to expose cans to He pressure at 45 psi for 30
min (30 min exposure period is in addition to time necessary
to reach 45 psi.). Tank pressure should be reduced to psi
within 5-10 min.
E. Preparation of Can for Helium Test
If sample is to be taken for microbiological testing, proceed
as in 972.44D.
For nonsterile opening of can, use opener, 972.44C(a) to
cut IV2 in. hole in can lid. Remove and discard portion of
contents.
Push down any sharp metal projections around 1.5 in. hole.
Wipe lid dry and lightly sand area where rubber disc will be
inserted. Pool cyanoacrylate glue around surface covered by
edge of rubber disc. Place disc over hole and smooth edges
with fingers to remove air bubbles. Place wt (>500 g) on disc
>1 h.
F. Collection and Analysis of Headspace Gas
Can piercing assembly is shown in Fig. 984.36. Before
piercing can, close gage valve and pull plunger on syringe to
remove air from silicone tubing. Close syringe valve and expel
air from syringe. Puncture can and open gage valve to read
vac. or pressure. Turn gage valve and syringe valve to release
gas into syringe. If gas sample is >5.0 mL, withdraw this amt
(as shown in Fig. 984.36) and inject into port of gas chro-
matograph. If gas sample is <5.0 mL, force collected gas back
into can. Close syringe valve to retain gas in tubing and can.
Use syringe to add 40 mL room air to can, and pump syringe
twice to mix gas. Let syringe equilibrate to atm. pressure and
record syringe vol. From this dil. gas, sample may be obtained
for gas chromatograph. Percent He measured should be di-
vided by diln factor to obtain correct percent He in headspace
gas. Use following formula to det. diln factor:
Diln factor - (equilibrated syringe vol. - 40 mL air
+ headspace vol.)/(equilibrated syringe vol.
+ headspace vol.)
For example: (43-40 + 9)/(43 + 9) - 12/52 = 0.23 diln fac-
tor.
% He in can = % He measured /diln factor
For example: 5% He/0.23 = 22% He in can.
Headspace vol. may be measured by piercing control can
that still has vac. Assume sample and control can same vol-
ume. Measure amt of vac. (in. Hg) and vol. air pulled in from
syringe.
Headspace vol. = measured vol. from syringe
x 30 in Hg/measured vac. in can (in. Hg)
For example, if 6 mL air is pulled into can and vac. is 20
in. Hg, then,
Headspace vol. - 6 mL X 30 in Hg/20 in. Hg = 9 mL
To perform addnl work on can, collected gas may be stored
in capped syringe 2-3 h without appreciable change in its
composition.
458
Microbiological Methods
AOAC Official Methods of Analysis (1 990)
G. Interpretation of Results
Report can as leaker if, after exposure to pressurized He,
can internal pressure is >8 psi or percentage He is >1%. Re-
port can as nonleaker if, after exposure to pressurized He, can
internal vac. is >5 in., or percentage He is <1%.
Ref.: JAOAC 67, 942(1984).
972.45 Thermophilic Bacterial
Spores in Sugars
Microbiological Method
First Action 1972
Final Action 1989
(Sugar, both beet and cane, may carry spores of all 3 groups
of thermophilic bacteria that are important as spoilage agents
in low-acid canned foods, i.e., flat sour bacteria (Bacillus
stearothermophilus) , thermophilic anaerobes not producing H 2 S
(Clostridium thermosaccharolyticum) , and sulfide spoilage
bacteria or thermophilic anaerobes producing H 2 S (C. nigri-
ficans). These bacteria are not of health significance, but ex-
cessive numbers may survive com. heat processes.)
A. Sampling
Take 225 g (0.5 lb) samples from 5 sep. bags or barrels of
shipment or Jot, place in clean containers, and seal.
Sample liq. sugar by drawing 5 sep. 200-250 mL (6-8 oz)
portions during pumping transfer from tank trucks to storage
tanks or at refinery during filling of tank trucks.
Number of samples will vary in relation to size of shipment
or lot. If there is significant variability in lot, this fact will
become evident, in majority of cases, thru individual tests on
the 5 samples.
B. Preparation of Sample
(a) Dry sugar. — Place 20 g sample in sterile 150-250 mL
erlenmeyer marked to indicate 100 mL. Add sterile H 2 to
100 mL mark. Bring rapidly to bp, and boil 5 min. Replace
liq. evapd with sterile H 2 0.
(b) Liquid sugar. — Add sample contg 20 g dry sugar, detd
on basis of °Brix (e.g., 29.41 g 68° Brix (%) liq. sugar is
equiv. to 20 g dry sugar), to sterile 250 mL flask and proceed
as in 972.45D(a).
C. Culture Media
(a) Glucose tryptone agar. — For detection of flat sour bac-
teria. Use com. stdzd dehydrated medium (Bacto- Dextrose
Tryptone Agar) preferably, or prep, as follows: Suspend 10.0
g tryptone, 5.0 g glucose, 15.0 g agar, and 0.04 g bromocresol
purple in 1 L H 2 0, and mix thoroly. Final pH, 6.7 ±0.1.
Autoclave 30 min at 121° and cool to 55°.
(b) Liver broth. — For detection of thermophilic anaerobes
not producing H 2 S (C. thermosaccharolyticum). Mix 500 g
chopped beef liver with 1 L H 2 0. Slowly boil mixt. 1 hr, ad-
just to ca pH 7.0, and boil addnl 10 min. Press boiled material
thru cheesecloth and dil. liq. to 1 L. To broth, add 10.0 g
peptone and 1 .0 g K 2 HP0 4 , and adjust to pH 7.0. To test tube,
add 1-2 cm previously boiled ground beef liver and 10-12
mL broth. Sterilize 20 min at 121°. Before using medium, un-
less freshly prepd, exhaust by subjecting to flowing steam >20
min, and, after inoculation, stratify with 5-6 cm layer of plain
nutrient agar (common formula) that has been cooled to 50°.
(c) Sulfite agar, modified. — For detection of sulfide spoil-
age bacteria. Suspend 10.0 g tryptone, 1.0 g Na 2 S0 3 , and 20.0
g agar in 1 L H 2 0, and mix thoroly. At time agar is added to
tube, place clean iron strip or nail in each tube. No adjustment
of reaction is necessary. Prep, medium and Na 2 S0 3 soln, if
used in place of solid Na 2 S0 3 , fresh weekly. Autoclave me-
dium 20 min at 121° and cool to 55°.
D. Culture Technic
(a) Flat sour spores. — into 5 sep. petri dishes, pipet 2 mL
boiled sugar soln. Cover, and mix inoculum with glucose tryp-
tone agar. Incubate plates 35-48 hr at 55° and, to prevent drying
of agar, humidify incubator. Combined count from 5 plates
represents number of spores in 2 g original sugar. Multiply
this count by 5 to express results in terms of number of spores/
10 g sugar.
Characteristic colonies are round, 1-5 mm in diam., with
typical opaque central "spot," and, usually, surrounded by
yellow halo in field of purple. This halo may be insignificant
or missing with certain low acid -producing types or if plate is
so thickly seeded that entire plate has yellow tinge. Typical
subsurface colonies are compact and may approach "pin point"
conditions.
If identity of subsurface colonies is doubtful, observe nature
of surface colonies, if they show reasonable purity of formed
flora, assume that subsurface colonies have been formed by
similar bacterial groups. If plate is heavily seeded, counts may
not be accurate and colony structure and size may be atypical.
If plates are so heavily seeded that counting is impractical, dil.
original soln and repeat procedure.
To det. if typical subsurface colonies are flat sour organ-
isms, apply streak from colonies to agar plates to det. surface
characteristics.
(b) Thermophilic anaerobes not producing hydrogen sul-
fide. — Divide 20 mL boiled sugar soln equally among 6 liver
broth tubes and stratify liq. medium with plain nutrient agar.
After agar has solidified, preheat to 55° and incubate 72 hr at
that temp.
Thermophilic anaerobes not producing H 2 S are identified by
splitting of agar, presence of acid, and, occasionally, cheesy
odor. Method is suitable as qual . test but provides only rough
estn; results cannot be expressed as number of spores/unit wt
sugar.
(c) Sulfide spoilage bacteria. — Divide 20 mL boiled sugar
soln equally among 6 freshly exhausted tubes contg modified
sulfite agar. Incubate 48 hr at 55°.
In sulfite agar, sulfide spoilage bacteria form characteristic
blackened spherical areas. Due to solubility of H 2 S and its fix-
ation by Fe, no gas is noted. Some thermophilic anaerobes not
producing H 2 S generate relatively large amts of H, which splits
agar and reduces sulfite, thereby causing general blackening
of medium. This condition, however, is readily distinguishable
from restricted blackened area mentioned above. Count black-
ened areas to obtain quant, results.
E. Reporting Results
Report flat sour and sulfide spoilage results as number of
spores/ 10 g sugar. Report thermophilic anaerobes not pro-
ducing H 2 S as number of tubes pos. or neg. (+ or -).
Refs.: JAOAC 19, 438(1936); 21, 457(1938); 55, 445(1972).
985.41 Sporeformers in Low-Acid
Canned Foods
Gas Chromatographic Method
First Action 1985
Final Action 1989
A. Principle
Two org. compds produced by spo reforming organisms in
low-acid canned foods, d-( - )-2,3-butanediol (BD) and bu-
tyric acid (BA), but not produced by nonsporeformers, are
AOAC Official Methods of Analysis (1990)
Clostridium
459
measured by gas chromatgy. Identification of BD and BA is
based on relative retention times (RRTs) to internal std, pro-
pionic acid. Identification of sporeforming organisms as cause
of spoilage is based on ratio of peak hts for BD and butyric
acid (in external std) and BA and butyric acid (in external std).
B. Apparatus
(a) Gas chromatography — Suitable for use with 2 heated
flash vaporizer injectors contg glass sleeves; equipped with flame
ionization detectors (FID); linked to data processor (if avail-
able) with printer/ plotter (Sigma Series Instrument with con-
sole and printer /plotter, Perkin-Elmer, or equiv.).
(b) Gas chromatographic columns. — (1) 1.8 m (6 ft) x 2
mm id glass column packed with 15% SP 1220/1% H 2 P0 4 on
100-120 mesh Chromosorb W(AW); (2) 1.8 m x 2 mm (id)
glass column packed with 0.3% CW 20M/0.1% H 3 P0 4 on
60-80 mesh Carbopack C (Supelco Inc.). Condition both col-
umns, using recommended procedures: purge column at am-
bient temp. >:30 min with carrier gas at 20 mL/min; then pro-
gram from 50° to 150° at 2°/min and hold overnight. Cool
column 1, attach to detector, and set column temp, at 118°.
Inject twenty 10 |xL portions of freshly boiled H 2 on column
2 at 150°. Then cool, attach to detector, and set column temp,
at 125°.
(c) Operating conditions. — Select 2 methods from avail-
able chromatgc data systems software (if data processor is
available) that provide relative retention time (RRT) = 1.00
for internal std. Column 1: injector 200°, detector 240°, col-
umn 118° for 12 min isothermal run; He carrier flow rate 24
mL/min; propionic acid elution time 2.70-3.30 min; elec-
trometer range 10, attenuation 4; chart speed 5 mm/min; in-
jection vol. 1 |jlL. For butyric acid, retention time - ca 6 min,
RRT - 1.7, and peak ht - 60% FSD (10 cm). Column 2:
injector 200°, detector 240°, column 125° for 25 min isother-
mal run; N carrier flow rate 15 mL/min; propionic acid elution
time 2.25-2.45 min; electrometer range 10, attenuation 2; chart
speed 10 mm/min; injection vol. 0.3 |xL. For butyric acid,
retention time = ca 6.5 min, RRT = 2.7, and peak ht - 60%
FSD (10 cm). Theoretical plates for each column >1600.
(d) Syringes.— I and 5 julL (Hamilton 7001 -N and 1705-N,
or equiv.).
(e) Centrifuge. — With adapters suitable to accept 5 mL mini-
vials, 1.0 and 5.0 mL capacity, with silicone stopper and screw
cap (95010 and 95050, Alltech Associates, Inc., 2051 Wau-
kegan Rd, Deerfield, IL 66015), or equiv.
(f) Disposable Pasteur pipets. — 7760-F 30 series (Thomas
Scientific), or equiv.
C. Reagents
(a) Water. — Distd H 2 that elutes with no detectable peaks
on 2 columns used for assay.
(b) External std solns.-(l) For column 1, WSFA-2 (Su-
pelco Inc.,), or equiv. It must contain aq. soln of propionic,
isobutyric, and butyric acids. Concn of butyric acid should be
0.1%. Concns of propionic and isobutyric acids should be suf-
ficient to produce peak hts ranging from detectable to 100%
scale deflection (SD).
(2) For column 2, dil. WSFA-2 with equal vol. H 2 or prep,
equiv. soln. It must contain aq. soln of propionic and butyric
acids. Concn of butyric acid should be 0.05%. Concn of pro-
pionic acid should be sufficient to produce peak ht ranging
from detectable to 100% SD.
(c) Internal std solns.-(l) For column 1, dil. 5 |xL reagent
grade propionic acid to 5 mL with H 2 0. For injection, take up
1 (xL H 2 into 5 jxL syringe, followed by 0. 1 /xL soln. Modify
proportions of 2 components, if necessary, to give ca 50%
FSD for propionic acid peak eluting in 2-3 min. (2) For col-
umn 2, dil. protion of internal std for column 1 with equal
vol. H 2 0. For injection, take up 0.3 (xL H 2 into 1 jxL sy-
ringe, followed by 0.02 p,L of this diln. Modify proportions
of 2 components, if necessary, to give ca 50% FSD for pro-
pionic acid peak eluting in 2-3 min.
D. Assay
Pierce each can aseptically by any microbiologically ac-
ceptable technic (see 972.44C(a), (c) and 972.44D). Transfer
portion of liq. can contents to 5 mL mini-vial, using sterile
Pasteur disposable pipet, or equiv. and store at ca —20° over-
night or longer. Warm 5 mL vials to room temp, and then
centrf. 15 min at 1000 X g or until phases sep. Transfer por-
tion of clear upper phase to 1 mL mini-vial, using sterile Pas-
teur disposable pipet, or equiv. Store this upper phase at -20°
overnight or until day of assay.
Let 1 mL mini-vials of upper phase of centrfd can contents
warm to room temp. Use syringe to remove (and discard) any
particulate matter from bottom of cone; then mix sample, us-
ing syringe. For assays on either column, assay external std
to ensure optimal instrumental conditions; propionic acid must
be eluted in time range such that RRT - 1.000; theoretical
plates should be ca ^1600; and butyric acid peak should be
ca 60% FSD. Then replace glass liner in injector and inject
sample of H 2 0, using sample syringe. Repeat H 2 injections,
if necessary, until chromatogram has no ghost peaks. For as-
says on column 1 , take up 1 |xL portion of sample into 5 jxL
syringe, followed by 0.1 (xL internal std soln 1 and inject into
gas chromatograph. For assays on column 2, take up 0.3 fxL
portion of sample into 1 |xL syringe followed by 0.02 (xL in-
ternal std soln 2 and inject into gas chromatograph.
E. Interpretation
For each sample, examine chromatograms from both col-
umns, together with chromatograms for external std assayed
same day. On column 1, BD elutes as tailing peak with re-
tention time between those for isobutyric and butyric acids; it
will not elute on column 2 in 25 min run. Sample contg BA
gives peak eluting in both sample chromatograms in retention
time range for butyric acid. Measure peak ht for BD peak and
divide by peak ht for butyric acid in external std 1 assayed on
column 1 . Measure BA peak ht from column 2 and divide by
peak ht for butyric acid in external std 2 assayed on column
2. Peak ht ratio for BD >0.39 or peak ht ratio for BA >0.30
indicates sporeformers as cause of spoilage. Record cause of
spoilage as from either sporeformer or nonsporeformer.
Ref.: JAOAC 68, 626(1985).
CLOSTRIDIUM
977.26 Clostridium botulinum
and Its Toxins in Foods
Microbiological Method
First Action 1977
Final Action 1979
(Caution: See safety notes on pipets.)
A. Principle
Mice injected intraperitoneal^ (IP) with food ext contg > 1
min. lethal dose (MLD) of botulinum toxin die within 72 hr
after exhibiting sequence of symptoms characteristic of botu-
linum intoxication. Homologous antitoxin will protect mice from
symptoms while other antitoxins will not, thus detg serological
type. Viable spores in food will grow in suitable culture me-
dium and produce toxin, which is detected and typed.
460
Microbiological Methods
AOAC Official Methods of Analysis (1990)
B. Apparatus
(a) Can opener.— See 972.44C(a).
(b) Anaerobic jars. — GasPak (BBL) or Case-nitrogen re-
placement.
(c) Petri dishes. — 100 mm diam. Dry prepd plates ca 24 hr
at 35° before streaking.
(d) Centrifuge. — High-speed, refrigerated.
(e) Syringes. — 1.0 or 3.0 mL with 25 gage 5 /$" needles for
inoculating mice.
C. Media and Reagents
(a) Cooked meat broth. — Use either liver or heart medium.
(I) Chopped liver broth. — Grind 500 g fresh beef liver into
800 mL H 2 0. Heat to bp and simmer 1 hr. Cool, adjust to pH
7.0, and boil 10 min. Filter thru cheesecloth, pressing out ex-
cess liq. To broth add 10 g peptone, 1 g K 2 HP0 4 , and 1 g sol.
starch. Adjust to pH 7.0 and dil. to 1 L with H 2 0. Filter thru
coarse paper. (If desired, broth and liver may be stored sep.
in freezer for future use.) To 18 or 20 x 150 mm test tubes,
add liver to ht of 1-2 cm and 10-12 mL liq. Autoclave 20
min at 121°. (2) Cooked meat medium. — Use com. medium
of following formula: beef heart 454 g, proteose peptone 20
g, dextrose 2 g, and NaCl 5 g. Suspend 12.5 g medium in
100 mL cold H z O. Mix thoroly and let stand until particles are
thoroly wetted (ca 15 min). (Alternatively, add 1.25 g solid
medium into test tubes, add 10 mL cold H 2 0, and mix thoroly
to wet all particles.) Autoclave 15 min at 121°. Final pH, 7.2
± 1.
(b) Trypticase-peptone-glucose-yeast extract broth with
trypsin (TPGYT). — Dissolve 50 g trypticase, 5 g Bacto-pep-
tone, 20 g yeast ext, 4 g dextrose, and 1 g Na thioglycollate
in 1 L H 2 0, and dispense 15 mL portions into 20 X 150 mm
culture tubes or 100 mL portions into 6 fl oz prescription bot-
tles. Autoclave 10 min (tubes) or \5 min (bottles) at 121°.
Final pH, 7.0 ± 0.1. Refrigerate, and discard if not used within
2 weeks. Immediately before use, steam or boil 10—15 min to
remove O, cool quickly, and aseptically add 1.0 mL trypsin
soln/15 mL broth.
Prep, trypsin soln by dissolving 1.5 g trypsin (Difco 1:250)
in 100 mL H 2 0. Sterilize by filtering thru 0.45 |u,m Millipore
or equiv. filter, and refrigerate.
(c) Liver-veal-egg yolk agar or anaerobic egg yolk agar. — 'r
(/) Liver-veal-egg yolk agar (LVEY). — Wash 2 or 3 eggs with
stiff brush, and drain. Soak eggs in 0.1% HgCl 2 soln 1 hr.
Drain HgCl 2 soln and replace with 70% alcohol, soaking 30
min. Remove eggs, crack aseptically, and discard whites. Re-
move yolk with syringe, place in sterile container, and add
equal vol. sterile 0.85% NaCl soln. Mix thoroly. To each 500
mL prepd sterile com. dehydrated liver veal agar at 50°, add
40 mL egg yolk-NaCl suspension. Mix thoroly and pour plates.
Dry plates 2 days at room temp, or 24 hr at 35°. Discard con-
taminated plates, and store sterile plates in refrigerator. (2)
Anaerobic egg agar. — Dissolve 5 g yeast ext, 5 g tryptone,
20 g proteose peptone, 5 g NaCl, and 20 g agar in 1 L H 2 0.
Adjust to pH 7.0, dispense 500 mL into 1 L flask, and au-
toclave 20 min at 121°. To 500 mL melted agar at 45-50°,
add 40 mL egg yolk-NaCl suspension, prepd as in (7). Mix,
and pour plates immediately. Dry and store sterile plates as in
(d) Gel-phosphate buffer.— pH 6.2. Dissolve 2 g gelatin and
4 g Na 2 HP0 4 in 1 L H 2 with gentle heat. Dispense into 100
mL milk diln bottle. Autoclave 20 min at 121°.
(e) Clostridium botulinum antitoxin preparations . — Types
A thru F or polyvalent A-F. Available from Centers for Dis-
ease Control, CID, Office of Official Services, Atlanta, GA
30333.
D. Preparation of Sample
(a) Preliminary examination. — Keep samples refrigerated.
Unopened canned foods, unless badly swollen and in danger
of bursting, need not be refrigerated. Record code and con-
dition of container. Clean and identify container.
(b) Solid foods. — Aseptically transfer portion, with little or
no free liq., to sterile mortar. Add equal amt sterile gel-phos-
phate buffer, (d), and grind with sterile pestle. Alternatively,
inoculate small pieces of sample with sterile forceps directly
into enrichment broth.
(c) Liquid foods. — Inoculate with sterile pipets directly into
enrichment broth.
(d) Canned foods. — Prep., disinfect with ale. I soln, and
open cans as in 972. 44D. If can has swelled, position can so
vertical side seam is away from operator. If can has buckled
ends, chill before opening, and flame cautiously to avoid
bursting can.
(e) Visual examination. — Note appearance, odor, and any
evidence of decomposition. DO NOT TASTE PRODUCT un-
der any circumstances.
(f) Reserve sample. — After culturing, aseptically remove
portion to sterile sample jars for further tests which may be
needed later.
E. Detection of Viable C. botulinum
(a) Enrichment. — Remove dissolved O from media before
inoculation by steaming 10—15 min and cooling quickly with-
out agitation. Inoculate 2 tubes of cooked meat broth, (a), with
1-2 g solid or 1-2 mL liq. food or ext/ 15 mL broth, intro-
ducing inoculum slowly beneath surface of broth. Incubate at
35°. Similarly inoculate 2 tubes of TPGYT broth, 977.26C(b),
and incubate at 26°.
(b) Examination. — After 5 days, examine cultures for tur-
bidity, gas production, digestion of meat particles, and odor.
Also examine microscopically by wet mount under high power
phase contrast or by bright field illumination of smear stained
by Gram stain, crystal violet, or methylene blue. Observe mor-
phology of organisms and note existence of typical clostridial
cells, occurrence and relative extent of sporulation, and lo-
cation of spores within cells.
(c) Further treatment. — Usually 5 day incubation produces
active growth and highest concn of toxin, as well as peak spor-
ulation. Retain culture in refrigerator for pure culture isolation.
If there is no growth after 5 days, incubate addnl 10 days to
detect possible delayed germination of C. botulinum spores be-
fore discarding culture as sterile.
F. Isolation of Pure Cultures
If good sporulation has occurred, C. botulinum is more readily
isolated from mixed flora in enrichment culture or from orig-
inal sample.
(a) Pretreatment. — Add equal vol. filter-sterilized absolute
alcohol to 1-2 mL culture or sample in sterile screw -cap tube.
Mix well and incubate at room temp. 1 hr. Alternatively, heat
1-2 mL enrichment culture 10-15 min at 80° to destroy veg-
etative cells. (Do not use heat treatment for nonproteolytic type
C. botulinum.)
(b) Plating. — With inoculating loop, streak 1 or 2 loopfuls
of alcohol or heat-treated cultures, dild if necessary, to either
or both liver- veal -egg yolk agar or anaerobic egg yolk agar
dried plates in manner to obtain isolated colonies. Incubate
plates ca 48 hr at 35° under anaerobic conditions of Case an-
aerobic jar or Gas-Pak systems, or equivs.
(c) Selection of colonies. — Typical colonies are raised or
flat, smooth or rough, and commonly show some spreading
and have irregular edge. On egg yolk media, colonies usually
AOAC Official Methods of Analysis (1 990)
Clostridium
461
exhibit surface iridescence when examined by oblique light.
This luster zone is referred to as "pearly layer." Zone usually
extends beyond and follows irregular contour of colony. Be-
sides pearly zone, colonies of types C, D, and E are ordinarily
surrounded by wide (2-4 mm) zone of yellow ppt. Colonies
of types A and B generally show smaller zone of pptn. Not
all typical colonies will produce toxin. Some members of ge-
nus Clostridium have typical morphological characteristics but
do not produce toxins.
(d) Cultures. — With sterile transfer loop, inoculate each of
10 selected colonies into tube of sterile medium: (V) TPGYT
broth for C. botulinum Type E, incubating 5 days at 26°; and
(2) cooked meat broth for other toxin types, incubating 5 days
at 35°. Use cultures for confirmation as in (e) and for detection
and identification of toxin as in 977. 26G.
(e) Confirmation. — Streak culture from (d) in duplicate on
egg yolk agar plates, incubating 1 plate anaerobically and other
plate aerobically at 35°. If colonies typical of C. botulinum are
found on anaerobic plate and no growth is found on aerobic
plate, culture may be pure. Failure to isolate C. botulinum from
S:l of selected colonies may indicate that its population rela-
tive to mixed flora h low. Repeated serial transfers thru addnl
enrichment steps, 977.26E(a), may increase numbers suffi-
ciently to permit isolation. Store pure culture, (d), either under
refrigeration, on glass beads, or lyophilized.
G. Detection of Toxin
(a) Preparation of sample. — Ext solid foods with equal vol.
gel-phosphate buffer, 977.26C(d), macerating with sterile,
prechilled mortar and pestle. Centrf. ext and liq. foods contg
suspended solids under refrigeration. Rinse empty containers
suspected of having held toxic foods with few mL gel-phos-
phate buffer. Use min. vol. to avoid diln of toxin.
(b) Trypsin treatment. — Toxins of nonproteo lytic types, if
present, may need trypsin activation to be detected. Do not
use trypsin treatment with TPGYT culture which already con-
tains trypsin. Further treatment may degrade any fully acti-
vated toxin present in culture.
Adjust portion of food supernate, (a), liq. food, or cooked
meat culture, if necessary, to pH 6.2 with IN NaOH or HO.
Prep, satd trypsin soln by dispersing 1 g trypsin (Difco 1:250)
in 10 mL H 2 in clean culture tube. Mix 0.2 mL trypsin soln
with 1.8 mL liq. to be tested. Incubate 1 hr at 37° with oc-
casional gentle agitation.
(c) Toxicity testing. — Conduct each test in duplicate, i.e.,
on trypsin treated and untreated materials. Dil. portions of un-
treated and treated food supernate, liq. food, or culture 1:2,
1:10, and 1:100, resp., with gel-phosphate buffer. Inject sep.
pairs of mice, ca 15-20 g, IP with original and dild fluids,
treated and untreated, using syringe, (e). Heat 1 .5 mL original
untreated fluid 10 min at 100° for control. Cool, and inject
pair of mice each with 0.5 mL heated fluid. These mice should
not die because botulinum toxin, if present, is inactivated by
this heat treatment.
Observe mice periodically for 72 hr, recording symptoms
and time of deaths. Typical symptoms of botulism usually be-
gin within 24 hr with ruffling of fur, followed in sequence by
labored breathing, weakness of limbs, and finally total paral-
ysis with gasping for breath, followed by death due to respi-
ratory failure. Death without symptoms of botulism is not suf-
ficient evidence that injected material contained botulinum toxin.
Deaths may occur from chems present in fluid or from trauma.
If after 72 hr, all but mice receiving heated prepn have died,
repeat toxicity test, using higher dilns of fluids. It is necessary
to have dilns that kill as well as dilns that do not kill to es-
tablish an end point or MLD (min. lethal dose) as est. of amt
of toxin present. MLD is contained in highest diln killing both
(or all) mice inoculated. Calc. MLD/mL.
H. Typing of Toxin
Dil. monovalent antitoxins to types A, B, E, and F in 0.85%
NaCl soln to concn of 1 International Unit/0.5 mL. Prep, enough
dild antitoxin to inject 0.5 mL into each of 2 mice for each
diln of prepn to be tested.
Use toxic prepn which gave greatest number of MLD, either
treated or untreated. If untreated, same prepn can be used as
was used for toxicity testing; if trypsinized prepn was most
lethal, prep, freshly trypsinized fluid since continued action of
trypsin may destroy toxin. Prep, dilns to cover range of at least
10, 100, and 1000 MLD below previously detd end point of
toxicity.
Inject several groups of mice IP, each mouse receiving 0.5
mL of 1 of dild antitoxins, 30-60 min before challenging them
with IP injection of toxic prepns.
Inject pairs of mice protected by specific monovalent anti-
toxin injection IP with each diln of toxic prepn. Also inject
pair of unprotected mice (no injection of antitoxin) with each
toxic diln as control. (This protocol requires 30 mice: 3 pairs
for each of the 4 monovalent antitoxins (A, B, E, and F), each
pair to receive challenge of 1 of the 3 dilns of toxic prepn (2
x 3 x 4 — 24) plus 1 pair of unprotected mice for each diln
of toxic material as control (2x3 — 6).
Observe mice 72 hr for symptoms of botulism and record
time of deaths. If results indicate that toxin was not neutzd,
repeat test, using monovalent antitoxins to types C and D, plus
polyvalent antitoxin pool of types A thru F.
A interpretation
Toxin in food means that product, if consumed without thoro
heating, could cause botulism. Presence of toxin in food is
required for botulism to occur. Viable C. botulinum but no
toxin in food is not proof that food in question caused botu-
lism. Ingested organisms may be found in alimentary tract, but
are considered to be unable to multiply and produce toxin in
vivo .
Presence of botulinum toxin and/or organisms in low-acid
(pH >4.6) canned foods means that items were underpro-
cessed or were contaminated thru post- processing leakage.
Swollen cans are more likely than flat cans to contain botu-
linum toxin since organism produces gas during growth. Pres-
ence of toxin in flat can may imply that seams were loose
enough to let gas escape. Toxin in canned foods is usually of
type A or of proteolytic type B strain, since spores of proteo-
lytics can be among more heat resistant bacterial spores. Spores
of nonproteolytics, types B, E, and F, generally are of low
heat resistance and would not normally survive even mild heat
treatment.
Protection of mice from botulism and death with 1 of mono-
valent botulinum antitoxins confirms presence of botulinum toxin
and dets serological type of toxin in sample.
If mice are not protected by 1 of monovalent antitoxins, there
may be too much toxin in sample, there may be more than 1
kind of toxin present, or deaths may be due to some other
cause. In such cases, retesting at higher dilns of test fluids is
required and mixts of antitoxins must be used in place of
monovalent antiserum. If mice are still not protected, some
other toxic material, which is not heat labile, could be re-
sponsible if both heated and unheated fluids cause death. It is
also possible that heat stable toxic substance could mask bo-
tulinum toxin.
Ref.: JAOAC60, 541(1977).
462
Microbiological Methods
AOAC Official Methods of Analysis (1990)
976.30 Clostridium perfringens
in Foods
Microbiological Method
First Action 1976
Final Action 1979
(Applicable to examination of outbreak foods in which rela-
tively small numbers of vegetative cells are expected to be
present)
A. Apparatus
(a) Pipets. — 1.0 mL serological with 0.1 mL graduations
and 10.0 mL with 1.0 mL graduations.
(b) Colony counter. — Quebec, or equiv., dark field model.
(c) High-speed blender. — Waring Blendor, or equiv., multi-
speed model, with low-speed operation at 13,000 rpm, and 1
L glass or metal blender jars with covers. One jar is required
for each sample.
(d) Anaerobic jars . — BBL GasPak jars equipped with Gas-
Pak H + C0 2 generator envelopes are recommended. Anaero-
jar (Pfizer Diagnostics, 1407 N Dayton St, Chicago, IL 60622)
with replacement of air by purified N or N-C0 2 (9 + 1) is
satisfactory.
(e) Freezer , ultra-low temperature . — REVCO Model ULT-
107 (Revco Scientific, Inc., 275 Aiken Rd, Asheville, NC
28804) or equiv., capable of maintaining temp, of —68°.
(f) Shipping container. — Heavy duty styrofoam, including
hermetically scalable metal canister (friction-fit paint can is
satisfactory).
B. Reagents
(a) Peptone dilution water. — Dissolve 2.0 g peptone (Difco
Bl 18) in 2 L H 2 for each sample, and adjust to pH 7.0 ±
0.1. Dispense enough vol. in 175 mL (6 oz) bottles to give
90 ± 1 mL and in 750 mL erlenmeyers to give 450 ± 5 mL
after autoclaving 15 min at 121°.
(b) Nitrite test reagents. — {!) Reagent A. — Dissolve 8 g
sulfanilic acid in 1 L 5N HOAc (2 + 5). (2) Reagent £.—
Dissolve 5 g a-naphthol in 1 L 5/V HOAc.
(c) Buffered glycerol- salt soln. — Dissolve 4.2 g NaCl in 900
mL H 2 0. Add 12.4 g anhyd. K 2 HP0 4 , 4.0 g anhyd. KH 2 P0 4 ,
and 100 mL glycerol. Mix well to dissolve, and adjust pH to
7.2. Autoclave 15 min at 121°. For double-strength glycerol
soln (20%), use 200 mL glycerol and 800 mL H 2 0.
C. Culture Media
(Sizes of culture media containers (test tubes, flasks, and petri
dishes) are specified for each medium. All media except tryp-
tose-sulfite-cycloserine (TSC) agar are incubated in air at 35°.
Media not used <4 hr after prepn must be heated 10 min in
boiling H 2 or flowing steam to expel O and cooled rapidly
in tap H 2 without agitation just before use.)
(a) Tryptose-sulfite-cycloserine agar. — 15.0 g tryptose, 20.0
g agar, 5.0 g soytone, 5.0 g yeast ext, 1.0 g Na metabisulfite,
and 1.0 g ferric ammonium citrate (NF Brown Pearls) dild to
1 L with H 2 (SEP agar base, Difco 0811-01, is satisfactory).
Adjust to pH 7.6 ± 0.1, dispense 250 mL portions into 500
mL flasks, and sterilize 15 min at 121°. Before plating, add
20.0 mL 0.5% filter- sterilized soln of D-cycloserine to each
250 mL sterile melted medium at 50°. To make egg yolk-contg
plates, add 20 mL 50% egg yolk emulsion, (c), to 250 mL
sterile medium contg D-cycloserine. Dispense 15 mL portions
into 100 X 15 mm sterile petri dishes. Cover plates with towel
and let dry overnight at room temp, before use.
(b) ^-Cycloserine soln. — Dissolve 1 g D-cycloserine (Sigma
Chemical Co. or Serva Feinbiochemicia, Heidelberg, West
Germany) without heating in 200 mL 0.05M phosphate buffer
(pH 8.0 ± 0.1) and sterilize by filtering thru 0.45 |xm mem-
brane filter.
(c) Egg yolk emulsion. — Wash fresh eggs with stiff brush
and drain. Soak 1 hr in 70% alcohol. Aseptically remove yolk
and mix with equal vol. sterile 0.85% NaCl soln. Store at 4°.
(d) Buffered motility-nitrate medium. — 3.0 g beef ext, 5.0
g peptone, 5.0 g KN0 3 , 2.5 g Na 2 HP0 4 , 3.0 g agar, 5.0 g
galactose, and 5.0 g glycerol dild to 1 L with H 2 0. Adjust to
pH 7.3 ± 0.1, dispense 11 mL portions into 150 x 16 mm
tubes, and sterilize 15 min at 121°.
(e) Lactose- gelatin medium. — 15.0 g tryptose, 10.0 g yeast
ext, 10.0 g lactose, 5.0 g Na 2 HP0 4 , 0.05 g phenol red, and
120.0 g gelatin dild to 1 L with H 2 0. Adjust to pH 7.5 ± 0.1
before adding lactose and phenol red. Dispense 10 mL por-
tions into 150 X 16 mm screw -cap tubes and sterilize 15 min
at 121°.
(f) Sporulation broth. — 15.0 g polypeptone, 3.0 g yeast ext,
3.0 g sol. starch, 0.1 g MgS0 4 , 1.0 g Na thioglycollate, and
11.0 g Na 2 HP0 4 dild to 1 L with H 2 0. Adjust to pH 7.8 ±
0.1, dispense 15 mL portions into 150 X 20 mm screw-cap
tubes, and sterilize 15 min at 121°.
(g) Polypeptone-yeast extract (PY) medium. — 20.0 g poly-
peptone, 5.0 g yeast ext, and 5.0 g NaCl dild to 1 L with H 2 0.
Adjust to pH 6.9 ± 0.1, dispense 9 mL portions into 125 X
16 mm screw-cap tubes, and sterilize 15 min at 121°.
(h) Fluid thioglycollate medium. — (BBL No. 1 1260 or Difco
No. 0256). Dispense 10 mL portions into 150 x 16 mm screw-
cap tubes. Sterilize 15 min at 121°, and cool quickly. Final
pH is 7.1 ± 0.1.
D. Preparation of Sample
(a) For storage and shipping. — Using aseptic technic,
transfer 50 g sample to sterile container such as Whirl-Pak
plastic bag and add 50 g sterile buffered glycerol-salt soln.
Mix well by kneading bag or stirring with sterile pipet. Let
soln penetrate solid foods 10 min before freezing. Treat liq.
samples such as beef juice or gravy with double-strength (20%
glycerol) soln to obtain final concn of 10% glycerol. Freeze
samples as quickly as possible in ultra-low temp, freezer at
—68° or, alternatively, by placing in scalable metal canister
and storing with solid C0 2 in insulated shipping container. To
ship samples, place in seal able metal canister and pack in well
insulated styrofoam shipping carton with sufficient solid C0 2
to keep samples frozen during transit. Ship by most rapid means
possible. Upon receipt, transfer samples to ultra-low temp,
freezer at -68° or replenish solid C0 2 in shipping carton to
maintain temp, at ca —56° until samples can be examined.
Thaw samples and proceed as in (b) without delay.
(b) For analysis. — Using aseptic technic, weigh 50 g food
sample into sterile blender jar. Add 450 mL peptone diln H 2
and homogenize 2 min at low speed (13,000 rpm). Use this
1:10 diln to prep, serial dilns from 10" 2 to 10~ 6 by transferring
10 mL of 1:10 diln to 90 mL diln blank, mixing well with
gentle shaking, and continuing until 10~ 6 diln is reached,
E. Plate Count Technic
Pour ca 5 mL TSC agar without egg yolk into each of ten
100 x 15 mm petri dishes and spread evenly by rapidly ro-
tating dish. When agar has solidified, label plates and asep-
tically pipet 1 mL of each diln of homogenate in duplicate onto
agar surface in center of dish. Pour addni 15 mL TSC agar
without egg yolk into dish and mix well with inoculum by
gently rotating dish.
Alternatively, with sterile glass rod spreader, spread 0.1 mL
diln over previously poured plates of TSC agar contg egg yolk
emulsion. Let plates absorb inoculum 5-10 min; then overlay
AOAC Official Methods of Analysis (1990)
Clostridium
463
with 10 mL TSC agar without egg yolk. (TSC agar contg egg
yolk is preferred for foods which may also contain other sul-
fite-reducing Clostridium sp.)
When agar has solidified, place plates in upright position in
anaerobic jar. Produce anaerobic conditions, and incubate jar
20 hr at 35 c for TSC agar without egg yolk and 24 hr at 35°
for TSC agar with egg yolk. After incubation, remove plates
from jar and observe microscopically for growth and black
colony production. Select plates showing estd 20-200 black
colonies. Using Quebec colony counter with piece of white
tissue paper over counting area, count black colonies and calc.
number of Clostridium sp./g food. C. perfringens colonies in
medium contg egg yolk are black and usually surrounded by
2-4 mm zone of white ppt due to lecithinase activity. How-
ever, since a few strains are weak or neg. for lecithinase, count
any black colonies suspected to be C. perfringens and confirm
identity as in 976.30F.
F. Confirmation Technic
Select 10 characteristic colonies from countable plates (20-
200 colonies), inoculate each into tube of fluid thioglycollate
medium, and incubate 18—24 hr at 35°. Make Gram-stained
smear of fluid thioglycollate cultures and check for purity and
presence of short, thick, Gram-pos. bacillus characteristic of
C. perfringens. Streak contaminated cultures on TSC agar contg
egg yolk and incubate plates anaerobically 24 hr at 35° to ob-
tain pure cultures. Stab- inoculate buffered motility-nitrate and
lactose gelatin media with 2 mm loopfuls of pure fluid thiogly-
collate culture or portion of isolated colony from TSC agar
plate. Inoculate sporulation broth with 1 mL fluid thioglycol-
late culture and incubate 24 hr at 35°. Examine tubes of buff-
ered motility-nitrate medium by transmitted light for type of
growth along stab. Nonmotile organisms produce growth only
in and along line of stab. Motile organisms produce diffuse
growth out into medium away from stab.
Test buffered motility-nitrate medium for presence of nitrite
by adding 0.5 mL Reagent A and 0.2 mL Reagent B. Orange
which develops within 15 min indicates presence of nitrites.
If no color develops, add few grains of powd Zn metal, and
let stand 10 min. No color change after addn of Zn indicates
that nitrates are completely reduced; change to orange indi-
cates that organism is incapable of reducing nitrates.
Examine lactose-gelatin medium for gas and color change
from red to yellow, indicating that lactose is fermented with
production of acid. Chill tubes 1 hr at 5° and check for gelatin
liquefaction. If medium solidifies, re incubate addnl 24 hr at
35° and repeat test for gelatin liquefaction. Make Gram-stained
smear from sporulation broth and examine microscopically for
spores. Report whether or not spores are produced. Store spor-
ulated cultures at 4° if further testing of isolates is desired.
Nonmotile, Gram-pos. bacilli which produce black colonies
in TSC agar, reduce nitrates to nitrites, produce acid and gas
from lactose, and liquefy gelatin within 48 hr are provisionally
identified as C. perfringens.
Organisms suspected to be C. perfringens that do not meet
criteria stated above must be confirmed by further testing.
Subculture into fluid thioglycollate medium isolates that do not
liquefy gelatin or which are atypical in other respects. Incubate
24 hr at 35°, make Gram-stained smear, and check for purity.
Inoculate 1 tube of FY medium, (g), contg 1% salicin and 1
tube contg 1% raffinose with 0.1 mL fluid thioglycollate cul-
ture. Incubate media 24 hr at 35° andj check PY-salicin for
acid and gas. Transfer 1.0 mL culture to test tube and add 1-
2 drops 0.04% phenol red. Yellow indicates acid is produced
from salicin. (Salicin usually is not fermented by C. perfrin-
gens but is rapidly fermented with production of acid and gas
by closely related species.) Reincubate media addnl 48 hr and
test both media for production of acid. Acid is usually pro-
duced from raffinose by C. perfringens but not by closely re-
lated species. Acid is produced from salicin in PY medium by
a few strains of C. perfringens.
Calc. number of C. perfringens in sample on basis of %
colonies tested that are confirmed as C. perfringens . (Exam-
ple: If av. plate count of 10~ 4 diln was 85, and 8 of 1 colonies
tested were confirmed as C. perfringens, number of C. per-
fringens /g food is 85 X (8/10) X 10,000 - 680,000.) (Note:
Diln factor with plates contg egg yolk is 10-fold higher than
diln plated.)
Refs.: JAOAC 59, 606(1976); 61, 785(1978).
974.38 Clostridium perfringens
in Foods
alpha-Toxin Estimation Method
First Action 1974
Final Action 1979
(Applicable to examination of outbreak foods in which pres-
ence of large numbers of vegetative cells are suspected but
which may no longer be viable)
A. Apparatus
(a) Centrifuge. — High-speed, preferably refrigerated, with
250 mL bottles.
(b) Seitz filter. — 100-250 mL with sterilizing filter pads.
(c) High-speed blender. — Waring Blendor or Omni -Mixer
homogenizer (DuPont Co., Sorvall Operations, Newtown, CT
06470), with blending vessels.
(d) Vacuum flask. — Sidearm 1 L erlenmeyer fitted with 1-
hole rubber stopper to receive 200 mm glass tubing with 125
cm of 6 mm od (3 mm id) rubber tubing attached.
(e) Tubing. — Stainless steel thin wall (No. 9 surgical), 3
(od) x 180 mm (Tubesales, 175 Tubeway St, Forrest Park,
GA 30051).
(f) Dialysis tubing. — 1.21" flat width (Fisher Scientific Co.,
No. 8667C).
B. Reagents
(a) N -2 -Hydroxy ethyl piper azine-N' '-2 -ethane sulfonic acid
(HEPES) buffer soln.— Dissolve 6.0 g HEPES (Calbiochem
Corp.) and 11.7 g NaCl in 500 mL H 2 0. Adjust to pH 8.0
with 3A^ NaOH and store at 4°.
(b) LecithovitelUn soln. — Mix 1 egg yolk with 250 mL sa-
line soln, (e), and clarify by centrfg 20 min at 14,000 Xg at
4°. Filter-sterilize supernate with Seitz filter and store at 4°.
(c) Saline agar base. — Add 15.0 g purified agar (Difco
Laboratories) and 8.5 g NaCl to I L H 2 0. Adjust to pH 7.0,
heat to dissolve agar, dispense in 100 mL portions, and au-
toclave 15 min at 121°.
(d) Washed red blood cells. — Wash packed human red blood
cells 3 times by mixing with 4 vols saline soln, (e). Centrf.
10 min at low speed (2500 rpm) to sediment cells. Remove
supernate with vac. flask. Resuspend cells in addnl saline soln
and repeat these steps twice. After final wash, mix cells with
equal vol. saline soln. Use sterile precautions.
(e) Sterile saline soln. — Dissolve 8.5 g NaCl in 1 L FLO.
Adjust to pH 7.0, dispense 250 mL portions into Pyrex con-
tainers, and autoclave 15 min at 121°.
(f) Polyethylene glycol soln. — 30%. Dissolve 120 g poly-
ethylene glycol (Carbowax Compound 20M, Union Carbide
Corp., PO Box 8361, S Charleston, 1 WV 25303) in 400 mL
H 2 0.
(g) Antiserum. — Clostridium perfringens Type A diagnostic
464
Microbiological Methods
AOAC Official Methods of Analysis (1990)
serum (Coopers Animal Health Inc.
City, MO 64141-6167).
PO Box 41967, Kansas
C. Preparation of Hemolysin Plates
Melt 100 mL saline agar base, (c), cool to 50°, and add 11
mL washed red cells, (d). Mix thoroly and dispense 7 mL into
15 x 100 mm sterile plastic petri dishes. Dry plates overnight
at room temp, and store at 4°. Just before use, cut test wells
by applying vac. to sterile stainless steel tube, (e), and plung-
ing tube into agar. Using template, space 9 test wells 3 cm
apart and 2 cm from edge, and place 2 addnl wells 3 cm apart
near center of plate.
D. Toxin Extraction
Homogenize 25 g food (do not include fat) in 100 mL HEPES
buffer soln, (a), 1 min in high-speed blender. Centrf. homog-
enate 20 min at 14,000- 20, 000 x g at 5°. Filter supernate thru
Whatman No. 31 paper, or equiv., to remove fat (chill ext
centrfd without refrigeration 1 hr at 4° before filtering). Dis-
card solids. Rinse Seitz filter pad with 15 mL saline soln. Dis-
card saline soln and filter- sterilize ext, rinsing filter pad with
10 mL saline soln.
E. Concentration
Soak 90 cm dialysis tubing 1 hr in H 2 0. Tie one end and
fill with saline soln. Check for leaks and rinse out twice with
saline soln. Transfer sterile ext to dialysis sack and cone, to
<10 mL by dialyzing 4-5 hr against 400 mL 30% polyeth-
ylene glycol, (f), at 4°. Rinse outside of sack with tap H 2
and collect coned ext in sterile tube.
F. Toxin Testing
Adjust vol. of coned ext to 10 ± 0.5 mL with saline soln.
Set up 10 sterile 13 x 100 mm test tubes and add 0.5 mL
saline soln to all tubes except first and last. Add 0.5 mL ext
to first and second tubes. Mix ext and saline soln in second
tube and transfer 0.5 mL to third tube, etc., to serially dil. ext
from to 1 + 255. Change pipet after 3 dilns to prevent ex-
cessive carry-over. Mix 0.25 mL ext, 0.25 mL saline soln,
and 0.1 mL antiserum, (g), in last tube. Fill 1 peripheral well
of duplicate hemolysin plates with each diln of ext, using fine-
tipped Pasteur pipet. Fill 1 center well of each plate with ext-
antiserum mixt. and the other with saline soln. Add 0.5 mL
lecithovitellin soln, (b), to remainder of dild ext in each tube,
including ext-antiserum mixt. Mix well, and incubate tubes
and plates (in plastic bag) 24 hr at 35°.
G. alpha-Toxin Titer
After incubation, refrigerate plates 2 hr at 4°, Measure he-
molytic zone (width from edge of well in mm). Last 3 dilns
before end point should exhibit ca 1 mm reduction in width
for each 2-fold diln. If not, repeat a-toxin test. Hemolytic zone
1 mm in width is end point of titrn.
Examine ext-lecithovitellin mixt. in tubes for lecithinase ac-
tivity and record results. Max. reaction (+ + + +) is white pel-
licle 4-5 mm thick over clear liq. Activity decreases with diln
to ( + ) reaction (opaque soln with no pellicle). This diln is end
point of lecithovitellin test. Hemolytic and lecithinase activi-
ties neutzd by antiserum are due to a-toxin.
H. Population Estimate
Compare titer of a-toxin present in ext with data in Table
974.38 to est. population of C perfringens. Hemolysin (HI)
plate titer is preferred for this because lecithovitellin (LV) test
is less sensitive with some food exts.
Ref.: JAOAC57, 91(1974).
Table 974.38 Correlation Between Population Levels of C.
perfringens and Amount alpha-Toxin Produced
in Food a
a-
Toxin Titer b
Estd
C. perfringens
Population/g
Hl Plate
LV Test
x 10 6
Undild
1.2
1 + 1
Undild
2.5
1+3
1+1
6.5
1+7
1+3
9.5
1 + 15
1+7
25
1+31
1+15
55
1+63
1+31
80
1 + 127
1 + 127
150
1+255
1+255
210
a Based on viable counts obtained with 6 strains in chicken broth.
b Diln which produces 1 mm zone of hemolysis in HI plate or one + re-
action in LV test.
BACILLUS
980.31 Bacillus cereus in Foods
Enumeration and Confirmation Microbiological Methods
First Action 1980
Final Action 1981
A. Apparatus
(a) Pipets. — 1.0 mL with 1 mL graduations; also 5.0 mL
and 10.0 mL with 1.0 mL graduations.
(b) Colony counter. — Quebec, or equiv., dark field model.
(c) High-speed blender . — Waring blender, or equiv. 2 speed
model with high-speed operation at 18,000-21,000 rpm, and
1 L glass or metal blender jars with covers. One jar is required
for each sample.
(d) Anaerobic jar. — BBL GasPak jar equipped with GasPak
H + C0 2 generator envelopes, or equiv.
(e) Vortex mixer. — Vortex Genie, or equiv.
(f ) Sterile bent glass spreading rods. — Hockey stick or hoe
shape with fire polished ends; 3-4 mm diam. with 45-55 mm
spreading surface.
(g) Inoculating loops. — One each, 26 gage nichrome wire
with loop 2 mm id and one 24 gage nichrome wire loop 3 mm
id.
(h) Staining rack. — Rack must be accessible from below
for heating slides.
B. Media and Reagents
(a) Mannitol-egg yolk-poly my xin (MYP) agar. — 1.0 g beef
ext, 10.0 g peptone, 10.0 g D-mannitol, 10.0 g NaCl, 0.025
g phenol red (as soln), and 15.0 g agar dild to 900 mL with
H 2 0. Adjust to pH 7.2 ± 0.1, heat to dissolve, and dispense
225 mL portions into 500 mL flasks. Autoclave 15 min at 121°.
Cool to 50° in H 2 bath and add 12.5 mL egg yolk emulsion,
(b), and 2.5 mL polymyxin B soln, (c), to each 225 mL me-
dium. Mix well and dispense 18 mL portions into 100 x 15
mm sterile petri dishes. Dry plates 24 h at room temp, before
use.
(b) Egg yolk emulsion, — 50%. Wash fresh eggs with stiff
brush and drain. Soak 1 h in 70% alcohol. Aseptically remove
yolk and mix (1 + 1) with sterile 0.85% NaCl soln. (Difco
egg yolk enrichment 50% is satisfactory).
(c) Polymyxin B soln. — Dissolve 500,000 units sterile po-
lymyxin B sulfate in 50 mL sterile LLO.
(d) Trypticase-soy -polymyxin broth. — 17.0 g trypticase, 3.0
g phytone peptone, 5.0 g NaCl, 2.5 g K 2 HP0 4 , and 2.5 g
dextrose dild to 1 L with H 2 0. (Rehydrated trypticase soy broth
AOAC Official Methods of Analysis (1990)
Bacillus
465
is satisfactory.) Boil 2 min. Dispense 15 mL portions into 150
x 20 mm tubes and autoclave 15 min at 121°. Final pH 7.3
± 0. 1 . Just prior to use, add 0. 1 mL 0. 15% polymyxin B soln
to each tube of medium and mix well. To make polymyxin B
soln, dissolve 500,000 units sterile polymyxin B sulfate in 33.3
mL sterile H 2 0.
(e) Phenol red-dextrose broth. — J 0.0 g proteose peptone
No. 3, 1.0 g beef ext, 5.0 g NaCl, 0.018 g phenol red (as
soln), and 5.0 g dextrose dild to 1 L with H 2 0. (Phenol red
dextrose broth, Difco 0093, is satisfactory). Dispense 3 mL
portions into 100 X 13 mm tubes and autoclave 10 min at
121°. Final pH 7.4 ±0.1.
(f) Nitrate broth. — 3.0 g beef extract, 5.0 g peptone, and
1.0 g KN0 3 dild to 1 L with H 2 0. (Rehydrated nitrate broth
is satisfactory.) Adjust to pH 7.0 ± 0.1 and dispense 5 mL
portions into 125 X 16 mm tubes. Autoclave 15 min at 121°.
(g) Nutrient agar slants and plates. — 3.0 g beef ext, 5.0 g
peptone, and 15.0 g agar dild to I. L with H 2 (dehydrated
nutrient agar is satisfactory). Heat to dissolve, and dispense
6.5 mL portions into 125 X 16 mm screw-cap tubes. Auto-
clave 15 min at 121° and slant tubes until medium solidifies.
Final pH 6.8 ± 0. 1 . For plates, dispense 100-500 mL portions
in bottles or flasks and autoclave 15 min at 1.21°. Cool to 50°
in H 2 bath and dispense 18-20 mL portions in 100 x 15 mm
sterile petri dishes. Dry plates 24-48 h at room temp, before
use.
(h) Nutrient agar with l-tyrosine. — Prep, nutrient agar as
in (g) and dispense 100 mL portions into bottles. Autoclave
15 min at 121°. Cool to 45° in H 2 bath and add 0.5 g sterile
L-tyrosine suspended in 10 mL H 2 to each 100 mL of me-
dium. Mix thoroly by rotating or inverting bottle and aseptic-
ally dispense 3.5 mL portions of complete medium into sterile
100 x 13 mm tubes. Slant tubes and cool rapidly to prevent
sepn of tyrosine. To prep. L-tyrosine suspension, add 0.5 g to
150 x 20 mm tube and suspend in 10 mL H 2 with Vortex
mixer. Autoclave 15 min at 121°.
(i) Nutrient broth with lysozyme. — 3.0 g beef ext and 5.0
g peptone dild to 1 L with H 2 0. (Rehydrated nutrient broth,
Difco 0003, is satisfactory.) Dispense 99 mL portions in bot-
tles and autoclave 15 min at 121°. Final pH 6.8 ± 0.1. Mix
1 .0 mL 0.1% lysozyme soln with 99 mL broth and aseptically
dispense 2.5 mL complete medium into sterile 100 x 13 mm
tubes. To make lysozyme soln, dissolve 0.1 g lysozyme in 65
mL sterile 0.01/V HC1, boil for 20 min, and dil. to 100 mL
with sterile 0.017V HO. Alternatively, dissolve 0.1 g lysozyme
hydrochloride in 100 mL H 2 and sterilize with 0.45 fxm
membrane filter.
(j) Modified Voges-Proskauer (VP) medium. — 7.0 g pro-
teose peptone, 5.0 g dextrose, and 5.0 g NaCl dild to 1 L with
H 2 0. Dispense 5 mL portions into 150 X 20 mm tubes. Au-
toclave 10 min at 121°. Final pH 6.5 ± 0.1.
(k) Motility medium. — 10.0 g trypticase, 2.5 g yeast ext,
5.0 g dextrose, 2.5 g Na 2 HP0 4 , and 3.0 g agar dild to 1 L
with H 2 0. Heat to dissolve. Dispense 2 mL portions into 13
x 100 mm tubes, and autoclave 10 min at 121°. Final pH 7.4
±0.2. Alternatively, dispense 100 mL amts in 150 mL bottles
and autoclave 15 min at 121°. Cool at 50° and aseptically dis-
pense 2 mL into sterile 13 X 100 mm tubes. For best results,
store at room temp. 2-4 days before use to prevent growth
along side of medium.
(I) Trypticase- soy -sheep blood (TSSB) agar. — Dil. 15.0 g
trypticase, 5.0 g phytone peptone, 5.0 g NaCl, and 15.0 g agar
to I L with H 2 0. Adjust pH to 7.0 ± 0.2. Heat to boiling to
dissolve, and dispense 100-500 mL portions in bottles or flasks.
Autoclave 15 min at 121° and cool to 48° in H 2 bath. Add
5 mL sterile defibrinated sheep blood per 100 mL medium.
Mix well, and dispense 18-20 mL portions into 100 x 15 mm
petri dishes. (Trypticase-soy or tryptic-soy agar plates contg
5% sheep blood are satisfactory.)
(m) Butterfield's buffered phosphate diluent. — (1) Stock
soln. —Dissolve 34.0 g KH 2 P0 4 in 500 mL H 2 0, adjust to pH
7.2 with ca 175 mL IW NaOH, and dil. to 1 L with H 2 0.
Store in refrigerator. (2) Diluent. — Dil. 1.25 mL stock soln
to 1 L with H 2 0. Prep. 90 ± 1 mL diln blanks with this soln
and autoclave 15 min at 121°.
(n) Nitrite test reagents. — (1) Reagent A. — Dissolve 8 g
sulfanilic acid in 1 L 5N HOAc (2 + 5). (2) Reagent B.—
Dissolve 2.5 g a-naphthol in 1 L 5N HOAc.
(o) Voges-Proskauer (VP) test reagents.- — (7) Alpha-
naphthol soln. — 5%. Dissolve 5.0 g a-naphthol in 100 mL
absolute alcohol. (2) Potassium hydroxide soln. — 40%. Dis-
solve 40 g KOH in H 2 and dil. to 100 mL. (3) Creatine
crystals .
(p) Basic fuchs in stain. — Dissolve 0.5 g basic fuchsin in
20 mL alcohol and dil. to 100 mL with H 2 0. Filter soln if
necessary thru fine paper to remove excess dye particles. Store
in tightly stoppered container. (TB Carbol-fuchsin ZN stain is
satisfactory.)
C. Preparation of Food Homogenate
Using aseptic technic, weigh 50 g food sample into sterile
blender jar. Add 450 mL phosphate buffered diln H 2 and
homogenize 2 min at high speed (ca 20,000 rpm). Use this
1:10 diln to prep, serial dilns from 10~ 2 to 10~ 6 by transferring
10 mL of 1:10 diln to 90 mL diln blank, mixing well with
vigorous shaking, and continuing until 10~ 6 is reached.
D. Plate Count Technic
Inoculate duplicate MYP agar plates with each diln of ho-
mogenate by spreading 0.1 mL evenly onto each plate with
sterile glass rod spreader. Incubate plates 24 h at 30° and check
for colonies surrounded by ppt zone indicating lecithinase is
produced. B. cereus colonies usually are pink which becomes
more intense after addnl incubation. If reactions are not clear,
incubate plates for addnl 24 h before counting.
Select plates showing estd 15-150 eosin pink colonies sur-
rounded by lecithinase zones. Mark bottom of plates into zones
with black felt pen to facilitate counting and count colonies.
This is the presumptive count of B. cereus /g of food. Pick 5
or more colonies from plates counted and transfer to nutrient
agar slants for confirmation tests.
E. Most Probable Number Technic
(For foods containing <10 3 B. cereus /g)
Inoculate 3-tube most probable number (MPN) series in
trypticase-soy-polymyxin broth, (d), using 1 mL inocula of
1:10, 1:100, and 1:1000 dilns with triplicate tubes at each diln.
Incubate 48 ± 2 h at 30° and examine tubes for dense growth
typical of B. cereus. Streak pos. tubes on sep. MYP agar plates^
(a), and incubate 24-48 h at 30°. Pick 1 or more eosin pink
colonies surrounded by ppt zone due to lecithinase from each
plate and transfer to nutrient agar slants for confirmation tests.
Confirm as B. cereus and compute MPN of B. cereus /g using
Table 966.24 on basis of number of tubes in which B. cereus
was present.
F. Confirmation Technic
Pick >5 presumptive pos. colonies from MYP agar plates
and transfer to nutrient agar slants. Incubate 24 h at 30°. Make
Gram stained smears from slants and examine microscopi-
cally. B. cereus will appear as large Gram pos. bacilli in short
to long chains; spores are ellipsoidal, central to subterminal,
and do not swell sporangium.
Transfer 3 mm loopful culture from each slant to 100 x 13
466
Microbiological Methods
AOAC Official Methods of Analysis (1990)
mm tube contg 0.5 mL sterile phosphate buffered dim H 2
and suspend culture in diluent with Vortex mixer. Inoculate
following media with suspended culture:
(a) Phenol red dextrose broth, (e). — Inoculate broth with
2 mm loopful culture and incubate anaerobically 24 h at 35°
in Gas-Pak anaerobic jar. Shake tubes and check for growth.
Change from red to yellow indicates acid was produced from
dextrose anaerobically.
(b) Nitrate broth, (/). — Inoculate with 3 mm loopful cul-
ture and incubate 24 h at 35°. Test for presence of nitrite by
adding 0.25 mL nitrite test reagent A and 0.25 mL reagent B.
Orange which develops within 10 min indicates presence of
nitrites.
(c) Modified VP medium, (j). — Inoculate with 3 mm loop-
ful of culture and incubate 48 h at 35°. Transfer 1 mL culture
to empty tube to test for acetylmethylcarbinol. Add 0.2 mL
40% KOH soln, 0.6 mL 5% ale. a-naphthol soln, and few
crystals creatine. Let stand .1 h. Test is pos. if eosin pink de-
velops.
(d) Nutrient agar with L-tyrosine, (h). — Inoculate entire
surface of slant with 3 mm loopful of culture. Incubate 48 h
at 35°. Check for clearing of medium near growth indicating
tyrosine is decomposed. Check neg. tubes for growth and in-
cubate addnl 72 h before discarding.
(e) Nutrient broth with lysozyme, (i). — Inoculate nutrient
broth contg 0.001% lysozyme with 2 mm loopful of culture;
also inoculate control tube of plain nutrient broth. Incubate 24
h at 35° and record growth as + or -, Incubate neg. tubes
addnl 24 h before discarding.
(f) MYP agar, (a). — (Test may be omitted if reactions of
all isolates on MYP agar plates were typical.) Inoculate pre-
marked 4 sq. cm area of MYP agar plate by gently touching
surface with 2 mm loopful of culture. Let inoculum be ab-
sorbed and incubate 24 h at 35°. Check for lecithinase pro-
duction as indicated by zone of ppt surrounding growth. Man-
nitol fermentation is neg. if growth and surrounding medium
are eosin pink.
Large Gram pos. bacilli which produce lecithinase and are
neg. for mannitol fermentation on MYP agar, grow and pro-
duce acid from dextrose anaerobically, reduce nitrate to nitrite,
produce acetylmethylcarbinol, decompose L-tyrosine, and grow
in the presence of 0.001% lysozyme are provisionally identi-
fied as B. cereus. (These characteristics are shared by all
members of B. cereus group. See Differentiation of Members
of Bacillus cereus Group, 983.26.)
Calc. number of B. cereus in sample on basis of % colonies
tested that are confirmed as B. cereus. {Example: If av. plate
count with 10" 4 diln of sample was 65 and 4 of 5 colonies
tested were confirmed as B. cereus, number of B. cereus /g
food is 65 X (4/5) x 10,000 x 10 = 5,200,000.) (Diln factor
is 10-fold higher than sample diln because only 0.1 mL was
tested.)
Ref.: JAOAC 63, 581(1980).
983.26 Differentiation of Members
of Bacillus cereus Group
Microbiological Method
First Action 1983
Final Action 1984
(Typical strains of B. cereus isolated from foods by 980.31
can be differentiated from other members of 5. cereus group
including: (1) insect pathogen/?, thuringiensis, (2) mammalian
pathogen B. anthracis, and (3) rhizoid strains ofB. cereus var.
mycoides .)
A. Apparatus
(a) Staining rack. — Rack must be accessible from below
for heating slides.
(b) Inoculating loops. — One each, 26 gage nichrome wire
with loop 2 mm id and one 24 gage nichrome wire loop 3 mm
id.
B. Media and Reagents
(a) Mannitol-egg yolk-poly my xin (MYP) agar. — 1.0 g beef
ext, 10.0 g peptone, 10.0 g D-mannitoI, 10.0 g NaCl, 0.025
g phenol red (as soln), and 15.0 g agar dild to 900 mL with
H 2 0. Adjust to pH 7.2 ± 0.1, heat to dissolve, and dispense
225 mL portions into 500 mL flasks. Autoclave 15 min at 121°.
Cool to 50° in H 2 bath and add 12.5 mL sterile 50% egg
yolk emulsion (b) and 2.5 mL polymyxin B soln contg 10 000
units per mL (if available) to each 225 mL medium. (Addn of
polymyxin B soln is optional when medium is to be used for
testing reactions of pure cultures.) Mix well and dispense 18
mL portions into 100 x 15 mm sterile petri dishes. Dry plates
24 h at room temp, before use. (Dehydrated mannitol-egg yolk-
polymyxin (MYP) agar contg 50% egg yolk enrichment is sat-
isfactory.)
(b) Egg yolk emulsion. — 50%. Wash fresh eggs with stiff
brush and drain. Soak 1 h in 70% alcohol. Aseptically remove
yolk and mix (1 + 1) with sterile 0.85% NaCl soln. (50% egg
yolk enrichment is satisfactory.)
(c) Nutrient agar slants and plates. — 3.0 g beef ext, 5.0 g
peptone, and 15.0 g agar dild to 1 L with H 2 (dehydrated
nutrient agar is satisfactory). Heat to dissolve, and dispense
6.5 mL portions into 125 x 16 mm screw-cap tubes. Auto-
clave 15 min at 121° and slant tubes until medium solidifies.
Final pH 6.8 ± 0.2. For plates, dispense 100-500 mL portions
in bottles or flasks and autoclave 15 min at 121°. Cool to 50°
in H 2 bath and dispense 18-20 mL portions in 100 X 15 mm
sterile petri dishes. Dry plates 24-48 h at room temp, before
use.
(d) Motility medium. — 10.0 g trypticase, 2.5 g yeast ext,
5.0 g dextrose, 2.5 g Na 2 HP0 4 , and 3.0 g agar dild to 1 L
with H 2 0. Heat to dissolve. Dispense 2 mL portions into 13
X 100 mm tubes, and autoclave 10 min at .121°. Final pH 7.4
± 0.2. Alternatively, dispense 100 mL amts in 150 mL bottles
and autoclave 15 min at 121°. Cool to 50° and aseptically dis-
pense 2 mL into sterile 13 x 100 mm tubes. For best results,
store at room temp. 2-4 days before use to prevent growth
along side of medium.
(e) Trypticase-soy-sheep blood {TSSB) agar. — Dil. 15.0 g
trypticase, 5.0 g phytone peptone, 5.0 g NaCl, and 15.0 g agar
to 1 L with H 2 0. Adjust pH to 7.0 ± 0.2, Heat to boiling to
dissolve, and dispense 100-500 mL portions in bottles or flasks.
Autoclave 15 min at 121° and cool to 48° in H 2 bath. Add
5 mL sterile defibrinated sheep blood per 100 mL medium.
Mix well, dispense 18-20 mL portions into 100 X 15 mm
petri dishes. (Trypticase-soy or tryptic-soy agar plates contg.
5% sheep blood are satisfactory.)
(f) Basic fuchsin stain. — Dissolve 0.5 g basic fuchsin in 20
mL alcohol and dil. to 100 mL with H 2 0. Filter soln if nec-
essary thru fine paper to remove excess dye particles. Store in
tightly stoppered container. (TB Carbol -fuchsin ZN stain is
satisfactory.)
(g) Butterfield's buffered phosphate diluent.— (I) Stock
soln.— Dissolve 34.0 g KH 2 P0 4 in 500 mL H 2 0, adjust to pH
7.2 with ca 175 mL IN NaOH, and dil. to 1 L with H 2 0.
Store in refrigerator. (2) Diluent. — Dilute 1.25 mL stock soln
to 1 L with H 2 0. Prep. 90 ± 1 mL diln blanks with this soln
and autoclave 15 min at 121°. Dispense 0.5 mL portions sterile
diluent into sterile 13 X 100 mm tubes for preparing suspen-
sion of cultures to be tested.
AOAC Official Methods of Analysis (1990)
Salmonella
467
(h) Methanol fixative. — Dispense undild methanol in plas-
tic squeeze bottle for use in fixing slides.
C. Differential Tests
(a) Preparing test inoculum. — Inoculate sep. nutrient agar
slants with each culture to be tested. Incubate slants 18-24 h
at 30° and transfer 3 mm loopful of culture from each slant to
100 x 13 mm tube contg 0.5 mL sterile phosphate buffered
diluent. Suspend culture in diluent with vortex mixer. Alter-
natively, inoculate 5 mL trypticase-soy broth and incubate tubes
18 h at 30°. Mix culture well and use for performing differ-
ential tests. Latter procedure is preferred for rhizoid strains and
other strains which do not disperse well in phosphate buffer.
(b) Reaction on MYP agar. — Mark bottom of MYP agar
plate into 6-8 equal segments with black felt pen as indicated
in Fig. 983.26 and label each section. Place plate in upright
position on piece of white paper and inoculate one or more of
the prelabeled sections by gently touching surface of agar with
2 mm loopful of culture. Let inoculum be absorbed and in-
cubate plates in upright position 24-48 h at 30-35°. Check
for lecithinase production as indicated by zone of ppt sur-
rounding growth. Mannitol fermentation is neg. if growth and
surrounding medium are eosin pink. These reactions should be
observed with all organisms of B. cereus group except rare
lecithinase-neg. variants.
(c) Motility tests. — Inoculate BC motility medium by stab-
bing down center with 3 mm loopful of culture. Incubate 18-
20 h at 30° and examine for type of growth along stab. Motile
strains produce diffuse growth into medium away from stab.
Nonmotile strains except B. cereus var. mycoides grow only
in and along stab. Strains of B. cereus var. mycoides often
produce "fuzzy" growth in semisolid media resulting from cel-
lular expansion but are not motile by means of flagella. Re-
check doubtful results by alternative microscopic motility test
as follows: Add 0.2 mL sterile H 2 to nutrient agar slant and
inoculate with 3 mm loopful of culture. Incubate slant 6-8 h
at 30°, and mix small loopful of liq. culture from base of slant
with drop of sterile FLO on microscope slide. Apply cover
glass and examine immediately for signs of motility. B. cereus
and B. thuringiensis cultures are usually actively motile by
means of peritrichous flagella. B. anthracis and typically rhi-
zoid strains of B. cereus var. mycoides are nonmotile.
(d) Rhizoid growth, — Inoculate predried nutrient agar plate
by touching medium surface near center with 2 mm loopful of
culture. Let inoculum be absorbed, and incubate plate in up-
right position 24-48 h at 30°. Check plate for rhizoid growth
characterized by root or hairlike structures which may extend
several cm from point of inoculation. Many B. cereus strains
FIG. 983.26 — Diagram of template for marking and inoculating
B. cereus confirmatory plates. Each section is labeled and in-
oculated in the center, as indicated by arrow
produce rough irregular colonies that should not be confused
with rhizoid growth. This property is characteristic only of strains
which are classified as B. cereus var. mycoides.
(e) Hemolytic activity. — Mark bottom of trypticase-soy- sheep
blood agar plate into 6-8 equal segments (see Fig. 983.26)
with black felt marking pen. Label each segment and inoculate
one or more segments near center by gently touching agar sur-
face with 2 mm loopful of culture. Let inoculum be absorbed,
and incubate plates 24 h at 30-32°. Check plates for hemolytic
activity as indicated by 2-4 mm zone of complete (beta) he-
molysis surrounding growth. B. cereus is usually strongly he-
molytic, whereas B. thuringiensis and B. cereus var. mycoides
are often weakly hemolytic and produce complete hemolysis
only underneath colonies. B. anthracis is usually nonhemo-
lytic after 24 h of incubation. Caution: Nonmotile, nonhem-
olytic cultures could be B. anthracis. See precautions under
interpreting test results, (g).
(f) Detection of toxin crystals. — Inoculate nutrient agar slant
with loopful of culture. Incubate slant 24 h at 30° and hold at
room temp. 2-3 days. Make smear on microscope slide with
sterile H 2 0. Air-dry and briefly heat- fix by passing slide slowly
over burner flame; let cool, and place slide on staining rack.
Flood slide with methanol, wait 30 s, and pour off methanol.
Dry thoroughly by passing through burner flame. Return slide
to staining rack, and flood completely with 0.5% aq. soln of
basic fuchsin or TB Carbol-fuchsin ZN stain. Heat slide gently
from below with micro burner until steam is seen. Wait 1-2
min and repeat this step. Let stand 30 s, pour off stain, and
rinse slide thoroughly in 1 L clean tap H 2 0. Dry slide without
blotting and examine microscopically under oil immersion for
presence of free spores and darkly stained tetragonal (dia-
mond-shaped) toxin crystals. Free toxin crystals are usually
abundant after 3 days but will not be detectable unless spor-
angia have lysed. Therefore, if free spores are not seen, leave
cultures at room temp, for a few more days and repeat test.
B. thuringiensis produces protein toxin crystals that usually
can be detected by staining, but are not produced by other
members of B. cereus group.
(g) Interpreting test results. — On basis of test results, iden-
tify asB. cereus those isolates which are actively motile, strongly
hemolytic, and do not produce rhizoid growth or protein toxin
crystals. Nonmotile strains of B. cereus may be encountered
and a few are weakly hemolytic. These strains can be differ-
entiated from B. anthracis by their resistance to penicillin and
to gamma bacteriophage. Caution: Nonmotile, nonhemolytic
strains could be B. anthracis, and should be handled with spe-
cial care and submitted to pathology laboratory such as Centers
for Disease Control for identification or destroyed by auto-
claving. Noncrystalliferous variants of B. thuringiensis and
nonrhizoid strains derived from B. cereus var. mycoides can-
not be differentiated from B. cereus by tests described.
ReL: J AOAC 65, 1134(1982).
SALMONELLA
967.25 Salmonella in Foods
Preparation of Culture Media and Reagents
First Action 1967
Final Action 1970
(Applicable to the detection and identification of Salmonella
from dried active yeast, dried whole egg, dried egg yolk, and
dried egg white, edible casein, milk chocolate, nonfat dry milk
and dry whole milk, and onion and garlic powders. Method
described is minimal. Depending upon history of sample, addnl
468
Microbiological Methods
AOAC Official Methods of Analysis (1990)
types of examinations may be applied. Use Edwards and Ew-
ing's Identification of Enter obacteriaceae , W. H. Ewing, El-
sevier Science Publishing Co., Inc., New York, NY 10017,
4th ed., 1986, as guide for further study of isolated microor-
ganisms. For food sampling plans and initial sample handling,
refer to Chapter 1 , Bacteriological Analytical Manual, 6th ed. ,
1984.)
A. Preparation
(Sizes of culture media containers (test tubes, flasks, and petri
dishes) are specified in prepn of each medium. Different size
containers may be used if they give identical results. All media
containers must have covers, caps, or plugs which prevent
contamination but maintain aerobic conditions unless other-
wise directed.)
(a) Lactose broth, —See 940.36A(f). Dispense 225 mL
portions into 500 mL flasks. Autoclave 15 min at 121°. Asep-
tically det. vol. and adjust, if necessary, to 225 mL. Final pH,
6.7 ± 0.2.
(b)(7) Selenite cystine broth. — Suspend 5.0 g tryptone or
polypeptone, 4.0 g lactose, 10.0 g Na 2 HP0 4 , 4.0 g NaHSe0 3 ,
and 0.01 g L-cystine in 1 L H 2 and mix thoroly. Heat with
frequent agitation. Dispense 10 mL portions into sterile 16 x
150 mm test tubes. Heat 10 min in flowing steam. Do not
autoclave. Final pH, 7.0 ± 0.2. Medium is not sterile. Use
same day as prepd.
(2) Selenite cystine broth (North and Bar tram). — Prep, as
in (/), using 5.0 g polypeptone or 4.0 g tryptone, 4.0 g lac-
tose, 4.0 g NaHSe0 3 , 5.5 g Na 2 HP0 4 , 4.5 g KH 2 P0 4 , and 1
mL 1% L-cystine (10 mg) soln prepd by dissolving 1.0 g l-
cystine in 15 mL \N NaOH and dilg to 100 mL with sterile
H 2 0,
(c) Tetrathionate broth {with iodine and brilliant green). —
Suspend 5.0 g polypeptone, 1.0 g bile salts, 10 g CaC0 3 , and
30 g Na 2 S 2 3 .5H 2 in 1 L H 2 0, mix thoroly, and heat to bp.
(Ppt will not dissolve completely.) Cool to <45° and store at
5-8°. Prep. 1-Kl soln by dissolving 5 g KI in 5 mL sterile
H 2 0, adding 6 g resublimed 1, dissolving, and dilg to 20 mL
with sterile H 2 0. Prep, brilliant green soln by dissolving 1
g dye in sterile H 2 and dilg to 100 mL. On day medium is
used, add 20 mL 1-Kl soln and 10 mL brilliant green soln per
1 L basal broth, Resuspend ppt by gentle agitation and asep-
tically dispense 10 mL portions into 20 or 16 X 150 mm sterile
test tubes. Do not heat medium, after addn of I-Kl and dye
so Ins.
(d) Xylose lysine desoxycholate agar (XLD). — Suspend in-
gredients (/) or (2) (varies with mfgr of formula) in 1 L H 2
and mix thoroly. Heat with frequent agitation just until me-
dium boils. Do not overheat. Cool in H 2 bath and pour 20
mL portions into 15 x 100 mm petri dishes. Let dry ca 2 h
with covers partially removed; then close plates. Final pH, 7.4
± 0.2. Do not autoclave.
(/) 3.5 g xylose, 5.0 g L-lysine, 7.5 g lactose, 7.5 g su-
crose, 5.0 g NaCl, 3.0 g yeast ext, 0.08 g phenol red, 2.5 g
Na desoxycholate, 6.8 g Na thiosulfate, 0.8 g ferric ammo-
nium citrate, and 13.5 g agar.
(2) 3.75 g xylose, 5.0 g L-lysine, 7.5 g lactose, 7.5 g su-
crose, 5.0 g NaCl, 3.0 g yeast ext, 0.08 g phenol red, 2.5 g
Na desoxycholate, 6.8 g Na thiosulfate, 0.8 g ferric ammo-
nium citrate, and 15 g agar.
(e) Hektoen enteric agar (HE). — Suspend ingredients (/) or
(2) (varies with mfgr of formula) in .1 L H 2 and mix thoroly.
Heat to boiling with frequent agitation and let boil few mo-
ments. Do not overheat. Cool in H 2 bath and pour 20 mL
portions into 15 x 100 mm petri dishes. Let dry ca 2 h with
covers partially removed; then close plates. Final pH, 7.6 ±
0.2. Do not autoclave.
(J) 12.0 g thiotone peptone, 3.0 g yeast ext, 9.0 g bile salts,
12.0 g lactose, 12.0 g sucrose, 2.0 g salicin, 5.0 g NaCL 5.0
g Na thiosulfate, 1.5 g ferric ammonium citrate, 0.064 g brom-
thymol blue, 0.1 g acid fuchsin, and 13.5 g agar.
(2) 12.0 g proteose peptone, 3.0 g yeast ext, 9.0 g bile salts
No. 3, 12.0 g lactose, 12.0 g sucrose, 2.0 g salicin, 5.0 g
NaCl, 5.0 g Na thiosulfate, 1.5 g ferric ammonium citrate,
0.065 g thymol blue, 0.1 g acid fuchsin, and 14.0 g agar.
(f) Bismuth sulfite (BS) agar (Wilson and Blair). — Suspend
10 g polypeptone or peptone, 5.0 g beef ext, 5.0 g glucose,
4.0 g Na 2 HP0 4 , 0.3 g FeS0 4 , 8.0 g Bi 2 (S0 3 ) 3 indicator, 0.025
g brilliant green, and 20 g agar in 1 L H 2 0, mix thoroly, and
heat with occasional agitation. Boil ca I min to obtain uniform
suspension. (Ppt will not dissolve.) Cool to 45-50°. Suspend
ppt by gentle agitation and pour 20 mL portions into 15 X 100
mm petri dishes. Let dry ca 2 hr with covers partially removed;
then close plates. Final pH, 7.6 ± 0.2. Do not autoclave. Pre-
pare plates day before streaking and store in dark at room temp.
Selectivity of plates decreases 48 hr after prepn.
(g) Triple sugar iron agar (TSI agar). — Suspend ingredi-
ents (J) or (2) in 1 L H 2 0, mix thoroly, and heat with occa-
sional agitation. Boil ca 1 min until ingredients dissolve. Fill
16 X 150 mm tubes 7.3 full and cap or plug so that aerobic
conditions are maintained during use. Autoclave 12 min at 121°.
Before medium solidifies, place tubes in slanted position so
that deep butts (ca 3 cm) and adequate slants (ca 5 cm) are
formed on solidification.
(/) 20 g polypeptone, 5.0 g NaCl, 10 g lactose, 10 g su-
crose, 1 g glucose, 0.2 g Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 0, 0.2 g Na 2 S 2 3 ,
0.025 g phenol red, and 13 g agar. Final pH, 7.3 ± 0.2.
(2) 3.0 g beef ext, 3.0 g yeast ext, 15 g peptone, 5.0 g
proteosepeptone, 1.0 g glucose, 10 g lactose, 10 g sucrose,
0.2 g FeS0 4 , 5.0 g NaCl, 0.3 g Na 2 S 2 3 , 0.024 g phenol red,
and 12 g agar. Final pH, 7.4 ± 0.2.
(h) Tryptophane broth.— See 940.36A(h). Use 16 or 20 X
150 mm test tubes.
(i) Buffered glucose broth (MR-VP medium). — See
940.36 A(b). Use 16 or 20 x 150 mm test tubes.
(j) Simmons' citrate agar.— Dissolve 2.0 g Na citrate, 5.0
g NaCl, 1.0 g K 2 HP0 4 , 1 .0 g NH 4 H 2 P0 4 , 0.2 g MgS0 4 , 0.08
g bromothymol blue, and 15 g agar in 1 L H 2 0, and heat
gentlv with occasional agitation. Boil 1-2 min until ingredi-
ents dissolve. Final pH, 6.9 ± 0.2. Fill 13 x 100 or 16 x
150 mm test tubes V3 full and cap or plug so that aerobic con-
ditions are maintained during use. Autoclave 15 min at 121°.
Before medium solidifies, place tubes in slanted position so
that deep butts (ca 2 or 3 cm, resp.) and adequate slants (ca
4 or 5 cm, resp.) are formed on solidification.
(k)(l) Urea broth. — Dissolve 20 g urea, 0.1 g yeast ext,
9.1 g KH 2 P0 4 , 9.5 g Na 2 HP0 4 , and 4.0 mL 0.25% phenol
red (10 mg) soln in 1 L H 2 0. Do not heat. Sterilize by filtra-
tion and aseptically dispense 1.5—3 mL portions into 13 x 100
mm sterile test tubes. Final pH, 6.8 ± 0.2.
(2) Rapid urea broth. — Prep, as in (7), using 0.091 and
0.095 g phosphate salts, resp,
(1) Malonate broth. — Dissolve 1.0 g yeast ext, 2.0 g
(NH 4 ) 2 S0 4 , 0.6 g K 2 HP0 4 , 0.4 g KH 2 P0 4 , 2.0 g NaCl, 3.0 g
Na malonate, 0.25 g glucose, and 0.025 g bromothymol blue
in 1 L H 2 0, heating if necessary until dissolved. Dispense 3
mL portions into 13 x 100 mm test tubes and autoclave 15
min at 121°. Final pH, 6.7 ± 0.2.
(m)(7) Lysine iron agar (Edwards and Fife). — Dissolve 5.0
g gelysate or peptone, 3.0 g yeast ext, 1 .0 g glucose, 10 g l-
lysine, 0.5 g ferric ammonium citrate, 0.04 g anhyd. Na 2 S 2 3 ,
AOAC Official Methods of Analysis (1990)
Salmonella
469
0.02 g bromocresol purple, and 15 g agar in 1 L H 2 0, heating
until dissolved. Dispense 4 mL portions into 13 x 100 mm
test tubes and cap or plug so that aerobic conditions are main-
tained during use. Autoclave 12 min at 121°. Before medium
solidifies, place tubes in slanted position so that 4 cm butts
and 2.5 cm slants are formed on solidification. Final pH, 6.7
± 0.2.
(2) Lysine decarboxylase broth (Falkow). — Dissolve 5.0 g
gelysate or peptone, 3.0 g yeast ext, 1.0 g glucose, 5.0 g l-
lysine, and 0.02 g bromocresol purple in 1 L H 2 0, heating
until dissolved. Dispense 10 mL portions into 16 X 125 mm
screw-cap test tubes. Autoclave, loosely capped, 15 min at
121°. Screw caps on tightly for storage and after inoculation.
Final pH, 6.5-6.8.
(n) Motility test medium {semisolid medium). — Dissolve 3.0
g beef ext, 10 g peptone or gelysate, 5.0 g NaCl, and 4.0 g
agar in 1 L H 2 and heat gently with occasional agitation. Boil
1-2 min to dissolve. If medium is to be stored, dispense 20
mL portions into screw-cap containers, replacing caps loosely.
Autoclave 15 min at 121°. Cool to 45°. To store, screw caps
on tightly and refrigerate at 5-8°. To use, remelt in boiling
H 2 or flowing steam and cool to 45°. Aseptically dispense
20 mL portions into 15 x 1 00 mm petri dishes and let solidify
with dish completely covered. Use plates same day as prepd.
Final pH, 7.4 ± 0.2.
(o) Potassium cyanide (KCN) broth. — Dissolve 3.0 g pro-
teose peptone No. 3 or poly peptone, 5.0 g NaCl, 0.225 g
KH 2 P0 4 , and 5.64 g Na 2 HP0 4 in 1 L H 2 0. Autoclave 15 min
at 121°. Cool and refrigerate at 5-8°. Final pH, 7.6 ± 0.2.
Dissolve 0.5 g KCN in 100 mL cold (5-8°) sterile H 2 0. Using
sterile bulb pipet or sterile syringe {do not pipet by mouth),
aseptically add 15 mL cold KCN soln per L cold, sterile basal
broth. Mix thoroly with gentle agitation and aseptically dis-
pense 1 .0—1.5 mL portions into sterile 13 X 100 mm test tubes.
Using aseptic technic, immediately stopper tubes with No. 2
corks impregnated with paraffin. Prep, corks by boiling in par-
affin ca 5 min. Place corks in tubes so that paraffin does not
flow into broth but forms good seal between rim of tube and
cork. Medium stored at 5-8° is usually stable ca 2 weeks.
(p)(7) Phenol red carbohydrate broth. — Dissolve 10 g
trypticase or proteose peptone No. 3, 5.0 g NaCl, 1.0 g beef
ext (optional), and 7.2 mL 0.25% phenol red (18 mg) soln in
1 L H 2 and heat with gentle agitation until dissolved. Dis-
solve 5 g dulcitol, 10 g lactose, or 10 g sucrose (as specified
in title of test) in this basal broth. Dispense 2.5 mL portions
into 13 x 100 mm test tubes contg inverted 6 X 50 mm fer-
mentation tubes. Autoclave 10 min at 1 18° (12 psi). Final pH,
7.4 ± 0.2. Alternatively, dissolve ingredients, omitting car-
bohydrate, in 800 mL H 2 with heat and occasional agitation.
Dispense 2.0 mL portions into 13 X 100 mm test tubes contg
inverted fermentation tubes. Autoclave 15 min at 118° and let
cool. Dissolve carbohydrate in 200 mL H 2 and sterilize by
passing soln thru bacteria-retaining filter. Aseptically add 0.5
mL sterile filtrate to each tube of sterilized broth after cooling
to <45°. Shake gently to mix. Final pH, 7.4 ± 0.2.
(2) Purple carbohydrate broth. — Prep, as in (/), using as
basal broth 10 g proteose peptone No. 3 or gelysate, 5.0 g
NaCl, and 0.015 or 0.020 g bromocresol purple. Final pH,
6.8 ± 0.2.
(q) MacConkey agar. — Suspend 3.0 g proteose peptone or
polypeptone, 17 g peptone or gelysate, 10 g lactose, 1.5 g bile
salts No. 3 or bile salts mixt., 5.0 g NaCl, 3.0 mL 1% neutral
red (30 mg) soln, 1 mL 0.1% crystal violet (1.0 mg) soln, and
13.5 g agar in 1 L H 2 and mix thoroly until homogeneous.
Heat, with occasional agitation, and boil 1-2 min until ingre-
dients dissolve. Autoclave 15 min at 121°. Cool to 45-50° and
pour 20 mL portions into 15 x 100 mm petri dishes. Let dry
>2 hr with plates covered. Do not use wet plates. Final pH,
7.1 ± 0.2.
(r) Brain-heart infusion broth. — Dissolve infusion from 200
g calf brain and from 250 g beef heart, 10.0 g proteose peptone
or gelysate, 5.0 g NaCl, 2.5 g Na 2 HP0 4 . 12H 2 0, and 2.0 g
glucose in 1 L H 2 0, heating gently if necessary. Dispense 5
mL portions into 16 X 150 mm test tubes and autoclave 15
min at 121°. Final p'H, 7.4 ± 0.2.
(s) Trypticase soy -tryptose broth. — Combine 15 g com. de-
hydrated trypticase soy broth medium (contg 17.0 g trypticase,
3.0 g phytone, 5.0 g NaCl, 2.5 g K 2 HP0 4 , and 2.5 g glucose),
13.5 g com. dehydrated tryptose broth medium (contg 20 g
tryptose, 5 g NaCl, and 1.0 g glucose), 3 g yeast ext, and 1
L H 2 0. Heat, if necessary, until dissolved. Dispense 5 mL
portions into 16 X 150 mm test tubes and autoclave 15 min
at 121°. Final pH, 7.2 ± 0.2.
(t) Trypticase {tryptic) soy broth. — Suspend 17.0 g trypti-
case or tryptose (pancreatic digest of casein), 3.0 g phytone
(papaic digest of soya meal), 5.0 g NaCl, 2.5 g K 2 HP0 4 , and
2.5 g glucose in 1 L H 2 0. Heat gently to dissolve completely.
Dispense 225 mL portions into 500 mL flasks. Autoclave 15
min at 121°. Aseptically det. vol. and adjust, if necessary, to
225 mL. Final pH, 7.3 ± 0.2.
(u) Lauryl sulfate tryptose broth. — Prep, as 966.23A(b),
but without inverted fermentation tubes, 10 x 75 mm.
(v) Reconstituted nonfat dry milk with brilliant green dye
{NFDM-BG).— Suspend 100 g dehydrated NFDM in 1 L H 2 0;
mix by swirling until dissolved. Autoclave 15 min at 121°.
Add brilliant green dye soln, 966.24(n), after blending sam-
ple/broth mixture as in 978.23A(e).
(w) Brilliant green (BG) water. — Prep, sterile H 2 as
966.24(m) and add 2 mL 1% aq. brilliant green dye, 966.24(n),
per L sterile H 2 and mix well.
B. Diagnostic Reagents
(a) Kovacs reagent for indole test. — Dissolve 5 g p-dime-
thylaminobenzaldehyde in 75 mL amyl alcohol and slowly add
25 mL HC1.
(b) Voges-Proskauer {VP) test reagents. — (7) Alpha-
naphthol soln. — 5%. Dissolve 5.0 g a-naphthol in 100 mL
absolute alcohol.
(2) Potassium hydroxide soln.-— 4-0%. Dissolve 40 g KOH
in H 2 and dil. to 100 mL.
(c) Sodium hydroxide soln. — IN. Dissolve 42.11 g 95% re-
agent NaOH in sterile H 2 and dil. to 1 L.
(d) Hydrochloric acid soln. — IN. Dil. 89 mL to 1 L with
sterile H 2 0.
(e) Methyl red indicator. — Dissolve 0.10 g Me red in 300
mL alcohol and dil. to 500 mL with H 2 0.
(f) Sterile physiological saline soln. — See 940.36B(c).
(g) Formalinized physiological saline soln. — Add 6 mL
HCHO soln (36-38%) to 1 L sterile saline soln, (f), mix, and
store in tightly stoppered containers.
(h) Salmonella polyvalent somatic {O) antiserum* . — ("Ser-
ological Identification of the Salmonella Serotypes," No. 1229,
Difco Laboratories, November 1977, p. 13, or equiv.) Con-
tains agglutinins for at least somatic (O) antigens 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 19, 22, 23, 24, 25,
34, and Vi. They are agglutinins for somatic (O) groups: A,
B, C,, C 2 , D, E,, E 2 , E 3 , E 4 , F, G,, G 2 , H, I, and Vi.
(i) Salmonella individual somatic {O) antisera* . — {See ref.
* Conform to specifications issued by Diagnostic Products Evaluation Branch,
Biological Products Div., Bureau of Laboratories, Centers for Disease Control,
Atlanta, GA 30333.
470
Microbiological Methods
AOAC Official Methods of Analysis (1990)
in (h).) For at least each of the somatic (O) groups listed in
(h).
(j) Salmonella polyvalent flagellar (//) antiserum. Poly a-
z*. — (See p. 12 of ref. in (h).) Contains agglutinins for at least
the following flagellar (H) antigens: a, b, c, d, e, f, g, h, i,
k, 1, m, n, p, q, r, s, t, u, v, w, x, y, z, z 4 , z 6 , z 10 , z J3 , z 15 ,
Z93 , Z24 . Z28 ? Z29 , Z32 •> 1. £•, j, o , / .
(k) Salmonella "Spicer-Edwards" flagellar (H) anti-
sera*. — (From pp. 11 and 12 of ref. in (h).) Consists of 7
pooled or polyvalent antisera which react as in Table 967.25.
(1) pH Test paper. — Min. range 6.0-7.6 with max. gra-
dations of 0.4 pH unit per color change.
(m) Sterile distilled water. — Dispense 1 L H 2 into 2 L
wide-mouth flask or wide-mouth jar; plug or cap loosely. Au-
toclave 20 min at 121°.
(n) Brilliant green dye soln. — 1%. Dissolve 1 g in sterile
H 2 and dil. to 100 mL. (Since some batches of dye are un-
usually toxic, test all batches of dye before use and use only
those producing satisfactory results when tested with known
pos. and neg. test organisms.)
(o) Bromcre sol purple soln. — 0.2%. Dissolve 0.2 g in ster-
ile H 2 and dil. to 100 mL.
Refs.: JAOAC 50, 753(1967); 51, 870(1968); 52, 455(1969);
56, 1027(1973); 59, 731(1976); 62, 499(1979); 64,
893(1981); 64, 899(1981); 65, 356(1982).
967.26 Salmonella in Foods
Detection
First Action 1967
Final Action 1974
A. Preparation of Sample
(a) Dried whole egg, dried egg yolk, and dried egg white. —
Aseptically open sample container and aseptically weigh 25 g
sample into sterile, empty, wide- mouth, screw-cap pt (500 mL)
jar. Add ca 15 mL sterile lactose broth, 967.25A(a). Stir with
sterile glass rod, sterile spoon, or sterile tongue depressor to
smooth suspension. Add 3 addnl portions lactose broth, 10,
10, and 190 mL for total of 225 mL. Stir after each addn until
sample is suspended without lumps. Cap jar securely and let
Table 967.25 Spicer-Edwards Salmonella H Antisera and H
Antigens with Which Each Antiserum Reacts
Spicer-Edwards Salmonella H Antisera
H Antigens
1
2
3
4
a
+
+
+
-
b
+
+
-
+
c
+
+
-
-
d
+
-
+
+
eh
+
-
+
-
G Complex 3
+
-
-
+
i
+
~
-
—
k
-
+
+
+
r
-
+
-
+
y
-
+
_
_
z
_
-
+
+
z 4 Complex* 7
-
-
+
~
Z10
—
-
-
+
z 29
-
+
+
_
H Antigens
Salmonella H Antisera
enx, enz 15
EN complex
lv,
lw, lz 13 ,
lz 2 8
L
complex
1,
2; 1, 5;
1, 6; 1
7
1
complex
a The G complex component of Spicer-Edwards Salmonella H antisera 1 and
4 reacts with antigens f, g, m, p, q, s, t, and u.
b The z 4 complex component reacts with z 4 , z 23 , z 24 , and z 32 .
(From Difco Laboratories)
stand at room temp. 60 min. Mix well by shaking, and det.
pH with test paper, 967.25B(I). Adjust pH, if necessary, to
6.8 ± 0.2 with sterile IN NaOH or HCI, 967.25B(c) or (d),
capping jar securely and mixing well before detg final pH.
Loosen jar cap ca i U turn and incubate 24 ± 2 hr at 35°.
(b) Dry whole milk. — Aseptically weigh 25 g sample into
sterile, wide-mouth screw-cap 500 mL (1 pt) jar. Add 225 mL
sterile H 2 and mix well. Cap jar securely and let stand 60
min at room temp. Mix well by swirling and det. pH with test
paper, 967.25B(I). Adjust pH, if necessary, to 6.8 ± 0.2 with
sterile IN NaOH or HCI, 967.25B(c) or (d). Add 0.45 mL of
1% aq. brilliant green dye soln, 967.25B(n), and mix well.
Loosen jar cap ca l U turn and incubate 24 ± 2 hr at 35°.
(c) Dried active yeast. — Aseptically weigh 25 g sample into
sterile, empty, wide-mouth, screw-cap pt (500 mL) jar. Add
225 mL sterile trypticase (tryptic) soy broth, 967.25 A(t), and
let yeast form smooth suspension. Cap securely and let stand
60 min at room temp. Det. pH with test paper, 967.25B(1).
Adjust pH, if necessary to 6.8 ± 0.2 with sterile 17V NaOH
or HCI, 967.25B(c) or (d), capping jar securely and mixing
well before detg final pH. (If pH is adjusted before yeast is
evenly suspended, final pH will be less than desired.) Incubate
24 ± 2 hr at 35°, with jar cap loosened l / 4 turn.
(d) Onion powder and garlic powder (First Action 1979;
Final Action 1980). — Aseptically weigh 25 g sample into ster-
ile, wide-mouth, screw-cap 500 mL (1 pt) jar. Sample is pre-
enriched in trypticase (tryptic) soy broth, 967.25A(t), with added
K 2 S0 3 (5 g/L) for final 0.5% K 2 S0 3 concn. Autoclave 225
mL portions in 500 mL flasks for 15 min at 121°. Aseptically
det. vol. and adjust, if necessary, to 225 mL. Add 225 mL
sterile trypticase (trypic) soy broth, 967.25A(t), with K 2 S0 3 ,
to sample, and mix thoroly using sterile glass rod or spoon.
Let stand 60 min and det. pH with test paper, 967.25B(1).
Adjust pH, if necessary, to 6.8 ± 0.2 with sterile 1A" NaOH
or IN HCI, 967.25B(c) or (d). Incubate 24 ± 2 hr at 35°, with
jar cap loosened l / 4 turn.
(e) Milk chocolate and casein. — Aseptically weigh 25 g
sample into sterile blender jar. Add 225 mL sterile reconsti-
tuted NFDM, 967.25A(v), to chocolate samples, and add 225
mL lactose broth, 967.25A(a), to casein samples. Blend each
sample/broth mixt. 2 min at high speed and decant blended
homogenate into sterile 500 mL jar. Cap jar securely and let
stand 60 min at room temp. Mix well by shaking, and det. pH
with test paper, 967.25B(I). Adjust pH, if necessary, to 6.8
± 0.2 with sterile IN NaOH or HCI, 967.25B(c) or (d), cap-
ping jar securely and mixing well before detg final pH. To
chocolate-reconstituted NFDM samples, add 0.45 mL 1% aq.
brilliant green dye, 967.25B(n), and mix well. Loosen jar caps
V4 turn and incubate jar 24 ± 2 h at 35°.
(f) Instant nonfat dry milk (NFDM) (First Action 1984). —
Aseptically open sample container and aseptically weigh 25 g
sample into sterile beaker (250 mL) or other appropriate con-
tainer. Cover with sterile foil cover or sterile cap to prevent
contamination. Using sterile glass or paper (made with tape to
withstand autoclaving) funnel, pour 25 g analytical unit gently
and slowly over surface of 225 mL brilliant green H 2 0,
967.25A(w), contained in sterile 500 mL erlenmeyer or other
appropriate container. Let container with sample-pre-enrich-
ment broth stand undisturbed 60 ± 5 min. Incubate loosely
capped container, without mixing or pH adjustment, for 24 ±
2 h at 35°.
B. Isolation
(a) Growth in selective broth. — Gently shake incubated
sample mixt., 967.26A, and transfer 1 mL to 10 mL selenite
cystine broth, 967.25A(b)(7) or (2), and addnl 1 mL to 10 mL
tetrathionate broth, 967.25A(c). Incubate 24 ± 2 h at 35°. (For
AOAC Official Methods of Analysis (1 990)
Salmonella
471
dried active yeast, substitute lauryl sulfate tryptose broth,
967.25A(u), for selenite cystine broth, 967.25A(b)(7) or (2).
Vortex-mix, and streak 3 mm loopful of incubated selenite
cystine broth on selective media plates of xylose lysine des-
oxycholate agar, 967.25A(d), Hektoen enteric agar, 967.25A(e),
and BS agar, 967. 25 A (f). Repeat with 3 mm loopful of in-
cubated tetrathionate broth. Incubate plates 24 ± 2 h at 35°.
(b) Appearance of typical Salmonella colonies. — (J) On
xylose lysine desoxycholate agar. — Pink colonies with or
without black centers. Many Salmonella may have large, glossy
black centers or may appear as almost completely black col-
onies. Atypically, a few Salmonella cultures produce yellow
colonies with or without black centers.
(2) On Hektoen enteric agar. — Blue-green to blue colonies
with or without black centers. Many Salmonella cultures may
have large glossy black centers or may appear as almost com-
pletely black colonies.
(3) On bismuth sulfite agar. — Brown, gray, or black,
sometimes with metallic sheen. Surrounding medium is usu-
ally brown at first, turning black with increasing incubation
time. Some strains produce green colonies with little or no
darkening of surrounding medium.
Examine XLD and HE agar plates for typical or suspicious
Salmonella colonies after 24 ± 2 h incubation at 35°. BS agar
plates should be examined for typical or suspicious Salmonella
colonies after 24 ± 2 h and 48 ± 2 h incubation at 35°.
C. Treatment of Typical or Suspicious Colonies
(a) Inoculation of triple sugar iron (TSf) agar and lysine
iron agar (LI A). — Pick with needle 2 or more typical or sus-
picious colonies, if present, from each xylose lysine desoxy-
cholate, Hektoen enteric, and BS agar plates having growth.
Inoculate TSI agar slant, 967.25A(g), with portion of each col-
ony by streaking slant and stabbing butt. After inoculating TSI
agar with needle, do not obtain more inoculum from colony
and do not heat needle, but inoculate LI A, 967. 25 A (m) (7), as
in 967.27C(a). Store picked selective plates at 5-8° or at room
temp, (ca 26°).
(b) Presumptive reactions. — Incubate TSI and LIA slants
at 35° for 24 ± 2 h and 48 ± 2 h, resp. Cap tubes loosely to
maintain aerobic conditions while incubating slants to prevent
excessive H 2 S production. Salmonella cultures typically have
alk. (red) slant and acid (yellow) butt, with or without H 2 S
(blackening of agar) in TSI agar. In LIA, Salmonella cultures
typically have alk. (purple) reaction in butt. Consider only a
distinct yellow coloration in butt of tube as an acidic (neg.)
reaction. Do not eliminate cultures that produce discoloration
in butt solely on this basis. Most Salmonella cultures produce
H 2 S in LIA. Retain all presumptive pos. Salmonella cultures
on TSI (alk. slant and acid butt) agar for biochem. and ser-
ological tests whether or not corresponding LIA reaction is
pos. (alk. butt) or neg. (acid butt). Do not exclude a TSI cul-
ture that appears to be non-Salmonella if the reaction in LIA
is typical (alk. butt) for Salmonella. Treat these cultures as
presumptive pos. and submit them to further examination. LIA
is useful in detection of S. arizonae and atypical Salmonella
strains that utilize lactose and/or sucrose. Discard only ap-
parent non-Salmonella TSI agar cultures (acid slant and acid
butt) if corresponding LIA reactions are not typical (acid butt)
for Salmonella. Test retained presumptive pos. TSI agar cul-
tures as directed in 967.26C(c) to det. if they are Salmonella
sp., 967.27D(e)(7), or S. arizonae organisms, 967.27D(e)(2).
If TSI slants fail to give typical Salmonella reactions, pick
addnl suspicious colonies from selective medium plate not giv-
ing presumptive pos. culture and inoculate TSI and LIA slants
as in (a).
(c) Selection for identification. — Apply biochem. and ser-
ological identification tests to 3 presumptive pos. TSI agar cul-
tures picked from selective agar plates streaked from selenite
cystine broth and to 3 presumptive pos. TSI agar cultures picked
from selective agar plates streaked from tetrathionate broth.
If 3 presumptive pos. TSI agar cultures are not isolated from
1 set of selective agar plates, test other presumptive pos. TSI
agar cultures, if isolated, by biochem. and serological tests.
A min. of 6 TSI cultures are examined for each 25 g sample
tested.
Refs.: JAOAC 50, 753(1967); 51, 870(1968); 52, 455(1969);
56, 1027(1973); 59, 731(1976); 61, 401(1978); 62,
499(1979); 64, 893(1981); 64, 899(1981); 65, 356(1982);
67, 807(1984); 69, 277(1986).
967.27 Salmonella in Foods
Identification
First Action 1967
Final Action 1968
A. Cultures
Pure cultures on TSI agar are required for inoculation of
biochem. test media.
(a) Pure cultures.— Proceed to 967. 27B.
(b) Mixed cultures. — Streak any culture that appears to be
mixed on MacConkey agar, 967.25A(q), or xylose lysine de-
soxycholate agar, 967.25A(d), or Hektoen enteric agar,
967.25A(e). Incubate 24 ± 2 h at 35°.
(c) Appearance of Salmonella colonies. — (I) On Mac-
Conkey agar. — Typical colonies appear transparent and col-
orless, sometimes with dark centers. Salmonella will clear areas
of pptd bile caused by other organisms sometimes present in
medium .
(2) On xylose lysine desoxycholate agar. — See 967. 26B
(h)(1).
(3) On Hektoen enteric agar. —See 967.26B(b)(2).
Pick with needle ^2 typical or suspicious colonies and in-
oculate TSI slants by streaking the slant and stabbing the butt
as in 967.26C(a). Retest purified cultures as in 967.26C(b),
and proceed with identification.
As alternative to conventional tube system for Salmonella,
any one of the 4 commercial biochem. systems (API, Enter-
otube, Minitek, or Micro-ID) may be used for presumptive
generic identification of foodborne Salmonella. See 978.24 and
989.12.
B. Subcultures
(a) Urease test. — Subculture small amt of growth from pre-
sumptive pos. TSI agar culture to urea broth, 967.25A(k)(7),
and incubate 24 ± 2 hr at 35° or inoculate rapid urea broth,
967. 25 A (k) (2) with two 3 mm loopful s of growth from each
presumptive-pos. TSI agar slant culture, and incubate 2 hr in
H 2 bath at 37 ± 0.5°. Discard all cultures that give pos. test
(purple-red color). Salmonellae are urease neg. (no change in
orange color of medium).
(b) Serological flagellar (H) screening test. — To reduce
number of presumptive pos. TSI agar cultures carried thru
identification tests, perform serological flagellar (H) screening
test by transferring one 3 mm loopful of each urease-neg. TSI
agar culture to either:
(J) Brain-heart infusion broth, 967.25 A(r), (for test on same
day) and incubate at 35° until visible growth occurs (ca 4-6
hr); or
(2) Trypticase soy- tryptose broth, 967.25A(s), (for test on
following day) and incubate 24 ± 2 hr at 35°.
472
Microbiological Methods
AOAC Official Methods of Analysis (1990)
To 5 mL of each of the 6 broth cultures add ca 2.5 mL
formalinized physiological saline soln, 967.25B(g). Select 2
formalinized broth cultures and test with Salmonella flagellar
(H) antisera, 967.25B( j) or (k), as in 967.28C or D
If selected fonnalinized broth cultures are pos., perform addnl
tests on these cultures, beginning with 967. 27C, except step
967.27C(d) may be omitted.
If both formalinized broth cultures are neg., perform ser-
ological test on the 4 additional broth cultures (967.27B(b)(V)
or (2)) to obtain, if possible, 2 pos. cultures for addnl testing,
967.27C.
If all urease-neg. TSI cultures from sample are Salmonella
serological flagellar (H) test neg., then perform addnl tests,
beginning with 967. 27C, on these cultures.
C. Testing Urease-Negative Cultures
Using needle, transfer portion of presumptive pos. TSI agar
culture to lysine iron agar medium and small amt of growth
from the TSI agar culture to each of other media:
(a) Lysine iron agar, 967.25A(m)(7). — Stab butt twice and
then streak slant. Replace tube cap loosely and incubate 48 ±
2 hr at 35°. Examine at least every 24 hr. Most salmonellae
give purple color of alk. reaction thruout medium (final color
is slightly darker than original purple color of medium). If H 2 S
is produced, butt of medium is blackened. Neg. test is purple
or red slant and yellow butt, if LIA test, 967.26C(a), was
satisfactory, it need not be repeated. Use lysine decarboxylase
broth for final detn of lysine decarboxylase if culture gives
doubtful LIA reaction.
If liq. medium is preferred, inoculate tube of lysine decar-
boxylase broth, 967.25A(m)(2). Close tube cap tightly after
inoculation and incubate 48 ± 2 hr at 35°. Examine at least
every 24 hr. Salmonellae give purple color of a Ik. reaction
thruout broth (final color is slightly darker than original purple
color of medium). Sometimes tubes which have yellow color
after 8-12 hr of incubation change to purple later. Neg. test
is permanent yellow color thruout broth. If medium appears
to be discolored (neither purple nor yellow) add few drops of
0.2% bromcresol purple dye 967.25B(o), and re-read tube re-
actions.
(LIA is incubated loosely capped so that aerobic conditions
are maintained, while lysine decarboxylase broth is incubated
tightly closed to exclude air.)
(b) Phenol red dulcitol broth, 967.25 A(p)(7). — Incubate
48 ± 2 hr at 35°. Examine at least every 24 hr. Most sal-
monellae give pos. test indicated by gas formation (displace-
ment of liq. in inverted tube) and/or acid reaction (yellow).
Neg. test is alk. reaction (red) and no gas formation.
(Purple broth base with dulcitol, 967.25A(p)(2), may be
substituted. Pos. test is acid reaction (yellow) and gas. Neg.
test is alk. reaction (purple).)
(c) Tryptophane broth, 967.25A(h). — Incubate 24 ± 2 hr
at 35° and test as follows:
(/) Transfer 3 mm loopful, excluding all solid particles, to
KCN broth, 967.25 A(o). Heat rim of tube to form good seal
when restoppered. Incubate 48 ± 2 hr at 35°. Salmonellae do
not grow in this broth as shown by lack of turbidity (neg. test).
(2) Transfer 3 mm loopful to malonate broth, 967.25A(1),
and incubate 48 ± 2 hr at 35°. Salmonellae give neg. test as
shown by green color (unchanged). Pos. test (alk. reaction) is
shown by blue color.
(J) Transfer 5 mL to empty test tube and add 0.2-0.3 mL
Kovacs reagent, 967.25B(a). Pos. test for indole is shown by
deep red color in reagent on surface of broth. Most salmo-
nellae are indole-neg.
(d) Brain-heart infusion broth, 967.25A(r), or trypticase soy-
tryptose broth, 967,25A(s). — Incubate brain-heart infusion broth
until visible growth occurs (ca 4-6 hr) or incubate trypticase
soy-tryptose broth 24 ± 2 hr at 35°. To 5 mL broth culture
add ca 2.5 mL formalized physiological saline soln, 967.256(g).
Refrigerate formalized broth at 5-8° if test is to be performed
on another day. Perform Salmonella serological flagellar (H)
test, 967.28C, or "Spicer-Ed wards" flagellar (H) test tube test,
967. 28D, using formalized broth culture as flagellar (H) an-
tigen to be tested.
(e) Tests indicating absence of Salmonella. — Discard, as
not Salmonella, cultures that show either:
(V) Pos. indole test (red) and neg. Salmonella serological
flagellar (H) test.
(2) Pos. KCN broth test (growth) and neg. lysine decar-
boxylase test (yellow).
(f) Testing of TSI agar cultures. — Use Salmonella sero-
logical somatic (O) test, 967.28 A.
(g) Classification. — Classify as Salmonella sp. cultures that
have all characteristics shown in Table 967. 27A. If 1 TSI cul-
ture from 25 g sample is classified as Salmonella sp., further
testing of other TSI cultures from same 25 g sample is un-
necessary.
(h) Special cases. — Cultures that contain demonstrable Sal-
monella antigens as shown by pos. Salmonella serological so-
matic (O) test and pos. flagellar (H) test but do not have
biochem. characteristics of salmonellae should be purified as
in 967.27A(b) and retested, beginning with 967.27B.
D. Additional Biochemical Tests
Perform addnl tests on cultures that do not give identical test
results as in Table 967. 27 A and do not classify as Salmonella
sp. Transfer 1 loopful of culture from each unclassified TSI
agar slant to each of following media:
(a) Phenol red lactose broth, 967.25 A(p)(/).— Incubate 48
± 2 hr at 35°. Examine inoculated broth at least every 24 hr.
Pos. test is shown by gas formation (displacement of liq. in
inverted tube) and acid reaction (yellow). Most salmonellae
give neg. test shown by alk. reaction (red) and no gas for-
mation.
Discard, as not Salmonella, cultures that give pos. phenol
red lactose broth test, except: (7) Cultures described in
967.26C(b), and (2) cultures that also give pos. malonate broth
test. Cultures that are phenol red lactose broth pos. or neg.
and malonate broth pos. are tested further to det. if they are
S. arizonae, 967.27D(e)(2).
(Purple lactose broth, 967.25A(p)(2), may be substituted.
Pos. test is acid reaction (yellow) and gas. Neg. test is alk.
reaction (purple) and no gas formation.)
(b) Phenol red sucrose broth, 967.25A(p)(7). — Incubate
and read as in (a) above. Discard, as not Salmonella, cultures
that give pos. test, except cultures described in 967.26C(b).
(Purple sucrose broth may be substituted and read as in (a)
above.)
Table 967.27A Characteristics of Salmonella
Test or Substrate
Results*
Urease, 967.27B(a)
Lysine decarboxylase, 967.27C(a)
Phenol red dulcitol broth, 967.27C(b)
KCN broth, 967.27C(c)(J)
Malonate broth, 967.27C(c)(2)
Indole test, 967.27C(c)(3)
Polyvalent flagellar test, 967.27B(b),
967.27C(d)
Polyvalent somatic test, 967.27C(f)
Negative (orange-red)
Positive (alk.; purple thruout
medium)
Positive (yellow and/or gas) b
Negative (no growth)
Negative (unchanged green) c
Negative (no red color)
Positive (visible agglutination)
Positive (visible agglutination)
3 +, 2:90% pos. in 1-2 days; -, >90% neg. in 1-2 days.
b Majority of S. arizonae cultures are neg.
c Majority of S, arizonae cultures are pos.
AOAC Official Methods of Analysis (1 990)
Salmonella 473
Table 967.27B Biochemical and Serological Reactions of Salmonella
Test or Substrate
Positive
Negative
Salmonella
reaction 3
Glucose (TSI), 967.26C(b)
H 2 S (TSI), 967.26C(b)
Urease, 967.27B(a)
Lysine decarboxylase broth, 967.27C(a}
Phenol red dulcitol broth, 967.27C(b)
KCN broth, 967.27C(c)(f)
Malonate broth, 967.27C(c)(2)
Indole test, 967.27C(c)(3)
Polyvalent flagellar test, 967.27B(b), 967.27C(d)
Polyvalent somatic test, 967.27C(f)
Phenol red lactose broth, 967.27D(a)
Phenyl red sucrose broth, 967.27D(b)
Voges-Proskauer test, 967.27D(c){7)
Methyl red test, 967.27D(c)(2)
Simmons' citrate, 967.27D(d)
yellow butt
blackening
purple-red
purple
yellow and /or gas
turbidity
blue
violet at surface
agglutination
agglutination
yellow and/or gas
yellow and/or gas
pink to red
diffuse red
growth; blue
red butt
no blackening
no color change
yellow
no gas; no color change
no turbidity
no color change
yellow at surface
no agglutination
no agglutination
no gas; no color change
no gas; no color change
no color change
diffuse yellow
no growth; no color change
3 +, =>90% pos. in 1-2 days; -, >90% neg. in 1-2 days; v, variable.
1 Majority of S. arizonae cultures are neg.
; Majority of S. arizonae cultures are pos.
(c) Buffered glucose broth (MR-VP medium, 967.25 A(i)). —
Incubate 48 ± 2 hr at 35°.
(7) Perform Voges-Proskauer (VP) test at room temp, by
transferring 1 ml 48-hr culture to test tube and adding 0.6 mL
a-naphthol soln, 967. 25B (b)(7), and 0.2 mL 40% KOH soln,
967.25B(b)(2). Shake after each addn. To intensify and speed
reaction, add few creatine crystals to test medium. Read re-
sults 4 hr after adding reagents. Pos. VP test is development
of eosin pink color. Salmonellae give neg. test.
(2) Incubate remainder of MR-VP medium addnl 48 ± 2 hr
at 35°. Perform Me red test by transferring 5 mL culture to
test tube and adding 5-6 drops Me red soln, 967.25B(e), and
read results immediately. Salmonellae give pos. test (red). Neg.
test is indicated by yellow color.
(d) Simmons' citrate agar, 967. 25 A (j). — Inoculate by
streaking slant and stabbing butt. Incubate 96 ± 2 hr at 35°.
Salmonellae usually give pos. test shown by growth and color
change from green to blue (alk.). Color change usually appears
first on slant and then spreads thru medium. Neg. test is in-
dicated by no or very little growth and no change in color of
medium.
(e) Classification. — Classify cultures according to results
listed in Table 967.27B. If 1 TSI culture from 25 g sample is
classified as Salmonella sp., further testing of other TSI cul-
tures from same 25 g sample is unnecessary.
(7) Salmonella sp. — Cultures that have reaction patterns of
Table 967.27B.
(2) Salmonella arizonae.— Cultures that have reaction pat-
tern of Table 967.27B, except footnote reactions b and c .
[3) Non-Salmonella sp. — Discard, as not Salmonella, cul-
tures that give results listed in any 1 subdivision of Table
967.27C.
E. Summary of Classification of Non-Salmonella Cultures
Classify, by performing addnl tests described in Edwards
and Ewing's Identification of Enterobacteriaceae, any culture
that is not clearly identified as Salmonella sp. or 5. arizonae
by classification schemes in Tables 967. 27 A and B or not
eliminated from these groups by test reactions listed in Table
967.27C.
If neither of 2 TSI cultures carried thru biochem. tests,
967.27C and D and Tables 967.27A-C, confirms as Salmo-
nella, perform biochem. tests, beginning with 967. 27C, on
remaining urease-neg, TSI cultures from same 25 g sample.
Refs.: JAOAC 50, 753(1967); 51, 870(1968); 52, 455(1969);
56, 1027(1973); 59, 731(1976); 62, 499(1979); 64,
893(1981); 64, 899(1981); 65, 356(1982).
Table 967.27C Criteria for Discarding Uon-Salmonella Cul-
tures
Test(s) or Substrate(s)
Results
(a) Urease test, 967.27B(a)
(b) Indole test, 967.27C(c)(3)
Polyvalent flagellar test, 967.27B(b),
967.27C(d), or Spicer- Edwards
flagellar (H) test, 967.28D
(c) Lysine decarboxylase test,
967.27C(a)
KCN broth, 967.27C(c)(7)
(d) Phenol red lactose broth 3 , 967.27D(a)
(e) Phenol red sucrose broth, 967.27D(b)
(f) KCN broth, 967.27C(c)( 1)
Voges-Proskauer test, 967.27D(c)(/)
Methyl red test, 967.27D(c)(2)
Positive (purple-red)
Positive (red/or violet at
surface)
Negative (no
agglutination)
Negative (yellow)
Positive (growth)
Positive (yellow and/or gas) b
Positive (yellow and/or gas) 6
Positive (growth)
Positive (red)
Negative (yellow)
3 Malonate broth positive cultures are tested further to det. if they are
Salmonella arizonae, 967.27D(e){2)
b Do not discard pos. broth cultures if corresponding LIA cultures give typical
Salmonella reactions; test further to det. if they are Salmonella sp. See
967.26C(a)
978.24 Salmonella sp. in Foods
Biochemical Identification Kit Method
Final Action
(Use of com. biochem. kit as alternative to conventional
biochem. testing in 967.27B-E is based upon demonstration in
analyst's laboratory of adequate correlation between biochem.
kit intended for use and conventional biochem. tests in 967.27B-
E. Com. biochem. kits should not be used as a substitute for
serological tests as described in 967.27B-E, 967.28.)
A. Kits
(a) API 20E. — Available from Analytab Products Inc., 200
Express St, Plain view, NY 11803. Kit is series of 20 plastic
microtubes contg biochem. test substrate affixed to plastic strip
for conducting following 22 tests: urease; oxidase; tryptophan
deaminase; o-nitrophenyl-p-D-galactosidase (ONPG); lysine and
ornithine decarboxylase; arginine dihydrolase; gelatinase; ci-
trate utilization; H 2 S production; indole production; acetoin
production (Voges-Proskauer or VP test); nitrate reduction; and
fermentation of amygdalin, arabinose, glucose, inositol, man-
nitol, melibiose, rhamnose, sorbitol, and sucrose. Required re-
agents include Kovacs reagent, 967.25B(a); 10% FeCl 3 soln
(for phenylalanine deaminase test); VP test reagents (5% ct-
naphthol soln and 40% KOH soln), 967.258(b); nitrate re-
474
Microbiological Methods
AOAC Official Methods of Analysis (1990)
duction reagents (solns of sulfanilic acid and M/V-dimethyl-a-
naphthyl amine); sterile mineral oil; oxidase test reagents (1%
MA^,yV',A^'~tetramethyl-/?-phenylenediamine.2HCl soln and 0.2%
ascorbic acid soln); sterile H 2 0; and 1.5% H 2 2 .
(b) Enterotube II®. — Available from Roche Diagnostics
Systems, Div. of Hoffmann-La Roche, Inc., One Sunset Ave,
Montclair, NJ 07042-5188, order No. 43128. Consists of self-
contained sterile compartmental plastic tube contg 12 different
conventional media and enclosed inoculating needle for con-
ducting following 15 tests: lysine and ornithine decarboxylase;
phenylalanine deaminase; urease; Voges-Proskauer (VP); ci-
trate utilization; H 2 S production; indole production; and utili-
zation of dulcitoi, lactose, adonitol, arabinose, sorbitol, and
glucose (acid and gas). Kovacs reagent, 967.25B(a) (for in-
dole test) and VP reagents, 967.25B(b), are also required.
(c) Enterobacteriaceae II Set. — (Formerly Minitek sys-
tem.) Available from BBL Microbiological Systems, No. 25147.
Consists of system for differentiation of microorganisms by
observation of their effect upon chem. substrates impregnated
into paper disks for conducting following 25 tests: urease; o-
nitrophenyl-p-D-galactosidase (ONPG); phenylalanine deami-
nase; lysine and ornithine decarboxylase; arginine dihydrolase;
nitrate reduction; citrate utilization; H 2 S production; indole
production; malonate utilization; Voges-Proskauer (VP) test;
and fermentation of adonitol, arabinose, dulcitoi, esculin, glu-
cose, inositol, lactose, mannitol, raffinose, rhamnose, salicin,
sorbitol, and sucrose. In addition to inoculum broth, required
reagents include Kovacs reagent, 967.25B(a), 10% FeCl 3 soln
(for phenylalanine deaminase test); VP test reagents (5% a-
naphthol soln and 40% KOH soln), 967.25B(b); nitrate re-
duction reagents (solns of sulfanilic acid and 7V\Af-dimethyl-a-
naphthylamine); and sterile mineral oil. Required apparatus in-
cludes Minitek pipetter, disposable pipet tips, color compar-
ator cards, disk dispenser, plastic multiwell plates, humidor
with sponges for incubation of disks in plates after adding in-
oculum broth contg test culture, and paper disks impregnated
with individual substrates for performing biochem. tests.
Systems (a)-(c) are also available from Fisher Scientific Co.
Systems (b) and (c) are also available from Scientific Prod-
ucts, Inc., and VWR Scientific, Inc.
B. Isolation
Prep, samples and isolate presumptive cultures by 967.26.
C. Identification
Assemble supplies and prep, reagents required for utilizing
kit. Inoculate each unit according to directions supplied by
manufacturer, incubating for time and temp, specified. Add
reagents, observe, and record results. For presumptive iden-
tification, classify cultures according to flow charts and tables
supplied by manufacturer as Salmonella or non-Salmonella sp.
For confirmation of cultures presumptively identified as Sal-
monella sp., perform Salmonella serological somatic (O) test,
967.28A, and Salmonella serological flagellar (H) test, 967.28C,
or "Spicer-Edwards" flagellar (H) test, 967. 28D, and classify
cultures according to following guidelines:
(a) Cultures classified as presumptive Salmonella sp. with
com. biochem. kits are confirmed as Salmonella sp. when cul-
ture demonstrates pos. Salmonella somatic (O) test and pos.
Salmonella (H) test.
(b) Cultures classified as presumptive non-Salmonella sp.
with com. biochem. kits are discarded as non-Salmonella sp.
when the cultures conform to manufacturer's criteria for clas-
sifying cultures as non-Salmonella sp.
(c) Cultures which do not conform to (a) or (b) should be
classified according to addnl tests specified in 967.27B-E,
967,28 or addnl tests specified in Edwards and Ewing's Iden-
tification of Enterobacteriaceae, or sent to ref. typing labo-
ratory for definitive serotyping and identification.
Refs.: JAOAC 61, 1043(1978); 64, 408(1981).
989.12 Salmonella sp., Escherichia coli,
and Other Enterobacteriaceae in Foods
Biochemical Identification Kit Method
First Action 1989
Use of com. biochem. kit as alternative to conventional
biochem. testing in 966.24(a)-(f) (E. coli) and 967.27 (Sal-
monella sp.) is based on demonstration in analyst's laboratory
of adequate correlation between biochem. kit intended for use
and conventional biochem. tests in 966. 24(a)- (f) and 967.27.
Com. biochem. kit should not be used as substitute for sero-
logical tests for Salmonella as described in 967 '.27 ', 967.28.
Com. biochem. kit can be used for presumptive identification
of other Enterobacteriaceae isolated from foods.
A. Principle
Method uses kit in which inoculum contains preformed en-
zymes at levels detectable in 4 h by means of sensitive indi-
cator system. Kit contains filter paper discs impregnated with
reagents which detect presence of specific enzymes and/or
metabolic products produced by certain microorganisms. These
reagents include substrate to be acted on by bacterial enzyme,
and detection system which reacts with metabolic end product
to yield readily identifiable color change. Precise quantities of
substrate and/or detection reagents are supplied to each disc
so that chem. incompatible materials are sepd until tray is in-
oculated. Tests included are Voges-Proskauer (VP), nitrate re-
ductase, phenylalanine deaminase, H 2 S, indole, ornithine
decarboxylase, lysine decarboxylase, malonate utilization,
urease, esculin hydrolysis, p-galactosidase, and arabinose,
adonitol, inositol, and sorbitol fermentations.
B. Method Performance
Results
Percent
Agreement
95% Confidence
Range (Approx.)
Salmonella sp.
Escherichia coli
Other enterics 2
98.8
97.7
84.6
97.2-100
94.6-100
81.2-88.0
Agreement with conventional biochem. tests {AOAC methods).
2 Enterobacteriaceae correctly identified to genera other than Salmonella and
E. coli
C. Apparatus, Culture Media, and Reagents
Use distd or deionized H 2 0.
(a) Plate count agar (standard methods agar) slants. — 5.0
g tryptone, 2.5 g yeast ext, 1.0 g dextrose, and 15.0 g agar.
Suspend ingredients in 1 L H 2 0. Heat to boiling to dissolve
medium completely. Dispense 8-10 mL portions into 16 x
150 mm tests tubes. Autoclave 15 min at 121°. Before medium
solidifies, place tubes in slanted position so that adequate
slants are formed.
(b) Physiological saline. — Dissolve 8.5 g NaCl in 1 L H 2 0.
Final pH must be 6.0 ± 0.5. Do not use saline prepns contg
preservatives such as Na azide or other bacterial growth in-
hibitors. Saline does not need to be sterile but should be freshly
prepd.
(c) 20% KOH soln. — Slowly add 20 g KOH pellets to 60
mL H 2 0. Dissolve by stirring. Add sufficient H 2 to prep.
100 mL soln. Keep KOH soln in tightly closed container when
not in use. Caution: Caustic reagent. Handle with care.
AOAC Official Methods of Analysis (1990)
Salmonella 475
(d) Test tubes. — 16 X 100 mm or larger. One test tube is
required for each isolate to be identified.
(e) Pipets. — 1 mL and 5 mL serological, with cotton plug.
(f) Pathotec cytochrome oxidase test. — No. 34191 (Or-
ganon Teknika Corp., 100 Akzo Ave, Durham, NC 27704);
or equiv.
(g) MICRO -ID identification kit and manual. — No. 34146
(Organon Teknika Corp.).
(h) Support rack. — To hold test kit units (Organon Teknika
Corp., No. 34147).
D. Preparation of Inocula
(1) Select isolated colony from agar medium. Transfer col-
ony to plate count agar slant. Incubate 18-24 h at 35°. Note:
Cultures older than 30 h may give false neg. results.
(2) Perform cytochrome oxidase test on portion of growth
from slant. Cytochrome oxidase-neg. rods should be further
tested.
(3) Pipet ca 3.5 mL physiological saline (b) into 16 x 100
mm test tube for each isolate to be identified. Transfer growth
from each slant into tube of saline until density of suspension
of organisms is equiv. to Mc Far! and No. 2.0. Note: Sterile
test tubes are not required.
E. General Instructions
Components and procedures of test kit have been stdzd for
use in MICRO-ID identification system. Components or pro-
cedures other then those supplied by Organon Teknika Corp.
may yield unsatisfactory results, and should be pretested.
F. Inoculation and Reading of Unit
(J) Open sealed, moisture-proof, foil package and remove
test unit. Do not remove clear plastic tape that covers test wells.
(2) Record sample no. and other required information on
area provided on right side of cover.
(3) Open cover and let unit lie flat on laboratory bench.
(4) Pipet ca 0.2 mL of organism suspension into each in-
oculation well at top of unit.
(5) Close cover and stand tray upright in support rack. (Make
sure that organism suspension is in contact with all substrate
discs. DO NOT moisten detection discs.)
(6) Incubate 4 h at 35-37°. DO NOT use C0 2 incubator.
(7) After 4 h incubation, place each unit flat on bench, open
lid, and add 2 drops (ca 0.1 mL) of 20% KOH soln (c) to
inoculation well of VP test ONLY. Do not add KOH to any
other inoculation well. Close lid and hold tray upright. Be cer-
tain that KOH flows down into VP test soln.
(8) Rotate unit clockwise ca 90° so upper discs in first 5
wells become wet. Hold tray upright and tap gently on bench
to dislodge any suspension trapped under upper disc. Be cer-
tain that each upper disc in reaction chambers 1-5 is mois-
tened by this procedure.
(9) Read all reactions immediately, except VP test, as pos.
or neg. according to color changes listed below. Let color de-
velop in VP well for ca 10 min, and then read. Read color of
upper disc for first 5 tests; read color of organism suspension
for remaining 10 tests. Record result for each biochem. test
on encoding forms supplied with system.
Test
Positive
Reaction
Negative
Reaction
Voges-Proskauer
pink to red
light yellow
Nitrate reductase
red
colorless 1o
light pink
Phenylalanine
green 1
light yellow
deaminase
H 2 S
brown to black 2
white
Indole
pink to red
light yellow
to orange
Positive
Negative
Test
Reaction
Reaction
Ornithine
purple to
amber to yellow
decarboxylase
red-purple
Lysine
purple to
amber to yellow
decarboxylase
red-purple
Malonate
green to blue
yellow
utilization
Urease
orange to
red-purple
yellow
Esculin
brown to black
no color change
hydrolysis
or beige
p-Galactosidase
light yellow
to yellow
colorless
Fermentations:
Arabinose
yellow to amber
red-purple
to purple
Adonitol
yellow to amber
red-purple
to purple
Inositol
yellow to amber
red-purple
to purple
Sorbitol
yellow to amber
red-purple
to purple
1 In phenylalanine deaminase test, any green color in organism suspension
also indicates pos. reaction.
2 Pos. H 2 S reaction might vary from thin, dark line at bottom of detection
disc to entire disc turning black. It is often advisable to read this disc be-
fore it has been wetted.
(10) Use MICRO-ID identification manual (g) to det. 5 digit
octal no. for each isolate, and record identification of isolate.
G. Confirmation (Salmonella sp. Only)
For confirmation of cultures presumptively identified as Sal-
monella sp., see 978. 24C.
Ref.: JAOAC71, 968(1988).
967.28 Salmonella in Foods
Serological Tests
First Action 1967
Final Action 1968
(Follow manufacturer's instructions forreconstitution, mixing,
diln, and operation of Salmonella antisera. Dil. and pretest all
Salmonella serological antisera with known test cultures to en-
sure reliability of results with unknown cultures. Caution:
Handle viable cultures carefully to prevent contaminating en-
vironment.)
A. Polyvalent Somatic (O) Slide or Plate Test
Using wax pencil, mark off 2 sections ca 1 x 2 cm on inside
of glass or plastic petri dish. Place ] / 2 of 3 mm loopful of
culture from 24- or 48-hr TSI agar slant on dish in upper part
of each marked section. Add 1 drop saline soln, 967.256(f),
to lower part of one section only. Add 1 drop Salmonella po-
lyvalent somatic (O) antiserum, 967.25B(h), to other section
only. With clean, sterile transfer loop or needle, emulsify cul-
ture in saline soln for one section and repeat for other section
contg antiserum. Tilt mixt. in both sections back and forth I
min and observe against dark background. Any degree of ag-
glutination is pos. reaction.
Classify polyvalent somatic (O) test as:
(a) Positive. — Agglutination in culture-saline-serum mixt.
and no agglutination in culture-saline mixt.
(b) Negative. — No agglutination in culture-saline-serum mixt.
(Polyvalent somatic antisera do not contain agglutinins for an-
tigens of some salmonellae isolated from foods. Neg. somatic
reactions occur with Salmonella serotypes whose correspond-
ing agglutinins are not contained in the antisera, i.e., S. cerro,
476
Microbiological Methods
AOAC Official Methods of Analysis (1990)
group K(18); S. minnesota, group L(2I); S. alachua, group
0(35).)
(c) Non-specific. — Both mixts agglutinate. Requires addnl
testing as in Edwards and Ewing' s Identification of Entero-
bacteriaceae .
B. Determination of Somatic Grouping (Optional)
Perform serological somatic (O) test on culture as in 967.28A,
using individual group somatic (O) antiserum (including Vi),
967.25B(i), instead of Salmonella polyvalent somatic (O) anti-
serum. Repeat test, using each group somatic antiserum or un-
til culture reacts with specific group antiserum.
Suspend cultures pos. with Vi antiserum by emulsifying
growth from slant surface in J mL physiological saline soln,
967.25B(f ), to make heavy suspension. Heat in boiling H 2
20-30 min and let cool. Retest heated suspension, using so-
matic group D, C], and Vi antisera. Vi-pos. cultures which
react with somatic group D antiserum are probably S. typhi,
and Vi-pos. cultures which react with somatic group Cj anti-
serum are probably S. paratyphi C. For these cultures to be
classified as Salmonella sp. , they must have characteristics of
salmonellae as in Table 967. 27 A or B. Heated Vi-pos. cultures
which do not react with any individual somatic serum but con-
tinue to react with Vi antiserum probably belong to Citrobac-
ter and are not Salmonella. Confirm conclusion by biochem,
tests listed in Table 967. 27A.
Cultures that give pos. somatic (G) test with any individual
somatic (O) antiserum are recorded as pos. for that somatic
(O) group; cultures that do not react with any individual so-
matic (O) antiserum are recorded as neg. for individual group
somatic (O) test.
C. Polyvalent Flagellar (H) Test Tube Test
Place 0.5 mL appropriately dild Salmonella polyvalent fla-
gellar (H) antiserum, 967.25B(j), in 10 x 75 or 13 x 100
mm serological test tube and add 0.5 mL antigen to be tested:
formalinized bra in -heart infusion broth, 967.27B(b)(7), or for-
malinized trypticase soy-tryptose broth, 967.27B(b)(2) or
967.27C(d). If formalinized culture contains granular parti-
cles, pellicles, or sediment, also prep, saline control by mixing
0.5 mL formalinized saline soln, 967.25B(g), with 0.5 mL
formalinized trypticase soy-tryptose or brain-heart infusion broth
culture in same size serological test tube. Incubate mixts 1 hr
in H 2 bath at 48-50°. Observe preliminary results at 15 min
intervals and read final results at 1 hr.
Classify polyvalent flagellar (H) test as:
(a) Positive. — Agglutination in culture-formal inized saline-
serum mixt. and no agglutination in culture-formalinized sa-
line mixt.
(b) Negative. — No agglutination in culture-formalinized
saline-serum mixt. (Polyvalent flagellar antiserum does not
contain agglutinins for antigens of some salmonellae isolated
from foods. Neg. flagellar reactions occur with Salmonella
serotypes whose corresponding agglutinins are not contained
in the antisera (i.e., S. simsbury, z 27 ; S. chittagong, z 35 )).
(c) Non-specific. — Both mixts agglutinate. Requires addnl
testing as in Edwards and Ewing' s Identification of Enter o-
bacteriaceae .
Cultures that give typical biochem. results as salmonellae
but do not agglutinate in Salmonella flagellar (H) antisera must
be tested to det. if enough flagellar (H) antigens are present.
Test motility of culture as follows:
Inoculate petri dish contg motility test medium, 967.25 A(n),
with 3 mm loopful TSI culture by stabbing medium once, 10
mm from edge of plate to depth of 2-3 mm. (Do not stab to
bottom of plate with inoculum.) Do not inoculate any other
portion of plate. Incubate 24 hr at 35°. When organism has
migrated 40 mm or more toward other side of plate, it is suf-
ficiently motile to retest.
Transfer 3 mm loopful of growth which migrated farthest
from inoculation point into tube of trypticase soy-tryptose broth,
967.27C(d). Incubate and retest this culture by adding l / 2 vol.
formalinized physiological saline soln, 967.25B(g), and repeat
Salmonella serological flagellar (H) test, 967.28C or D.
Incubate cultures that are not motile after first 24 hr incu-
bation for addnl 24 hr at 35°. If still neg., incubate 5 days at
25° before classifying as nonmotile (flagellar (H) antigen not
detected) .
Cultures that are non-motile or cultures that are Salmonella
serological flagellar (H) test-neg. , when retested, are classified
according to results of other tests in Edwards and Ewing' s
Identification of Enterobacteriaceae .
D. "Spicer-Edwards" Flagellar (H) Test Tube Test
(Alternative to polyvalent flagellar (H) test tube test, 967. 28C,
to det. presence or absence of flagellar (H) antigens)
Test each culture, using each of the 7 "Spicer-Edwards" fla-
gellar (H) antisera, 967.25B(k). Perform test as in 967. 28C,
using 1 of the 7 "Spicer-Edwards" (H) antisera for each test
instead of Salmonella polyvalent flagellar (H) antiserum. Since
there are 7 "Spicer-Edwards" antisera, each culture must be
tested 7 times.
Pos. agglutination indicates presence of flagellar (H) anti-
gen. Identify by comparing pattern of agglutination reactions
obtained with agglutinins known to be present in each of the
7 "Spicer-Edwards" (H) antisera. Results of these reactions
are shown in Table 967.25.
If culture produces pos. agglutination when tested with each
of the 4 "Spicer-Edwards" antisera 1, 2, 3, and 4 (4 plus pat-
tern), then results indicate presence of non-specific antigen other
than Salmonella antigen or presence of more than single Sal-
monella H antigen which cannot be identified with this antisera
until antigens are sepd.
Refs.: JAOAC 50, 753(1967); 51, 870(1968); 52, 455(1969);
56, 1027(1973); 59, 731(1976); 62, 499(1979); 64,
893(1981); 64, 899(1981); 65, 356(1982).
975.54 Salmonella in Foods
Fluorescent Antibody (FA) Screening Method
First Action 1975
Final Action 1977
A. Precautions
Method is screening test for presence of Salmonella; it is
not confirmatory test, since conjugate will react with some other
members of Enterobacteriaceae.
Enrichment broths from samples pos. by FA method must
be streaked on selective media as in 967. 26B and typical or
suspicious colonies identified as in 967. 26C, 967.27, 967.28.
Method must be followed rigorously since errors in prepn
of sample, smears, conjugate, and other reagents can lead to
invalid results. Microscopic observation of stained smears must
be performed with critically aligned and properly functioning
equipment.
Visual estimation of degree of fluorescence of stained cells
is somewhat subjective and should be conducted by analyst
with prior training or experience in both FA methodology and
in cultural technic for detection of Salmonella.
If sample prepn does not normally include pre-enrichment
step (as with meat, poultry, and certain environmental sam-
ples), 4 hr post-enrichment incubation period may not be suf-
AOAC Official Methods of Analysis (1990)
Salmonella
477
ficient for development of number of Salmonella cells required
for detection by FA method. Therefore, include pre-enrich-
ment step or extend post-enrichment incubation time. In some
cases when pre-enrichment step is not used, sample is not ad-
equately dild and carryover of debris into post-enrichment broth
may interfere with observation of FA stained cells.
B. Apparatus
(a) Multiwell coated slides. — Clean thin (1 .0-1.2 mm) slides
thoroly with detergent and rinse with distd H 2 and alcohol.
Apply double row of 4 sep. drops of glycerol (8 drops total)
to each of series of slides and spray with fluorocarbon coating
material (Fluoroglide, Ace Scientific Co. , Inc. , 1420 E Linden
Ave, Linden, NJ 07036). After few min, rinse off each slide
individually under tap and then with distd H 2 0, and stand on
end in rack to dry. (Prepd slides are available from Cell-Line
Associates, PO Box 35, Newfield, NJ 08344 and Clinical Sci-
ences, Inc., 30 Troy Rd, Whippany, NJ 07981.)
(b) Fluorescence microscope. — With exciter filter with
wavelength transmission of 330-500 nm and barrier filter with
wavelength reception >400 nm.
C. Reagents
(a) Phosphate-buffered saline (PBS) soln. — pH 7.5; O.OlAf;
0.85% NaCl. Dissolve 12.0 g anhyd. Na 2 HP0 4 , 2.2 g Na-
H 2 P0 4 .H 2 0, and 85.0 g NaCl in H 2 and dil. to 1 L. Dil. 100
mL this soln to 1 L with H 2 0. Adjust pH to 7.5 with 0AN
HC1 or 0AN NaOH, if necessary.
(b) Carbonate buffer.— pH 9.0. Mix 4.4 mL 0.5M Na 2 C0 3
(5.3 g in 100 mL H 2 0) with 100 mL 0.5M NaHC0 3 (4.2 g in
100 mL H 2 0). pH should be 9.0; if not, adjust by addn of
0.5MNa 2 CO 3 .
(c) Glycerol saline soln. — pH 9.0. Mix 9 mL glycerol with
1 mL carbonate buffer, (b). pH decreases on storage; prep.
weekly .
(d) Salmonella polyvalent fluorescent antibody conju-
gate. — Fluorescein isothiocyanate-labeled Salmonella OH
globulin, polyvalent, contg antibodies for all antigens within
Salmonella O groups A-S, and meeting specifications of Cen-
ters for Disease Control, Atlanta, GA 30333 (1975). (Avail-
able from Difco Laboratories (FA Salmonella Poly); Clinical
Sciences, Inc., 30 Troy Rd, Whippany, NJ 07981). Before
use, titer each lot to det appropriate routine test diln (RTD).
Use pure cultures of Salmonella representative of several so-
matic groups. Prep. 5 dilns (1:2, 1:4, 1:8, 1:16, and 1:32) of
conjugate in PBS soln, (a). Stain duplicate smears from cul-
tures with each diln and det. intensity of fluorescence. RTD
is that diln one less than highest diln giving 4+ fluorescence
with representative Salmonella cultures. Store stock (undild)
conjugate of known titer frozen, and dil. when needed. Dild
conjugate can be stored at 4° for few weeks as long as control
cultures remain pos.
D. Determination
(a) Pre-enrichment. — Pre-enrich product in noninhibitory
broth to initiate growth of salmonellae. Methods used vary with
product as in (7)-(9). In all cases loosen jar caps l U turn and
incubate 24 ± 2 hr at 35°. Except where selenite cystine and
tetrathionate broths, 967.25A(b)(7) or (2) and (c), resp., have
already been used ((2)(b) and (5)), transfer 1 mL incubated
mixts to selenite cystine broth and tetrathionate broth for se-
lective enrichment as in 967.26B(a). Where these broths have
already been used ((2)(b) and (5)), proceed directly to post-
enrichment, (b).
(1) Dried yeast (inactive). — Weigh 25 g into sterile, wide-
mouth, screw-cap, 500 mL (pt) jar, add 225 mL sterile tryp-
ticase (tryptic) soy broth, 967. 25 A (t), and mix well to form
smooth suspension. Cap jar securely and let stand 60 min at
room temp. If pH is <6.6, adjust to 6.8 ± 0.2 with IN NaOH.
(2) Meats, animal substances, glandular products, and fish
meal. — (a) Heated, processed, and dried products. — Weigh
25 g into sterile blending jar, add 225 mL sterile lactose broth,
940.36 A(f), and blend 2 min at 8000 rpm. If product is powd.,
ground, or comminuted, blending may be omitted. Transfer
aseptically to sterile, wide-mouth, screw-cap, 500 mL (pt) jar
and adjust pH to 6.8 ± 0.2 with \N NaOH. If product contains
large amt of fat, add 2.2 mL of steamed (15 min) Tergitol
Anionic 7 (Na heptadecyl sulfate, Union Carbide Corp.).
(b) Raw and highly contaminated products. — Weigh du-
plicate 25 g samples into sep. sterile blending jars. Add 225
mL of selenite cystine broth to one jar and 225 mL of tetra-
thionate broth to other, and blend 2 min. Transfer aseptically
to sterile, wide-mouth, screw cap, 500 mL (pt) jars.
(c) Raw frog legs. — Aseptically place 2 legs into single
sterile, wide-mouth, screw cap, 500 mL (pt) jar contg 225 mL
sterile lactose broth, 940.36A(f).
(3) Dry whole milk. — Weigh 25 g into sterile, wide-mouth,
screw cap, 500 mL (pt) jar, add 225 mL sterile distd H 3 0, and
mix well. Adjust pH to 6.8 ± 0.2 with IN NaOH, if neces-
sary. Add 0.45 mL 1% aq. brilliant green soln and mix well.
(4) Dried whole eggs, yolks, and whites; pasteurized liquid
and frozen eggs; prepared powdered mixes (cake, cookie, donut,
biscuit, and bread); and infant formula . — If product is frozen,
thaw rapidly at <45° for < 15 min or overnight at 5-10°. Weigh
25 g into sterile, wide-mouth, screw cap jar. Add 225 mL
lactose broth, little at time with mixing, cap jar, and let stand
at room temp. 60 min. Mix well and adjust to pH 6.8 ±0.2
with IN NaOH or HC1.
(5) Nonpasteurized frozen egg products. — Thaw as in (4).
Weigh duplicate 25 g samples into sep. sterile, wide-mouth,
screw cap, 500 mL (pt) jars. Add 225 mL selenite cystine broth
to one jar and 225 mL tetrathionate broth to other, and mix
well. Adjust pH to 6.8 ± 0.2 with lA r NaOH.
(6) Egg-containing foods (noodles, egg rolls, etc.) — Pro-
ceed as in (2)(a).
(7) Coconut. — Proceed as in (2)(a), using Tergitol Anionic
7, but omitting blending.
(8) Candy and candy coatings. — Weigh 25 g into sterile
blending jar. Add 225 mL sterile reconstituted nonfat dry milk,
967.25A(v), but without brilliant green dye, and blend 2 min.
Adjust pH to 6.8 ± 0.2 with IN NaOH, if necessary. Add
0.45 mL 1% aq. brilliant green soln and mix well.
(9) Nonfat dry milk. — Examine as in 967.26A(f).
(b) Post-enrichment. — Transfer 1 mL of incubated selenite
cystine enrichment broth to 10 mL of sterile selenite cystine
broth as post-enrichment. (Other vols may be used if 1:10 diln
ratio is maintained.) Take aliquot from upper third of selective
enrichment cultures to minimize product carryover. Similarly,
transfer 1 mL of incubated tetrathionate enrichment broth to
10 mL of sterile selenite cystine broth. Incubate 4 hr in 35°
H 2 bath.
(c) Staining. — Transfer ca 0.0075 mL of each post-enrich-
ment medium with sterile 2 mm loop into sep. wells of mul-
tiwell coated slide, and dry thoroly in air at room temp. Fix
by immersion in bath of alcohol-CHCJ 3 -formalin (60 + 30 +
10) 3 min. Rinse 2 or 3 times in alcohol, and air dry at room
temp. Change alcohol periodically to prevent cell carryover
(250 mL alcohol will rinse 5—10 slides). Slides may also be
fixed and rinsed by flooding. Apply solns to one end of slide
and allow to flow into wells.
Cover dried smears with titered Salmonella polyvalent FA
conjugate and let stain in moist chamber 15-30 min. FA con-
jugate must not dry on smear. (Covered plastic petri dish contg
piece of filter paper moistened with H 2 is excellent staining
chamber.) Drain excess conjugate by standing slide on edge
478
Microbiological Methods
AOAC Official Methods of Analysis (1990)
few sec. (Avoid mixing conjugate from one well on slide to
another.) Immediately rinse slides in PBS soln, 975.54C(a).
Then soak slides 10 min in fresh PBS soln and rinse briefly
with H 2 0. Air-dry smears again at room temp, and then mount
by placing drop of glycerol saline soln, (c), directly onto each
smear and covering with No. 1 glass cover slip. Add enough
glycerol saline soln to smear to ensure adequate, but not ex-
cessive, coverage of all wells after cover slips have been placed.
Do not trap air bubbles under cover slip.
(d) Examination. — Examine smears with fluorescent mi-
croscope. Scan entire smear using 40-50 X oil immersion ob-
jective to locate fluorescent cells. When found, change objec-
tive to 100X oil immersion lens for definitive detn of cell
morphology and fluorescence. Objectives with iris diaphragm
for adjusting numerical aperture are helpful for control of con-
trast between cells and background. Estimate degree of fluo-
rescence of cells on scale of neg. to 4+ as follows:
4+ - Max. fluorescence; brilliant yellow-green; clearcut cell
outline; sharply defined cell center.
3+ = Less brilliant yellow-green fluorescence; clearcut cell
outline; sharply defined cell center.
2+ = Definite but dim fluorescence; cell outline less well
defined.
1+ - Very subdued fluorescence; cell outline indistinguish-
able from cell center in most instances.
— = Negligible or complete lack of fluorescence.
Typical pos. smears for salmonellae exhibit >2 short to me-
dium rod-shaped cells per field, using lOOx objective. Cells
should be distributed thruout entire smear. Intensity of fluo-
rescence should be in range of 3+ to 4+. Occasionally cells
are observed with proper morphology and cell distribution, but
fluorescence is rated 2+. Sometimes 3+ to 44- fluorescence
is observed, but distribution is poor and not all fields contain
cells, due to improper processing of slides. Score both cases
pos. and subject to confirmatory tests.
Each time samples are tested, carry culture of known Sal-
\monella strain thru all cultural, staining, and observation steps
las control.
\ Report: (1) morphological characteristics of fluorescent cells;
(2) number of typical cells per field under 100x oil immersion
objective; and (3) degree of fluorescence of cells (1+ to 4+).
Ref.: JAOAC 58, 828(1975).
985.42 Salmonella in Foods
Hydrophobic Grid Membrane Filter Screening Method
First Action 1985
Final Action 1986
(Applicable to detection of Salmonella from chocolate, raw
poultry meat, pepper, cheese powders, powdered egg, and
nonfat dry milk)
A. Principle
Hydrophobic grid membrane filter (HGMF) uses membrane
filter imprinted with hydrophobic material in grid pattern. Hy-
drophobic lines act as barriers to spread of colonies, thereby
dividing membrane filter surface into sep. compartments of
equal and known size.
R Apparatus, Culture Media, and Reagents
(a) Hydrophobic grid membrane filter (HGMF) — Mem-
brane filter has pore size of 0.45 |xm and is imprinted with
nontoxic hydrophobic material in grid pattern. ISO-GRID
(available from QA Laboratories Ltd, 135 The West Mall, To-
ronto, Ontario, Canada, M9C 1C2) or equiv. meets these spec-
ifications.
(b) Filtration units for HGMF, — Equipped with 5 (xm mesh
pre filter to remove food particles during filtration. One unit is
required for each sample. ISO-GRID (available from QA Lab-
oratories Ltd) or equiv. meets these specifications.
(c) Pipets. — 1.0 mL serological with 0.1 mL graduations;
1.1 mL or 2.2 mL milk pipets are satisfactory.
(d) Blender. — Waring, or equiv. with high-speed operation
at 20,000 rpm, and 500 mL glass or metal blender jars with
covers. One jar is required for each sample.
(e) Vacuum pump. — H 2 aspirator vac. source is satisfac-
tory.
(f) Manifold or vacuum flask.
(g) Peptone diluent. — Dissolve 1 .0 g peptone (Difco 01.18)
in 1 L H 2 0. Dispense enough vol. into diln bottles to give 99
± 1 mL after autoclaving 15 min at 121°.
'(h) Lactose broth. — See 940.36A(f). Dispense 225 mL
portions into 500 mL flasks. Autoclave 15 min at 121°. Asep-
tically det. vol. and adjust if necessary to 225 mL, Final pH
6.7 ± 0.2.
(i) Trypticase (tryptic) soy broth. — Suspend 17.0 g trypti-
case or tryptose (pancreatic digest of casein), 3.0 g phytone
(papaic digest of soya meal), 5.0 g NaCl, 2.5 g K 2 HP0 4 , and
2.5 g glucose in 1 L H 2 0. Heat gently to dissolve completely.
Dispense 225 mL portions into 500 mL flasks. Autoclave 15
min at 121°. Aseptically det. vol. and adjust if necessary to
225 mL. Final pH 7.3 ± 0.2.
(j) Reconstituted nonfat dry milk with brilliant green dye
(NFDM-BG). —Suspend 100 g dehydrated NFDM in 1 L H 2 0;
mix by swirling until dissolved. Autoclave 15 min at 121°.
Add brilliant green dye soln after blending sample/broth mixt.
as described below.
(k) Tetrathionate broth (with iodine and brilliant green). —
Suspend 5.0 g polypeptone, 1.0 g bile salts, 10 g CaC0 3 , and
30 g Na 2 S 2 3 .5H 2 in 1 L H 2 0, mix thoroly, and heat to bp.
(Ppt will not dissolve completely.) Cool to <45° and store at
5-8°. Prep. I-KI soln by dissolving 5 g KI in 5 mL sterile
H 2 0, adding 6 g resublimed I, dissolving, and dilg to 20 mL
with sterile H 2 0. Prep, brilliant green soln by dissolving 0.1
g dye in sterile H 2 and dilg to 100 mL. On day medium is
used, add 20 mL I-KI soln and 10 mL brilliant green soln per
1 L basal broth. Resuspend ppt by gentle agitation and asep-
tically dispense 10 mL portions in 16 x 150 mm sterile tubes.
Do not heat medium after addn of I-KI and dye solns. Temper
to 25-35° before use.
(I) Selective lysine agar (SLA). — Suspend 5.0 g proteose
peptone No. 3, 3.0 g yeast ext, 10.0 g l- lysine. HO, 3.5 g
glucose, 1.5 g bile salts No. 3, 0.001 g crystal violet (1.0 mL
of 0.1% (w/v) aq. soln), 0.03 g bromocresol purple, 0.3 g
sulfapyridine, and 15.0 g agar in 1 L H 2 and heat to bp with
stirring to dissolve completely. Autoclave 15 min at 121°. Cool
to 45-50°. Dispense 20 mL vol. in 15 X 100 mm petri dishes.
Final pH, 6.8 ±0.1.
(m) Hektoen enteric agar (HE). — Suspend ingredients (J)
or (2) (varies with manufacturer of formula) in I L H 2 and
mix thoroly. Heat to boiling with frequent agitation and let boil
few moments. Do not overheat. Cool in H 2 bath and pour
20 mL portions into 15 x 100 mm petri dishes. Let dry ca 2
h with covers partially removed; then close plates. Final pH,
7.6 ± 0.2. Do not autoclave.
(1) 12.0 g thiotone peptone, 3.0 g yeast ext, 9.0 g bile salts,
12.0 g lactose, 12.0 g sucrose, 2.0 g salicin, 5.0 g NaCl, 5.0
g Na thiosulfate, 1 .5 g ferric ammonium citrate, 0.064 g brom-
othymol blue, 0.1 g acid fuchsin, and 13.5 g agar.
(2) 12.0 g proteose peptone, 3.0 g yeast ext, 9.0 g bile salts
No. 3, 12.0 g lactose, 12.0 g sucrose, 2.0 g salicin, 5.0 g
AOAC Official Methods of Analysis (1990)
Salmonella
479
NaCl, 5.0 g Na thiosulfate, 1.5 g ferric ammonium citrate,
0.065 g thymol blue, 0.1 g acid fuchsin, and 14.0 g agar.
(n) Triple sugar iron agar (TSI agar). — Suspend ingredi-
ents (/) or (2) in 1 L H 2 0, mix thoroly, and heat with occa-
sional agitation. Boil ca 1 min until ingredients dissolve. Fill
16 X 150 mm tubes Vs full and cap or plug so that aerobic
conditions are maintained during use. Autoclave 12 min at 121°.
Before medium solidifies, place tubes in slanted position so
that deep butts (ca 3 cm) and adequate slants (ca 5 cm) are
formed on solidification.
(J) 20 g polypeptone, 5.0 g NaCJ, 10 g lactose, 10 g su-
crose, 1 g glucose, 0.2 g Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 0, 0.2 g Na 2 S 2 3 ,
0.025 g phenol red, and 13 g agar. Final pH, 7.3 ± 0.2.
(2) 3.0 g beef ext, 3.0 g yeast ext, 15 g peptone, 5.0 g
proteose peptone, 1.0 g glucose, 10 g lactose, 10 g sucrose,
0.2 g FeS0 4 , 5.0 g NaCl, 0.3 g Na 2 S 2 3 , 0.024 g phenol red,
and 12 g agar. Final pH, 7.4 ± 0.2.
(o) Lysine iron agar (LI A) (Edwards and Fife). — Dissolve
5.0 g gelysate or peptone, 3.0 g yeast ext, 1.0 g glucose, 10
g L-lysine, 0.5 g ferric ammonium citrate, 0.04 g anhyd.
Na 2 S 2 3 , 0.02 g bromocresol purple, and 15 g agar in 1 L
H 2 0, heating until dissolved. Dispense 4 mL portions into 13
x 100 mm test tubes and cap or plug so that aerobic conditions
are maintained during use. Autoclave 12 min at 121°. Before
medium solidifies, place tubes in slanted position so that 4 cm
butts and 2.5 cm slants are formed on solidification. Final pH
6.7 ± 0.2.
(p) MacConkey agar (MAC). — Suspend 3.0 g proteose
peptone or polypeptone, 17 g peptone or gelysate, 10 g lac-
tose, 1.5 g bile salts No. 3 or bile salts mixt., 5.0 g NaCl,
3.0 mL 1% neutral red (30 mg) soln, 1 mL 0.1% crystal violet
(1.0 mg) soln, and 13.5 g agar in 1 L H 2 and mix thoroly
until homogeneous. Heat, with occasional agitation, and boil
1-2 min until ingredients dissolve. Autoclave 15 min at 121°.
Cool to 45-50° and pour 20 mL portions into 15 x 100 mm
petri dishes. Let dry ca 2 h with plates covered. Do not use
wet plates. Final pH 7.1 ± 0.2.
(q) Sodium, hydroxide soln. — IN. Dissolve 42.11 g 95%
reagent NaOH in sterile H 2 and dil. to 1 L.
(r) Hydrochloric acid soln. — IN. Dil. 89 mL to 1 L with
sterile H 2 0.
(s) pH Test paper. — Min. range 6.0-7.6 with max. gra-
dations of 0.4 pH unit per color change.
(t) Sterile distilled water. — Dispense 1 L H 2 into 2 L wide-
mouth flask or wide-mouth jar; plug or cap loosely. Autoclave
20 min at 121°.
(u) Brilliant green dye soln. — 1%. Dissolve 1 g in sterile
H 2 and dil. to 100 mL. (Since some batches of dye are un-
usually toxic, test all batches of dye before use, and use only
those producing satisfactory results when tested with known
pos. and neg. test organisms.)
(v) Brilliant green dye water. — Prep, sterile H 2 0, (t), and
add 2 mL of 1% aq. brilliant green dye, (u), per L sterile H 2
and mix well.
C. Preparation of Sample
(a) Powdered egg. — Aseptic ally open sample container and
aseptically weigh 25 g sample into sterile, empty, wide-mouth,
screw-cap pt (500 mL) jar. Add ca 15 mL sterile lactose broth.
Stir with sterile glass rod, sterile spoon, or sterile tongue de-
pressor to smooth suspension. Add 3 addnl portions lactose
broth, 10, 10, and 190 mL for total of 225 mL. Stir after each
addn until sample is suspended without lumps. Cap jar se-
curely and let stand at room temp. 60 min. Mix well by shak-
ing, and det. pH with test paper. Adjust pH, if necessary, to
6.8 ± 0.2 with sterile IN NaOH or HO, capping jar securely
and mixing well before detg final pH. Loosen jar cap ca l / 4
turn and incubate 18-24 h at 35°.
(b) Chocolate. — Aseptically weigh 25 g sample into sterile
blender jar. Add 255 mL sterile reconstituted NFDM-BG. Blend
2 min at high speed and decant blended homogenate into ster-
ile 500 mL jar. Cap jar securely and let stand 60 min at room
temp. Mix well by shaking, and det. pH with test paper. Ad-
just pH, if necessary, to 6.8 ± 0.2 with sterile IN NaOH or
HC1, capping jar securely and mixing well before detg final
pH. Add 0.45 mL 1% aq. brilliant green dye and mix well.
Loosen jar cap [ / 4 turn and incubate 18-24 h at 35°.
(c) Raw meat. — Aseptically weigh 25 g sample into sterile
blender jar. Add 225 mL sterile lactose broth and blend 2 min
at high speed. Cap jar securely and let stand 60 min at room
temp. Mix well by shaking and det. pH with test paper. Adjust
pH, if necessary, to 6.8 ± 0.2 with sterile IN NaOH or HO,
capping jar securely and mixing well before detg final pH.
Aseptically transfer sample to sterile 500 mL wide- mouth screw-
cap jar. Loosen jar cap l j 4 turn and incubate 18-24 h at 35°.
(d) Cheese powder. — Aseptically weigh 25 g sample into
sterile 500 mL wide-mouth screw-cap jar. Add 225 mL sterile
lactose broth and mix well. Cap jar securely and let stand 60
min at room temp. Mix well by shaking and det. pH with test
paper. Adjust pH, if necessary, to 6.8 ± 0.2 with sterile IN
NaOH or HO, capping jar securely and mixing well before
detg final pH. Loosen jar cap '/i turn and incubate 18-24 h
at 35°.
(e) Pepper. — Aseptically weigh 25 g sample into sterile 500
mL wide-mouth screw-cap jar. Add 225 mL sterile trypticase
soy broth and mix well. Cap jar securely and let stand 60 min
at room temp. Mix well by shaking and det. pH with test pa-
per. Adjust pH, if necessary, to 6.8 ± 0.2 with sterile IN
NaOH or HO, capping jar securely and mixing well before
detg final pH. Loosen jar cap V4 turn and incubate 18-24 h
at 35°.
(f ) Powdered milk. — Use sterile funnel to aseptically add
25 g sample slowly and gently to 225 mL sterile brilliant green
dye water in 500 mL wide-mouth screw-cap jar. Do not mix.
Allow to soak undisturbed 60 min at room temp. Do not mix
or adjust pH. Loosen jar cap l / 4 turn and incubate 1.8—24 h at
35°.
D. Isolation
(a) Selective enrichment. — Gently shake incubated sample
mixt. and transfer 0.1 mL to 10 mL tempered (25-35°) tetra-
thionate broth. Mix inoculated broth on vortex mixer or by
hand to disperse inoculum. Incubate in H 2 bath 6-8 h at 35
± 0.5°.
(b) Filtration and selective isolation. — Mix incubated te-
trathionate broth by hand or vortex-mixer to resuspend. For
raw meats, prep. 10~ 2 diln by transferring 1 .0 mL into 99 mL
sterile peptone diluent. Mix by shaking. For all other products,
use undild tetrathionate.
(See Figs 986. 32 A and 986. 32B). Turn on vac. source. Place
sterile filtration unit on manifold or vac. flask. Open clamp
A. Rotate back funnel portion C. Aseptically place sterile HGMF
on surface of base D. Rotate funnel forward. Clamp shut by
sliding jaws L of stainless steel clamp over entire length of
flanges B extending from both sides of funnel C and base D,
and rotating moving arm K into horizontal (locked) position.
Aseptically add ca 15-20 mL sterile H 2 to funnel. Pipet
1.0 mL of required tetrathionate diln into funnel. Apply free
end of vac. tubing E to suction hole F to draw liq. thru prefilter
mesh G. Aseptically add addnl 10-15 mL H 2 to funnel and
draw thru mesh as before. Close clamp A to direct vac. to base
of filtration unit and draw liq. thru HGMF.
480
Microbiological Methods
AOAC Official Methods of Analysis (1990)
Open clamp A. Rotate moving arm K of stainless steel clamp
into unlocked (ca 45° angle) position and slide jaws L off of
flanges B. Rotate back funnel C. Aseptically remove HGMF
and place on surface of pre-dried SLA. Avoid trapping air bub-
bles between filter and agar. For nonfat dry milk samples, in-
sert second sterile HGMF into same filtration unit, repeat fil-
tering procedure and place second HGMF on surface of pre-
dried HE. Incubate SLA 24 ± 2 h at 43 ± 0.5°, and HE 24
± 2 h at 35°. If HGMFs do not have typical or suspicious
colonies or do not contain growth, record as neg. test result.
(c) Appearance of typical Salmonella colonies. — (7) On
SLA. — Blue-green, blue, or purple colonies (lysine-pos, re-
action). Typically, Salmonella produces relatively flat colonies
which are neither watery nor mucoid. Lysine-neg. colonies are
typically yellow or yellow-green. However, this can be masked
if large no. of lysine-pos. colonies are present on HGMF.
(2) On HE. — Black, or green with black centers. Some Sal-
monella will produce yellow colonies with black centers or
green colonies with no blackening. H 2 S reaction can be par-
tially suppressed if very heavy growth is present on HGMF.
E. Treatment of Typical or Suspicious Colonies
(a) Inoculation ofTSI, LI A, and MAC or HE. — (/) Raw
meats. — Select 5 typical or suspicious colonies from each
HGMF.
(2) All other products. — Select 3 typical or suspicious col-
onies from each HGMF.
Using sep. sterile, completely cooled needle for each col-
ony, pick each selected colony and inoculate TSI slant with
portion of colony by stabbing butt and streaking slant. Without
heating needle or obtaining more inoculum, inoculate LI A with
portion of colony by stabbing butt in 2 places and streaking
slant. Without heating needle or obtaining more inoculum, streak
remainder of inoculum to MAC or HE. Incubate TSI, LI A,
and MAC or HE 24 ± 2 h at 35°. Cap tubes loosely to main-
tain aerobic conditions while incubating slants to prevent ex-
cessive H 2 S production.
(b) Presumptive positive reactions. — Salmonella cultures
typically have alk. (red) slant and acid (yellow) butt, with or'
without H 2 S (blackening of agar) in TSI agar. In LIA, Sal-
monella cultures typically have alk. (purple) reaction in butt.
Consider only distinct yellow coloration in butt of LIA tube
as acidic (neg.) reaction. Do not eliminate cultures that pro-
duce discoloration in butt solely on this basis. Most Salmo-
nella cultures produce H 2 S in LIA. Retain all presumptive pos.
Salmonella cultures on TSI agar (alk. slant and acid butt) for
biochem. and serological tests whether or not corresponding
LIA reaction is pos. (alk butt) or neg. (acid butt). Do not ex-
clude TSI culture that appears to be non-Salmonella if reaction
in LIA is typical (alk. butt) for Salmonella. Treat these cul-
tures as presumptive pos. and submit them to further exami-
nation. LIA is useful in detection of 5 arizonae and atypical
Salmonella strains that utilize lactose and /or sucrose. Discard
only apparent non-Salmonella TSI agar cultures (acid slant and
acid butt) if corresponding LIA reactions are not typical (acid
butt) for Salmonella .
F. Purification and Identification
(a) Appearance of Salmonella colonies. — (7) On MAC. —
Typical colonies appear transparent and colorless, sometimes
with dark centers. Salmonella will clear areas of pptd bile caused
by other organisms sometimes present in medium.
(2) On HE. — Blue-green to blue colonies with or without
black centers. Many Salmonella cultures may have large glossy
black centers or may appear as almost completely black col-
onies.
(b) Purification of mixed cultures. — Examine MAC or HE.
If pure, proceed with identification. If mixed culture, pick with
needle ^2 well isolated typical or suspicious colonies and in-
oculate TSI, LIA, and MAC or HE as described above. In-
cubate and examine for presumptive pos. reactions.
(c) Identification. — Carry out biochem. and serological
identification procedures on 3 presumptive pos. TSI cultures
from each HGMF as described in 967.27B-E, 967.28. As al-
ternative to conventional tube system for Salmonella, any one
of 4 commercial biochem. systems (API, Enterotube, Minitek,
or Micro-ID) may be used for presumptive generic identifi-
cation of foodborne Salmonella. See 978.24.
Ref.: JAOAC 68, 555(1985).
986.35 Salmonella in Foods
Colorimetric Monoclonal Enzyme Immunoassay
Screening Method
First Action 1986
Final Action 1988
Method is screening procedure for presence of Salmonella
in all foods; it is not a confirmatory test because monoclonal
antibodies used in test may cross-react with small percentage
of non-Salmonella.
Enrichment broths and M-broth from samples pos. by en-
zyme immunoassay (EIA) method must be streaked on selec-
tive media as in 967. 26B and typical or suspicious colonies
must be identified as in 967.26C, 967.27, 967.28.
Detn of pos. result is objective and must be performed using
filter photometer having 405-420 nm filter. Pos. result is valid
only when neg. and pos. controls possess acceptable optical
density readings.
A. Principle
Detection of Salmonella antigens is based on solid phase
immunoassay and uses mag. force to transfer solid phase from
one reaction mixt. to another. Monoclonal antibodies to Sal-
monella antigen are bound to surface of beads made of ferrous
metal. Beads are placed in sample to be assayed. If Salmonella
antigens are present in sample, they will attach to specific an-
tibody on beads. Beads are washed and then released into re-
action mixt. contg peroxidase-conjugated anti-Salmonella im-
munoglobulins. Conjugate will bind to Salmonella antigens if
they are attached to antibody molecules on surface of beads.
Beads are washed to remove unbound conjugate and then placed
in substrate soln. Appearance of color indicates presence of
Salmonella antigen in sample. Fig. 986. 35 A shows schematic
representation.
B. Method Performance
For all foods:
Results
Percent
95% Confidence
Range (Approx.)
Agreement 1
False neg. (BAM) 2
False neg. (EIA) 3
96.9
1.5
3.4
95.7-98.1
0.4-2.5
1.8-5.0
1 This rate reflects no. of samples read identically between AOAC/BAM
(Bacteriological Analytical Manual (1984) 6th ed., AOAC, Arlington, VA)
culture method and EIA.
2 This rate reflects no. of samples found to be pos. by EIA but detd to be
neg. by AOAC/BAM culture method.
3 This rate reflects no. of samples found to be pos. by AOAC/BAM culture
method but detd to be neg. by EIA.
Of 21 laboratories, 10 (48%) had complete agreement be-
tween culture method and EIA (153/153); 17 laboratories (81%)
AOAC Official Methods of Analysis (1990)
Salmonella 481
ANTIBODY "CAPTURE" or "SANDWICH"
Ab-COATED
SOLID PHASE
BACTERIAL Aqs
CD CKA
□ ~ So
"CAPTURE"
* QKHD'
I CP
HRP-LABELLED Ab
.*
****
"-flHKHHtf
PJ
"SANDWICH"
Ab = antibody
Ag - antigen
HRP = horseradish peroxidase
H 2 2 = hydrogen peroxide
ABTS = 2,2'-azino-di[3-ethylbenzthiazoline-sulfonate(6)]
FIG. 986.35A — Antibody "capture" or "sandwich"
showed agreement on >96% of samples; 20 (95%) showed
agreement on >93% of samples.
C. Reagents
Items (a)-(m) are available as Salmonella Bio-EnzaBead
Screen Kit (Organon Teknika Corp., 100 Akzo Ave, Durham,
NC 27704).
(a) Antibody-coated beads. — Monoclonal antibodies to Sal-
monella, 2 vials (48 beads/vial). Store bead vials tightly capped
at 2-8°. Beads are stable 14 days after opening.
(b) Control antigens. — Pos. control (heat-treated S. ja-
viana) which reacts with antibodies to Salmonella, 1 vial; neg.
control which is nonreactive with antibodies to Salmonella, 1
vial. Reconstituted control antigens are stable 28 days when
stored at 2-8°.
(c) Conjugate diluent. — 1 vial (24 mL/vial). Contains 1%
bovine serum in phosphate-buffered saline contg 0.05% Tween
20 and 0.01% thimerosal as preservative.
(d) Reagent water. — 1 bottle (125 mL/bottle). Store at room
temp, or warm to room temp, before use.
(e) Phosphate-buffered saline.— PBS, pH = 7.5 ± 0.2; 1
bottle (125 mL/bottle). Contains 1.2 g Na 7 HP0 4 , 0.22 g
NaH 2 P0 4 .H 2 0, and 8.5 g NaCl/L H 2 0.
(f) Peroxidase-conjugated antibodies to Salmonella. — 1 vial
(lyophilized). When reconstituted, conjugate is stable 28 days
when stored at 2-8°.
(g) Wash solution (50x). — 1 vial (2.5 mL). Contains 2.5%
surfactant.
(h) ABTS substrate. — 2 vials (lyophilized). After reconsti-
tution, each vial contains 0.03% 2,2'-azino-di(3-ethyl-benz-
thiazoline-sulfonate). Reconstituted substrate is stable 14 days
when stored tightly capped at 2-8°. Let reconstituted substrate
warm to room temp, before dispensing.
(i) Substrate diluent for ABTS. — 2 vials (30 mL/vial).
Contains H 2 2 .
(j) "Stop" soln. — 1 vial (5 mL/vial). Contains 1.25% NaF.
Caution: Avoid contact with skin. If contact occurs, wash area
with H 2 0.
(k) Microtitration plates. — Plate (3 5 /ie x 5 in.) possessing
96 wells, each having capability of holding >0.3 mL fluid.
g tryptone, 2.0 g d-
5.0 gK 2 HP0 4 , 0.14
These must be designed in 8 x 12 format which will fit into
mag. transfer device. Spaces between wells should be hol-
lowed out, and not filled in with plastic coming to top of well.
Available as "Accessory Package" (Organon Teknika Corp.),
or equiv. may be used. Note: Not all microti trn plates meet
these criteria.
(1) Package insert.
(m) Data record sheets.
(n) M -broth. — 5.0 g yeast ext, 12.5
mannose, 5.0 g Na citrate, 5.0 g NaCl,
g MnCl 2 , 0.8 g MgS0 4 , 0.04 g FeS0 4 , 0.75 g Tween 80.
Suspend ingredients in I L H 2 0, and heat to boiling for 1-2
min. Dispense 10 mL portions into 16 x 125 mm screw-cap
test tubes. Cap tubes loosely and autoclave 15 min at 121°.
Tighten caps securely for storage. Final pH should be 7.0 ±
0.2.
(o) Diagnostic reagents. — Necessary for cultural confir-
mation of presumptive pos. EIA tests; see 967. 25B.
D. Apparatus
Items (a)-(e) are available from Organon Teknika Corp.
(a) Magnetic transfer device. — Mag. app. which houses
microtitrn plates and is used to transfer metal beads from one
reagent to another as well as to wash metal beads.
(b) Incubator. — 37° with 100 rpm agitator.
(c) Enzyme immunoassay reader. — Photometer with 405-
420 nm screening filter which will read thru microtitrn plates.
Must be able to be set to zero while reading thru unreactive
substrate well (blank). Reader should be equipped with printer
so that records of analysis can be kept. Semiautomated Or-
ganon Teknika 30 or equiv. meets these specifications.
(d) Bead dispenser. — Either single bead dispenser or 96-
well bead dispenser, or suitable alternative. Places beads into
wells of microtitrn plate.
(e) Micropipet. — Capable of delivering accurate amts in range
50-300 fxL. Micropipets capable of delivering these vols to
multiple wells simultaneously (multichannel) or individually
(single channel) are needed.
(f ) Centrifuge. — Having min. capacity to spin centrf. tubes
(<20 mm diam.) at 1500 x g in swinging bucket rotor or 3000
482
Microbiological Methods
AOAC Official Methods of Analysis (1990)
X g in fixed angle rotor for 20 min. IEC Centra 7 or Centra
8 tabletop centrf. with IEC 216 horizontal rotor (available from
International Equipment Co., or equiv., meets these criteria.
(g) Boiling water bath. — Able to attain and maintain 100°.
Must be able to hold centrf. tubes upright. Microwave or au-
toclave set at 100° is acceptable alternative, as are generators
of flowing stream. Caution: H 2 baths which do not maintain
boiling conditions are unacceptable.
(h) Vortex mixer. — Capable of vigorous agitation of centrf.
tube, such that pellet at bottom of conical tube can be resus-
pended. S/P mixer (available from Scientific Products, Inc.)
or equiv. meets these criteria.
E. General Instructions
Include pos. control antigen and duplicate neg. controls with
each group of test samples. All controls must function properly
for test to be valid. One addnl well per group of test samples
should be left empty initially. This well, filled with substrate,
will be used to "blank" assay reader. See sample data record
sheet (Fig. 986.35B).
Use data record sheets to identify location of each test sam-
ple.
Do not use mag. transfer device without top and bottom plate
in position. Always insert plates in device with notched side
facing operator.
Do not reuse wells of a plate or the beads.
Use sep. pipets for each sample and kit reagent to avoid
cross-contamination. Take care not to contaminate substrate
with conjugate.
The components and procedures of this test kit have been
standardized for use in Bio-EnzaBead procedure. Use of com-
ponents or procedures other than those supplied by Organon
Teknika Corp. may yield unsatisfactory results.
F. Preparation of Sample
(a) P re-enrichment. — Pre-enrich product in non-inhibitory
broth to initiate growth of salmonellae. Methods used may vary
with product, and should be performed as indicated in 967. 26 A,
or in Bacteriological Analytical Manual, 6th ed., AOAC, Ar-
lington, VA, Chap. 7, section C, with the following exception:
Raw or highly contaminated products. — A septic ally weigh
25 g sample into sterile blender jar. Add 225 mL sterile lactose
broth. Blend 2 min at high speed (ca 20 000 rpm). Cap jars
securely and let stand at room temp. 60 min. Mix well by
shaking and det. pH with test paper. Adjust pH to 6.8 ± 0.2,
if necessary, using sterile IN NaOH or HC1, capping jar se-
curely and mixing well before detg final pH. Aseptically trans-
fer contents of each jar to sterile wide -mouth, screw -cap 500
mL jar. Loosen jar caps V4 turn and incubate 24 ± 2 h at 35°.
(b) Selective enrichment. — Transfer 1 mL incubated pre-
enrichment mixts to selenite cystine broth and 1 mL into te-
trathionate broth as in 967.26B(a). (For dried active yeast,
substitute lauryl sulfate tryptose broth 967.25A(u) for selenite
cystine broth.) Incubate 18-24 h at 35°.
(c) Post-enrichment. — Remove selective broths from in-
cubation and mix by hand or by vortex mixer. Remove 0.5
mL from tetrathionate tube and transfer to 10 mL tube of ster-
ile M-broth which has been warmed to 35°. Also remove 0.5
mL from selenite cystine tube and transfer to same tube of M-
broth and vortex-mix well. Incubate M-broth tube 6 h at 35°.
Return tetrathionate and selenite cystine tubes to 35° incubator
for addnl 6 h.
(d) Centrifugation and preparation of sample for El A anal-
ysis. — Remove M-broth from incubation and mix tube by hand
or vortex mixer. Pipet 10 mL into centrf. tube (<20 mm diam.)
and label tube. Refrigerate (2-8°) remaining M-broth and te-
trathionate and selenite cystine tubes from (c) above for cul-
tural confirmation of any enzyme immunoassay pos. samples.
These broths may be refrigerated, if necessary, for <18 h at
2-8°. Centrf. M-broth at min. speed of 1500 X g (swinging
bucket rotor) or 3000 x g (fixed angle rotor) for 20 min. Suc-
tion off supernate from tube, using trap flask filled with dis-
infectant. Resuspend pellet with 1 mL PBS. Vortex-mix tube
to mix well. Heat resuspended pellet in boiling H 2 bath or
FIG. 986.35B — Data record sheet for identifying location of test samples
AOAC Official Methods of Analysis (1990)
Salmonella
483
in flowing steam 20 min. Cool heated exts to 25-37° prior to
analysis by EIA. Mote: Heated exts which are not cooled to
this temp, can destroy monoclonal antibodies on metal beads.
G. Enzyme Immunoassay
(1) Reconstitute control antigens by adding 2.5 mL PBS to
each vial. Swirl gently to dissolve. Following record sheet,
add 0.2 mL aliquot of neg. and pos. control antigens and 0.2
mL aliquot of test samples into designated wells of 96-well
plate. Note: Use 2 neg. controls and one pos. control for each
group of samples. Label plate "antigen."
(2) Using bead dispenser or forceps, place antibody-coated
beads in wells of one of empty plates according to record sheet.
If using forceps, remove beads from vials by rolling beads into
cap or onto gauze pad. Do not put bead in substrate blanking
well.
(5) To start assay, simultaneously transfer beads to "anti-
gen" plate by using mag. transfer device as follows:
(a) Invert 96-well plate and insert it with notched end facing
operator into top slot until it snaps into position. Magnet should
be in UP position.
(b) Slide plate contg beads (with notched end facing oper-
ator) into lower slot until it snaps into position, centering it
under inverted top plate.
(c) To remove beads from bottom plate, lower magnet to
full DOWN position (all beads should now be in inverted plate).
Without disturbing top plate, remove lower plate (save for later
step) and slide plate contg samples under inverted plate.
(d) Raise magnet to allow all beads to drop into wells of
"antigen" plate.
(4) Remove bottom plate contg samples and beads. Incu-
bate plate with agitation (10-100 rpm) for 20 min at 37°. Dur-
ing incubation period, proceed to steps 5 and 6.
(5) If entire plate is being used. prep. IX wash soln by
adding 1 .5 mL 50 X wash soln to 75 mL reagent water in clean
glass or plastic screw-cap bottle. Mix by inverting bottle sev-
eral times. Add 0.3 mL lx wash soln into appropriate wells
of 96-well plate previously saved. Label plate "wash 1." Sim-
ilarly, fill second plate with 1 x wash soln and label "wash
2." If entire plate is not being used, calc. amt of wash soln
required by multiplying number of tests by 0.6 and prep, amt
of lx wash soln required based on 0.5 mL 50x wash soln to
25 mL reagent water.
(6) Prep, conjugate soln by adding 24 mL 1 x conjugate
diluent to lyophilized material in vial. Mix gently by inverting
bottle several times. Date vial. Add 0.2 mL conjugate into
appropriate wells of sep. plate. Label plate "conjugate."
(7) Following 20 min incubation (above), wash beads as
follows:
(a) Assure that 96-well plate (from step 5 a) is inverted in
top slot of mag. transfer device and that magnet is in UP po-
sition.
(b) Slide "antigen" plate contg beads under top plate and
lower magnet to DOWN position (all beads should now be in
inverted plate).
(c) Remove bottom "antigen" plate without disturbing top
plate and place it in container for proper disposal.
(d) Slide "wash 1" under top plate and wash beads 12 times
by raising and lowering magnet to extreme UP and full DOWN
positions (count UP and DOWN as 1 wash).
(e) With magnet in DOWN position (beads in top inverted
plate), remove wash plate without disturbing top plate and save
for step lie. Note: Proceed immediately with next step. Do
not let beads dry in top plate.
(8) Slide "conjugate" plate under top plate and raise magnet
to let beads drop into wells.
(9) Remove bottom plate contg conjugate and beads and in-
cubate with agitation (10-100 rpm) for 20 min at 37°.
(10) While beads are incubating, reconstitute ABTS sub-
strate by adding contents of substrate diluent vial to lyophi-
lized substrate. Mix gently by inverting bottle several times.
Date substrate. Add 0.2 mL room temp, substrate into each
appropriate well of unused plate. Also, put 0.2 mL into extra
well which will be used to "blank" EIA reader.
(11) Following conjugate incubation period, wash beads as
follows:
(a) Assure that 96-well plate (from step 5a) is still inverted
in top slot of mag. transfer device and that magnet is in UP
position.
(b) Slide "conjugate" plate contg beads under top plate and
lower magnet to DOWN position (all beads should now be in
inverted plate).
(c) Remove bottom plate and slide "wash 1" under top plate.
Raise magnet to extreme UP position, allowing beads to fall
into "wash I."
(d) Remove top plate and replace with unused top plate.
Lower magnet to remove beads from "wash 1." Wash beads
twice in "wash I" by raising and lowering magnet to its ex-
treme UP and full DOWN positions (count UP and DOWN as
1 wash).
(e) With magnet in DOWN position (beads in top plate),
remove "wash 1" and insert "wash 2."
(f) Wash beads 9 times by raising and lowering magnet to
extreme UP and full DOWN positions.
(g) With magnet in UP position (beads in "wash 2"), re-
place top inverted plate with unused plate. Note: This change
must be made to avoid contamination of substrate with con-
jugate.
(h) Lower magnet and remove bottom "wash 2" without
disturbing top plate.
(12) Immediately slide "substrate" plate under top plate and
raise magnet to let all beads drop into the wells.
(13) Remove bottom plate contg substrate and beads and
incubate uncovered at room temp. (20-25°) for 10 min. Do
not agitate plate.
(14) After 10 min, add 0.025 mL (25 |ulL) "stop" soln to
each well including substrate blanking well. Gently swirl beads
in plate to disperse colored reaction product forming at surface
of beads. Remove beads (be sure that 96-well plate is inverted
in top slot of mag. transfer device) by placing "substrate" plate
in mag. transfer device and lowering magnet.
(15) Remove "substrate" plate and release beads into used
plate by sliding plate into mag. transfer device and raising
magnet. Mix contents of substrate blanking well with pipet tip.
(16) Read results on EIA reader.
(17) Sterilize all used plates, tubes, etc., prior to disposal.
Tightly close and return unused reagents to 2-8° storage.
H. Reading
Insert 405 nm filter and bring reader to zero (blank reader)
on well contg only substrate and "stop" soln. Then read each
individual control and sample well. Average optical density
readings of the 2 neg. control wells. For test to be valid, pos.
control should read >0.200 and av. of neg. controls should
read <0.120. Record optical density (OD) of each well on data
sheet. Samples reading >0.200 should be considered pos.
Samples reading <0.200 should be considered neg.
I. Confirmation of Positive EIA Samples
Pos. EIA reading indicates that Salmonella may be present.
However, since antibodies may cross-react with a few other
organisms, cultural confirmations should be performed by
streaking HE, XLD, and BS plates from tetrathionate broth,
484
Microbiological Methods
AOAC Official Methods of Analysis (1990)
selenite cystine broth, and M-broth tubes as described in
967. 26B, and typical or suspicious colonies should be iden-
tified as in 967.26C, 967.27, 967.28.
Ref.: JAOAC 69, 786(1986).
987.11 Salmonella in Low-Moisture Foods
Colorimetric Monoclonal Enzyme Immunoassay
Screening Method
First Action 1987
Method is screening procedure for presence of Salmonella
in low-moisture foods.
Enrichment broths and M-broths from samples pos. by en-
zyme immunoassay (EIA) method must be streaked on selec-
tive media as in 967. 26B and typical or suspicious colonies
should be identified as in 967.26C, 967.27, 967.28.
Detn of pos. result is objective and must be performed using
filter photometer having 405 nm filter. Pos. result is valid only
when neg. and pos. controls exhibit acceptable optical density
readings.
A. Principle
See 986.35A.
B. Method Performance
Results
Percent
95% Confidence
Range
(Approx.)
Agreement 1
False neg. rate 2
97.0
3.3
95.8-98.2
1.7-4.9
1 This rate reflects no. of samples read identically between 302AOAC/
BAM (Bacteriological Analytical Manual (1984) 6th ed., AOAC, Arlington,
VA) culture method and EIA.
2 This rate reflects no. of samples found to be pos. by AOAC/BAM culture
method but detd to be neg. by EIA.
Of 15 laboratories, 7 (47%) had complete agreement be-
tween culture method and EIA (156/156); 14 laboratories (93%)
showed agreement on >97% of samples.
C. Reagents
See 986.35C.
D. Apparatus
See 986.35D.
E. General Instructions
See 986.35E.
F. Preparation of Sample
(a) P re-enrichment. — Pre-enrich low-moisture food prod-
uct in non- inhibitory broth to initiate growth of salmonellae.
Methods used may vary with product, and should be per-
formed as indicated in 967. 26 A, or in Bacteriological Ana-
lytical Manual (1984) 6th ed., AOAC, Arlington, VA, Chap.
7, section C, except incubation time is 18-24 h.
(b) Selective enrichment. — Transfer 1 mL incubated pre-
enrichment mixt. to selenite cystine broth and 1 mL to tetra-
thionate broth as in 967.26B(a). Pre- warm both broths to 35°
before inoculation. (For dried active yeast, substitute lauryl
sulfate tryptose broth, 967.25A(u), for selenite cystine broth.)
Incubate 6-8 h in 35° water bath.
(c) Post- enrichment. — Remove selective broths from in-
cubation and mix by hand or by vortex mixer. Remove 1.0
mL from tetrathionate tube and transfer to 10 mL tube of ster-
ile M-broth. Also remove 1.0 mL from selenite cystine tube
and transfer to another 10 mL tube of sterile M-broth. Incubate
both M-broths and remaining tetrathionate and selenite cystine
broths for 14-18 h at 35°.
(d) Preparation of sample for EIA analysis. — Remove the
2 tubes of M-broth (M-broth-Tet and M-broth-SC) from in-
cubation and mix tubes by hand or by vortex mixer. Remove
0.5 mL from M-broth-Tet tube and transfer to glass screw-cap
test tube. Also remove 0.5 mL from M-broth-SC tube and
transfer to same screw-cap test tube. Refrigerate (2-8°) re-
maining M-broths and tetrathionate and selenite cystine broths
from (c) for cultural confirmation of EIA-pos. samples. Heat
combined M-broths in boiling H 2 bath or in flowing steam
20 min. Cool heated exts to 25-37° prior to analysis by EIA.
Note: Heated exts which are not cooled to this temp, can de-
stroy monoclonal antibodies on metal beads.
G. Enzyme Immunoassay
See 986.35G.
H. Reading
See 986.35H.
/. Confirmation of Positive EIA Samples
Pos. EIA reading indicates that Salmonella may be present.
However, since antibodies may cross-react with a few other
organisms, culture confirmations should be performed by
streaking HE, XLD, and BS plates from tetrathionate broth,
selenite cystine broth, and the associated M-broth tubes as de-
scribed in 967. 26B, and typical or suspicious colonies should
be identified as in 967.26C, 967.27, 967.28.
Ref.: JAOAC 70, 530(1987).
989.14 Salmonella in Foods
Colorimetric Polyclonal Enzyme Immunoassay
Screening Method
First Action 1989
Method is screening procedure for presence of Salmonella
in all foods; it is not a confirmatory test because polyclonal
antibodies used in test may cross-react with small percentage
of non-Salmonella.
Enrichment broths and M-broths from samples pos. by en-
zyme immunoassay (EIA) method must be streaked on selec-
tive media as in 967. 26B and typical or suspicious colonies
must be identified as in 967.26C, 967.27, 967.28.
Detn of pos. result may be performed (I) visually by aid of
color comparator card where pos. result is valid when neg.
and pos. controls match those described on card or (2) instru-
mentally using filter photometer having 414 nm filter where
pos. result is valid only when neg. and pos. controls possess
acceptable optical density readings.
4. Principle
Detection of Salmonella antigens is based on enzyme im-
munoassay using highly purified antibodies prepd from anti-
gens unique to Salmonella. Polyclonal antibodies to Salmo-
nella antigen are adsorbed onto internal surface of 96- well
microtiter tray. Sample to be assayed is placed into well of
tray. If Salmonella antigens are present in sample, they will
attach to specific antibody adsorbed on well. All other material
in samples is washed away. Conjugate is added and will bind
to Salmonella antigens if they are attached to adsorbed anti-
body on surface of well. Wells are washed to remove unbound
conjugate, and enzyme substrate is added. Dark blue-green color
indicates presence of Salmonella antigen in sample.
AOAC Official Methods of Analysis (1990)
Salmonella
485
&. Method Performance
For all foods:
Results
Percent
95% Confidence
Range (Approx.)
Agreement 1
False neg. (BAM) 2
False neg. (EIA) 3
96.8
1.6
1.4
95.4-98.2
0.5-2.7
0.4-2.4
1 This rate reflects no. of samples read identically between AOAC/BAM (Bac-
teriological Analytical Manual, 1984, 6th ed., AOAC, Arlington, VA) culture
method and EIA.
^This rate reflects no. of samples found to be pos. by EIA but neg. by AOAC/
BAM culture method.
3 This rate reflects no. of samples found to be pos. by AOAC/BAM culture
method but neg. by EIA.
Of 14 laboratories, 3 had complete agreement between cul-
ture method and EIA method. Excluding 1 food group, turkey,
13 of the 14 laboratories had perfect agreement between BAM/
AOAC and EIA methods. Laboratory that did not have perfect
agreement had difference in each of pepper, nonfat dry milk,
and chocolate food groups.
C. Reagents
Items (a)-(m) are available as TECRA Salmonella Visual
Immunoassay (Bioenterprises Pty Ltd, 28 Barcoo St, Rose-
ville, NSW 2069, Australia). Substitutions must be pretested
for equivalency.
(a) Antibody adsorbed strips. — Removawell® (Dynatech
Laboratories, Inc.) strips. Polyclonal antobodies to Salmo-
nella, 96 wells. Store wells at 2-8° when not in use.
(b) Tray. — Sufficient to secure individual wells or strips.
(c) Control antigens. — Pos. control (lyophilized). Purified
Salmonella antigen, which reacts with antibodies to Salmo-
nella, .1 vial. Neg. control (lyophilized lactose), which is non-
reactive with antibodies to Salmonella, 1 vial. Reconstituted
control antigens are stable 28 days when stored at 2-8°.
(d) Controls diluent. — 1 vial (5 mL/vial). Contains 0.006
g Tris [tris(hydroxymethyl)aminomethane], 0.044 g NaCl,
0.0025 g Tween 20 (polyoxyethylene 20 sorbitan monolaur-
ate), and 0.005 g thimerosai in H 2 0.
(e) Conjugate. — 1 vial (lyophilized). Contains 147 ng anti-
Salmonella antibodies (from sheep) conjugated to horseradish
peroxidase, 0.00686 g Na 2 B 4 7> 0. 12 g Dextran T10, 0.06 g
hydrolyzed gelatin, 0.0024 g CaCl 2 , and 120 ng thimerosai.
Reconstituted conjugate is stable 28 days when stored at
2-8°.
(f) Conjugate diluent. — 1 vial (22 mL/vial). Contains 0.42
g Na 2 B 4 7 , 0. 193 g 'NaCl, 0.22 g hydrolyzed gelatin, and 0.0022
g thimerosai in H 2 0.
(g) Substrate. — 1 vial (lyophilized). Contains 0.011 g 2,2'-
azino-di(3-ethylbenzthiazoline sulfonate) and 0.123 g Na-
H2PO4. Reconstituted substrate is stable 28 days when stored
at 2-8°.
(h) Substrate diluent. — 1 vial (22 mL/vial). Contains 0.116
g citric acid, 0.001 1 g H 2 2 , and 0.0185 g NaOH in H 2 0.
(i) "Stop" soln. — \ vial (6 mL/vial). Vial contains 0.15 g
NaF in H 2 0. Caution: Avoid contact with skin. If contact oc-
curs, wash area with H 2 0.
(j) Wash soln concentrate. — 1 vial (25 mL/vial). Contains
] .45 g Tris, 7.03 g NaCl, 0.5 g Tween 20, and 0.0025 g thi-
merosai in H 2 0.
(k) Package insert.
(1) Data record sheet.
(m) Color comparator card. — For visual interpretation of
pos. and neg. tests.
(n) M -broth. — 5.0 g yeast ext, 12.5 g tryptone, 2.0 g D-
mannose, 5.0 g Na citrate, 5.0 g NaCl, 5.0 g K 2 HP0 4 , 0.14
g MnCl 2 , 0.8 g MgS0 4 , 0.04 g FeS0 4 , 0.75 g Tween 80.
Suspend ingredients in 1 L H 2 and heat to boiling for 1-2
min. Dispense 10 ml portions into 16 x 125 mm screw -cap
test tubes. Cap tubes loosely and autoclave 15 min at 121°.
Tighten caps securely for storage. Final pH should be 7.0 ±
0.2.
(o) Diagnostic reagents. — Necessary for culture confir-
mation of presumptive pos. EIA tests; see 967. 25B.
D. Apparatus
(a) Incubator. — 35-37°.
(b) Multipipets. — Capable of delivering accurate amts in
ranges 50-250 |xL and 5-50 \xL.
(c) Water bath. — Capable of maintaining 100°. Autoclave
set at 100° is acceptable alternative, as are generators of flow-
ing steam.
(d) Plastic squeeze bottle. — 500 mL, for dispensing wash
soln. Automatic washer may be used.
(e) Plastic film wrap or sealable plastic container. — To cover
wells during incubation.
(f) Enzyme immunoassay reader. — Optional. Photometer
with 414 ± 10 nm screening filter which will read thru mi-
crotiter plates.
E. General Instructions
Components of kit must be refrigerated when not in use. Kit
is intended for 1-time use only; do not reuse wells containing
sample, reagents, or wash solution.
Include duplicate pos. and neg. control antigens with each
group of test samples. All controls must function properly for
test to be valid.
Use data record sheet to identify location of each test sam-
ple.
Use sep, pipets for each sample and kit reagent to avoid
cross-contamination. If plastic troughs are used to dispense
conjugate and substrate, ensure that they are always kept sep-
arate.
Components in kit are intended for use as integral unit. Do
not mix components of different batch numbers.
F. Preparation of Sample
(a) Pre-enrichment. — Pre-enrich product in noninhibitory
broth to initiate growth of salmonellae. Methods used may vary
with product and should be performed as indicated in 967.26A,
or in Bacteriological Analytical Manual, 6th ed., AOAC,
Arlington, VA, Chap. 7, section C, with following exception:
Raw or highly contaminated products. — Aseptically weigh
25 g sample into sterile blender jar. Add 225 mL sterile lactose
broth. Blend 2 min at high speed (ca 20 000 rpm). Cap jars
securely and let stand 60 min at room temp. Mix well by shak-
ing and det. pH with test paper. Adjust pH to 6.8 ± 0.2, if
necessary, using sterile IN NaOH or HO, capping jar securely
and mixing well before detg final pH. Aseptically transfer con-
tents of each jar to sterile wide-mouth, screw-cap 500 mL jar.
Loosen jar caps l / 4 turn and incubate 24 ± 2 h at 35°.
(b) Selective enrichment. — Transfer I mL incubated pre-
enrichment mixts to selenite cystine broth and 1 mL to tetra-
thionate broth as in 967.26B(a). For all foods other than raw
or highly contaminated products, incubate 6-8 h at 35°. Se-
lective enrichments of raw or highly contaminated product must
be incubated 16-20 h at 35°.
(c) Post-enrichment. — Remove selective broths from in-
cubation and mix by hand or by vortex mixer. Remove 1 mL
from tetrathionate tube and transfer to 10 mL tube of sterile
M-broth which has been warmed to 35°. Also remove 1 mL
from selenite cystine tube and transfer to sep. tube of M-broth.
For all foods other than raw or highly contaminated products,
incubate M-broth tubes 14-18 h and return selective enrich-
486
Microbiological Methods
AOAC Official Methods of Analysis (1990)
ment broth tubes to 35° incubator and incubate for addnl 16-
18 h. For raw or highly contaminated products, incubate M-
broth tubes 6 h at 35° and return selective enrichment broth
tubes to 35° and incubate for addnl 6 h at 35°.
(d) Preparation of sample for EI A analysis. — Remove M-
broth tubes from incubation and mix tubes by hand or vortex
mixer. Combine 1 .0 mL from each M-broth tube in clean screw -
cap tube and heat in boiling H 2 bath or in flowing steam 15
min. Refrigerate (2-8°) remaining M-broth and tetrathionate
and selenite cystine tubes from (c) for culture confirmation of
any EI A pos. samples. Cool heated M-broths to 25-37° prior
to analysis by El A.
G. Enzyme Immunoassay
(1) Following reagents must be prepd prior to commencing
assay:
(a) Prep, working strength wash soln by di]g contents of 1
vial of wash soln cone, to I L with distd or deionized H 2
into plastic reagent bottle. Plastic squeeze bottle is ideal for
washing trays manually.
(b) Prep, reconstituted neg. control by transferring 2 mL
controls diluent to vial of lyophilized neg. control antigen; mix
thoroly. Similarly prep, reconstituted pos. control by transfer-
ring 2 mL controls diluent to vial of lyophilized pos. control
antigen; mix thoroly.
(c) Prep, reconstituted conjugate by adding 5 mL conjugate
diluent to vial of lyophilized conjugate. Let conjugate rehy-
drate at room temp., mix, and then pour contents of vial into
conjugate diluent vial. Finally, gently mix reconstituted con-
jugate.
(d) Prep, reconstituted substrate by adding vial of substrate
diluent to lyophilized substrate. Be sure substrate has dis-
solved and mixt. is room temp, prior to use. Reconstituted
substrate will appear pale green.
(e) Use stop soln as received. No reconstitution is required.
(2) Secure desired no. of test (Removawell) strips in tray,
allowing 1 well per food sample plus 4 wells for controls.
PRESS WELLS FIRMLY INTO PLACE. Remove sealing film
from top of wells to be used. Transfer 0.2 mL of each heated
M-broth sample to single well. Transfer 0.2 mL aliquots of
reconstituted neg. control into 2 wells and 0.2 mL aliquots of
reconstituted pos. control into 2 wells. Record sample position
on sample record sheet provided. Note: Be sure numbered tag
at end of each test strip has been removed.
(3) Cover tray and incubate 30 min at 35-37° in std labo-
ratory incubator. Tray must be covered to prevent evapn. Plas-
tic film or sealed plastic container may be used.
(4 ) After incubation, wash plate by hand using plastic squeeze
bottle contg working strength wash soln or use automatic washer
charged with working strength wash soln as follows:
(a) Quickly invert tray, emptying its contents into con-
tainer.
(b) Remove any residual liquid by FIRMLY tapping tray
face-down on paper towel several times.
(c) Completely fill each well with working strength wash
soln.
(d) Repeat (a) -(c) 2 more times.
(5 ) Empty tray according to 4(a) and (b); then add 0.2 mL
reconstituted conjugate to each well. Cover tray and incubate
30 min at 35-37°.
(6) Empty contents of tray and wash it thoroly 4 times ac-
cording to 4(a)~(c); then empty tray according to 4(a) and (b).
(7) Add 0.2 mL reconstituted substrate to each well. In-
cubate at room temp. (20-25°) until pos. control has reached
color equiv. to pos. control on color comparator card or to A
> 1.0. Because color development tends to cone, around edges
of wells, it is important to tap sides of plate gently to mix
contents prior to reading result. In this way, accurate readings
will be obtained.
(8) Add 0.02 mL stop soln to each well. Incubation time
should be ca 10-20 min. If >25 min has elapsed and A of 1.0
has not been attained, test is invalid.
H. Reading
Results of tests can be detd (J) visually or (2) with microtiter
tray reader.
(J) Place tray on white background, and then compare in-
dividual test wells with color comparator. Pos. control should
give strong blue-green color indicating that all reagents are
functional. If pos. control is lighter than "Positive Control" on
color comparator card, test is invalid; refer to "Troubleshoot-
ing Guide" in package insert (k). If neg. control is darker than
"Negative" on color comparator card, it is probable that tray
was inadequately washed, and assay must be repeated. Du-
plicate control antigens should appear equiv. by eye.
(2) A max. of blue-green end product occurs at 414 nm;
therefore, tray can be read at 414 ± 10 nm. For single and
dual wavelength readers, set reader to zero (blank) on air. For
dual wavelength readers, set second ref. wavelength at 490 ±
10 nm. A >0.3 indicates pos. result. A >0.25 for neg. control
indicates insufficient washing of tray. Pos. control should give
A >1.0.
/. Confirmation of Positive EIA Samples
Pos. EIA reading indicates that Salmonella may be present.
However, since antibodies may cross-react with a few other
organisms, culture confirmations must be performed by streak-
ing HE, XLD, and BS plates from tetrathionate broth, selenite
cystine broth, and M-broth tubes as described in 967. 26B, and
typical or suspicious colonies should be identified as in 967. 26C,
967.27, 967.28.
Ref.: JAOAC 71, 973(1988).
989.15 Salmonella in Foods
Fluorogenic Monoclonal
Enzyme Immunoassay Screening Method
First Action 1989
Method is screening procedure for presence of Salmonella
in all foods; it is not a confirmatory test because monoclonal
antibodies used in test may cross -react with small percentage
of non-Salmonella.
Enrichment broths and M-broths from samples pos. by en-
zyme immunoassay (EIA) method must be streaked on selec-
tive media as in 967. 26B and typical or suspicious colonies
must be identified as in 967.26C, 967.27, 967.28.
A. Principle
Detection of Salmonella antigens is based on enzyme im-
munoassay which measures Salmonella antigen in foods and
feeds. Monoclonal antibodies to Salmonella antigen are coated
on internal surface of plastic microtiter strip wells, and sample
to be assayed is added to strip well. If Salmonella antigens are
present in sample, they will be bound to antibody adsorbed
onto surface of well. All other material in sample is washed
away.
Salmonella antibody conjugated to alk. phosphatase is added
and will bind to Salmonella antigens if they are attached to
adsorbed antibody on surface of well. This forms antibody-
antigen-antibody complex. Unbound conjugate is removed by
washing and fluorescent substrate is added. Samples with flu-
orescent value greater than or equal to recommended cutoff
value are considered pos. for Salmonella antigens.
AOAC Official Methods of Analysis (1990)
Salmonella
487
B. Method Performance
For all foods:
Results
Percent
95% Confidence
Range (Approx.)
Agreement 1
False neg. (BAM) 2
False neg. (EIA) 3
98.5
1.1
0.8
97.9-99.1
0.8-1.4
0.0-2.7
1 This rate reflects no. of samples read identically between AOAC/BAM
(Bacteriological Analytical Manual (1984) 6th ed., AOAC, Arlington, VA) cul-
ture method and EIA.
2 This rate reflects no. of samples found to be pos. by EIA but neg. by
AOAC/BAM culture method.
3 This rate reflects no. of samples found to be pos. by AOAC/BAM culture
method but neg. by EIA.
Of 1 1 laboratories submitting usable data, 6 (55%) had com-
plete agreement between culture method and EIA; 10 labora-
tories (91%) showed agreement on >96% of samples; all lab-
oratories showed agreement on >93% of samples.
C. Reagents
Items (a)-(j) are available as Q-TROL Salmonella Detec-
tion Kit (Dynatech Laboratories, Inc., 14340 Sullyfield Circle,
Chantilly, V A 22021).
(a) Antibody coated microtiter wells. — Monoclonal anti-
body to Salmonella, eight 12- well strips. Stable 28 days, after
opening, when stored at 2-8°.
(b) Microtiter strip well holder. — Sufficient for securing
individual wells or strips.
(c) Control antigens . — Pos. control (lyophilized boiled sus-
pension of S. typhimurium) purified Salmonella antigen, which
reacts with antibodies to Salmonella, 1 vial; neg. control (ly-
ophilized boiled suspension of Proteus mirabilis), which is
nonreactive with antibodies to Salmonella, 1 vial. Reconsti-
tuted control antigens are stable 28 days when stored at 2-8°.
(d) Tween 20. — 1 vial. 25% Tween 20 (polyoxyethylene
(20) sorbitan monolaurate) in H 2 0. After opening, so In is sta-
ble 28 days when stored at 2-8°.
(e) Phosphate buffer-saline tablets. — For prepn of PBS-
Tween soln. Dissolve 1 tablet in 100 mL distd or deionized
H 2 to prep. 0.01M phosphate-buffered 0.85% saline. Add 8
drops Tween 20. PBS -Tween soln is used to rehydrate pos.
and neg. control antigens and for wash steps, and is stable 7
days when stored at 2-8°.
(f) Enzyme conjugate. — 1 vial contg antibody of Salmo-
nella conjugated to alk. phosphatase (lyophilized). Reconsti-
tuted conjugate is stable 28 days when stored at 2-8°.
(g) Conjugate diluent. — 1 vial (10 mL/vial). Contains 0.05M
tris buffer" (pH 8), 0.02% NaN 3 , 1 mM MgCl 2 , and 1% bovine
serum albumin.
(h) Substrate tablets. — 0.13 mg 4-methylumbelliferyl
phosphate (4-MUP) per tablet. Reconstituted substrate must be
used within 2 h.
(i) Substrate diluent. — 1 vial (31.5 mL/bottle). Aq. soln of
10% diethanolamine with 0.02% NaN 3 as preservative.
(j) Stop soln. — 1 vial (5.5 mL/vial). Aq. soln of 2% Na 3 P0 4 .
(k) M-brotk. — 5.0 g yeast ext, 12.5 g tryptone, 2.0 g d-
mannose, 5.0 g Na citrate, 5.0 g NaCl, 5.0 g K 2 HP0 4 , 0.14
g MnCl 2 , 0.8 g MgS0 4 , 0.04 g FeS0 4 , 0.75 g Tween 80.
Suspend ingredients in 1 L H 2 and heat to boiling for 1-2
min. Dispense 10 mL portions into 16 X 125 mm screw-cap
test tubes. Cap tubes loosely and autoclave 15 min at 121°.
Tighten caps securely for storage. Final pH should be 7.0 ±
0.2.
(I) Diagnostic reagents. — Necessary for cultural confir-
mation of presumptive pos. EIA tests; see 967. 25B.
D. Apparatus
(a) Fluorometer. — To measure relative fluorescence of
contents of microtiter well (Micro FLUOR® Reader, Dynatech
Laboratories, Inc.; or equiv.).
(b) Microtiter strip well-washer /aspirator . — With 12
channels to wash entire strip.
(c) Pipets. — Capable of delivering 50-200 fxL.
(d) Water bath. — Capable of maintaining 100°. Autoclave
set at 100° is acceptable alternative, as are generators of flow-
ing steam.
E. General Instructions
Components of kit must be refrigerated when not in use. Kit
is intended for 1-time use only; do not reuse wells contg sam-
ple, reagents, or wash soln.
Include 3 neg. and 1 pos. control antigens with each group
of test samples. All controls must function properly for test to
be valid.
Caution; Diluents for conjugate and substrate contain NaN 3
as preservative. Flush drains with H 2 if any solns contg NaN 3
are discarded in sink. Flushing will prevent formation of lead
or copper azide in plumbing, which may explode upon per-
cussion (such as hammering). 4-MUP diluent and stop soln
are basic and may cause skin irritation. If contact with skin
occurs, flush area with H 2 0.
Use data record sheet to identify location of each test sam-
ple.
Use sep. pipets for each sample and kit reagent to avoid
cross-contamination. If plastic troughs are used to dispense
conjugate and substrate, ensure that they are always kept sep-
arate.
Components in kit are intended for use as integral unit. Do
not mix components of different batch numbers.
F. Preparation of Sample
(a) Pre- enrichment. — Pre-enrich product in noninhibitory
broth to initiate growth of salmonellae. Methods used may vary
with product and should be performed as indicated in 967. 26A,
or in Bacteriological Analytical Manual, 6th ed., AOAC, Ar-
lington, VA, Chap. 7, sec. C, with following exception:
Raw or highly contaminated products. — Aseptically weigh
25 g sample into sterile blender jar. Add 225 mL sterile lactose
broth. Blend 2 min at high speed (ca 20 000 rpm). Cap jars
securely and let stand 60 min at room temp. Mix well by shak-
ing and det. pH with test paper. Adjust pH to 6.8 ± 0.2, if
necessary, using sterile IN NaOH or HC1, capping jar securely
and mixing well before detg final pH. Aseptically transfer con-
tents of each jar to sterile wide-mouth, screw-cap 500 mL jar.
Loosen jar caps 1 / 4 turn and incubate 24 ± 2 h at 35°.
(b) Selective enrichment. — Transfer 1 mL incubated pre-
enrichment mixts to selenite cystine broth and 1 mL into tetra-
thionate broth as in 967.26B(a). For all foods, incubate 6-8
h in 35° water bath.
(c) Post-enrichment. — Remove selective broths from in-
cubation and mix by hand or by vortex mixer. Remove 1 mL
from tetrathionate tube and transfer to 10 mL tube of sterile
M-broth which has been warmed to 35°. Also remove 1 mL
from selenite cystine tube and transfer to a separate tube of
M-broth. For all foods, incubate M-broth tubes for 14-18 h
and return selective enrichment broth tubes to 35° incubator
and incubate for addnl 16-18 h.
(d) Preparation of sample for EIA analysis. — Remove M-
broth tubes from incubation and mix tubes by hand or vortex
mixer. Combine 0.5 mL from each M-broth tube in clean screw-
cap tube and heat 20 min in boiling H 2 bath or flowing steam.
Refrigerate (2-8°) remaining M-broth and tetrathionate and se-
lenite cystine tubes from (c) for cultural confirmation of any
488
Microbiological Methods
AOAC Official Methods of Analysis (1990)
El A pos. samples. Cool heated M-broths to 20-30° prior to
analysis by E1A.
G. Enzyme immunoassay
(J) Following reagents must be prepd prior to commencing
assay:
(a) PBS-Tween soln. — For every two 12-well strips to be
used, dissolve 1 PBS tablet in H 2 and prep, soln as in (e).
(b) Reconstituted control antigens. — Transfer 3 mL PBS-
Tween soln to neg. control vial and mix contents thoroughly.
Transfer 3 mL PBS-Tween soln to pos. control vial and mix
contents thoroughly. These solns are reconstituted neg. and
pos. controls, resp.
(c) Reconstituted enzyme conjugate. — Add 10 mL (1 vial)
conjugate diluent to conjugate vial. Mix and let contents of
vial rehydrate at room temp.
(d) Stop soln. — No reconstitution is required. Warm soln at
35° if crystals are present.
(2) Turn on power to reader and printer. Allow at least 2 h
warm-up.
( 3) Remove necessary number of microtiter strips from Al
foil pouch, allowing 1 well per food sample plus 4 wells for
controls. Secure strips in strip well holder. Transfer 100 uJL
neg. control antigen into each of wells A-l, A-2, and A-3.
Transfer 100 |xL pos. control antigen into well designated
A-4. Transfer 100 fxL each heated M-broth sample to single
well. Record sample position on sample record sheet provided.
(4) Incubate tray for 60 min at 20-25°.
(5) After incubation, aspirate samples from wells and add
300 fxL PBS-Tween soln to each well by use of washer/as-
pirator.
(a) Repeat this step 4 more times.
(b) Aspirate last wash. Invert tray and firmly tap it on ab-
sorbent paper several times to remove last traces of fluid.
(6) Add 100 jxL reconstituted enzyme conjugate to bottom
of each well and incubate 40 min at 20-25°.
(7) During this incubation period, prep, substrate by adding
one 4-MUP substrate tablet to 5.2 mL substrate diluent. Dis-
solve 1 substrate tablet for every 2 microtiter strips to be used.
Swirl soln occasionally to dissolve tablet(s).
(8) Repeat steps 5(a) and (b).
(9) Add 200 |xL 4-MUP substrate to bottom of each well.
Incubate 20 min at 20-25°.
(10) Add 50 fxL stop soln to each well.
H. Reading
Place tray in reader. Read relative fluorescent units (RFU)
of each control and sample well. Calc. av. RFU of the 3 neg.
control wells. Individual neg. control values should be >0.85
av. RFU and <] .15 av. RFU. If 1 value is outside this range,
discard that value and recalc. mean. If 2 values are outside
range, test is invalid and must be repeated. Multiply av. valid
neg. controls by 2.3 to det. cutoff value. Any sample with
value at or above cutoff value is considered reactive.
If av. of neg. control values exceeds 1600 RFU, cutoff will
exceed dynamic range of reader and test is invalid. Poor wash-
ing and deterioration of substrate may result in high readings
of neg. control.
/. Confirmation of Positive EIA Samples
Pos. EIA reading indicates that Salmonella may be present.
However, since antibodies may cross-react with a few other
organisms, culture confirmations should be performed by
streaking HE, XLD, and BS plates from tetrathionate broth,
selenite cystine broth, and M-broth tubes as described in
967. 26B, and typical or suspicious colonies should be iden-
tified as in 967.26C, 967.27, 967.28.
Ref.: JAOAC72, 318(1989).
987.10 Salmonella in Foods
DNA Hybridization Screening Method
First Action 1987
Final Action 1989
Method is test procedure for presence of Salmonella in all
foods. Because a certain percentage of false pos. reactions are
expected, all pos. assays should be confirmed by standard cul-
ture methods. Enrichment broths and GN broths from samples
pos. by DNA hybridization method should be streaked to se-
lective media as in 967. 26B and typical or suspicious colonies
should be identified as in 967.26C, 967.27, 967.28.
A. Principle
Detection of Salmonella DNA in cultured food samples uses
specific DNA probes. Following pre-enrichment, selective en-
richment, and post-enrichment of test samples, bacteria are
collected on membrane filters by vac. filtration. Bacteria are
lysed, DNA is denatured, and resultant single-stranded DNA
is fixed to membrane filters. Filters are then incubated in hy-
bridization soln contg 32 P- labeled Salmonella-specific DNA
molecules. If Salmonella target DNA is present in test sample,
radiolabeled DNA probes will hybridize to target DNA se-
quences. Unbound probe is washed away and radioactivity on
filters is measured. Radioactivity on filter above threshold value
indicates presence of Salmonella in test sample.
B. Method Performance
For all foods:
Results
Percent
95% Confidence
Range
Agreement 1
False neg. (BAM) 2
False neg. (DNAH) 3
95.6
5.9
0.2
94.0-97.2
3.7-8.1
0.0-0.6
Rate reflects no. of samples read identically between AOAC/BAM (Bac-
teriological Analytical Manual (1984) 6th ed., AOAC, Arlington, VA) culture
method and DNA hybridization (DNAH) method.
2 Rate reflects no. of samples found to be pos. by DNAH method but
detected as neg. by AOAC/BAM culture method.
3 Rate reflects no. of samples found to be pos. by AOAC/BAM culture
method but detected as neg. by DNAH method.
Of II laboratories, 2 had complete agreement between cul-
ture and DNAH methods; 4 showed agreement on >97% of
samples; 7 showed agreement on >95% of samples; 10 showed
agreement on >93% of samples.
C. Reagents
Items (a)-(e) are available as GENE-TRAK® DNA Hy-
bridization Test for Detection of Salmonella (GENE-TRAK
Systems, Inc., 31 New York Ave, Framingham, MA 01701).
(a) Filter cups. — Filter cup assemblies contg 25 mm diam.
membrane filters (112 assemblies/box). Sufficient for 96 tests
plus controls.
(b) Soln set. — Contains (J ) 1 bottle (240 mL) denaturation
soln (0.2N NaOH and 0.6M Nad). (Caution: Contains NaOH.
If contact with skin occurs, wash skin thoroly with H 2 0.) (2)
1 bottle (240 mL) neutralization soln (1 .0M Tris, pH 7.0, and
0.6M NaCl). (3) 1 bottle (240 mL) fixation soln (95% EtOH).
(Caution: Flammable, contains alcohol.) (4 ) 1 bottle (120 mL)
pre-hybridization soln (0.9M NaCl, 0.09M Na citrate, 0.02%
Ficoll, 0.02% bovine serum albumin, 0.02% polyvinyl pyr-
rolidone, ImM Na 2 ethylenediamine tetraacetate, and 0.5% Na
dodecyl sulfate (SDS), pH 6.5-7.5). (5) 1 bottle (60 mL) hy-
bridization soln (same as soln 4, with 10% dextran sulfate).
(6) 3 bottles (240 mL each) wash soln (0.03M NaCl, 0.003M
Na citrate, and 0.5% SDS, pH 6.5-7.5).
AOAC Official Methods of Analysis (1990)
Salmonella
489
Sufficient for 96 tests plus controls. All solns are stable 6
months from date of manufacture when stored at 15-24°.
(c) Salmonella probe and controls set . — Contains (1 ) 1 bot-
tle (20 mL) pos. control soln (heat- killed S. typhimurium) . (2)
1 bottle (60 mL) neg. control soln (heat-killed Escherichia coli).
(3) 1 vial (0.75 mL, 75 p,Ci 32 P) 32 P-labeled Salmonella DNA
probe soln.
Sufficient for 96 tests plus controls. Shipped frozen on dry
ice; thaw at room temp. (15-24°) before use. Solns are stable
8 days from receipt when stored at 2-8°.
(d) Instruction manual.
(e) Data sheets.
(f) Gram negative (GN) broth. — 20.0 g tryptose, 1.0 g
dextrose, 2.0 g D-mannitol, 5.0 g Na citrate, 0.5 g Na des-
oxycholate, 4.0 g K 2 HP0 4 , 1.5 g KH 2 P0 4 , 5.0 g NaCl. Dis-
solve ingredients in 1 L H 2 0. Dispense 10 mL portions into
16 X 125 mm test tubes (or equiv.). Cap tubes loosely and
autoclave 15 min at 121°. Final pH should be 7.0 ± 0.2 at
25°.
(g) Diagnostic reagents. — Necessary for cultural confir-
mation of pos. DNA hybridization tests; see 967. 25B.
D. Apparatus
Items (a)-(e) are available from GENE-TRAK Systems, Inc.
(a) Manifold kit. — Vac. filtration manifold device consist-
ing of base with vac. control valve and top to house filter cup
assemblies.
(b) Bottle holder. — Plastic rack to hold soln bottles in water
bath.
(c) Shield. — 1 / 2 in. thick Lucite used to protect worker from
beta particle emissions from 32 P decay when radiolabeled DNA
probe soln is used.
(d) Vacuum pump. — Adjustable between 8 and 15 in. Hg.
Connected to manifold base thru trap consisting of 1 or 2 L
vac. filtration flask and requisite tubing.
(e) Beta detector. — Any instrument capable of measuring
radioactive decay of 32 P on dry, 25 mm membrane filters with
efficiency of 0.4-0.5, e.g., scintillation counter. Use of scin-
tillation counter requires appropriate carrier vials and insert
mini vials to hold filters upright.
(f) Heating water bath. — Capable of maintaining 65 ± 1.0°.
Able to accommodate bottle holder 12 x 6 x 4 l / 2 in. and H 2
level of 4 j /2 in.
(g) Micropipet. — Capable of delivering accurate amts in
range continually adjustable in 1 julL increments between 100
and 200 uX.
(h) Vortex mixer. — For mixing broth in culture tubes,
(i) Polypropylene screw-cap centrifuge tubes. — 50 mL,
conical bottom.
E. General Instructions
Test uses radioactive compd. Personnel must receive appro-
priate training in use of radioactive materials and have proper
facilities available for use of this substance. Facility must pos-
sess current, appropriate radioactive materials license issued
by U.S. Nuclear Regulatory Commission or other agency with
regulatory control.
Disposal of radioactive waste must be in accordance with
radioactive materials license of facility.
Treat all materials in contact with bacterial cultures or cul-
ture filtrates as biohazardous material and decontaminate by
appropriate methods.
Do not touch membrane filters. Handle with forceps only.
Return pos. and neg. controls and DNA probe soln to 2-8°
storage immediately after use.
Include 1 pos. control and 3 neg. controls with each group
of test samples .
Components and procedures of this test kit have been stan-
dardized for use in GENE-TRAK assay. Use of components
or procedures other than those supplied or recommended by
GENE-TRAK Systems, Inc., may yield unsatisfactory results.
F. Sample Preparation
(a) P re-enrichment. — Pre-enrich product in non-selective
medium to initiate growth of salmonellae. Procedure will vary
with product type and should be performed as indicated in
967.26A or in Bacteriological Analytical Manual (1984) 6th
ed., AOAC, Arlington, VA, Chap. 7, section C, with the fol-
lowing exception:
Raw meats and raw milk products: Aseptically weigh 25 g
sample into sterile blender jar. Add 225 mL sterile lactose broth.
Blend 2 min at high speed (ca 20 000 rpm). Cap jar securely
and let stand 60 min at room temp. Mix well by shaking and
det. pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2
using sterile IN NaOH or HC1; cap jar securely and mix well
before detg final pH. Aseptically transfer contents to sterile
wide-mouth, screw-cap 500 mL jar. Loosen jar cap i / 4 turn
and incubate 24 ± 2 h at 35°.
(b) Selective enrichment. — Transfer 1 mL incubated pre-
enrichment culture to tube contg 10 mL selenite cystine broth
and 1 mL to tube contg 10 mL tetrathionate broth (pre-warmed
to 35°) as in 967.26B(a). Incubate 6 h at 35° with the following
exception:
Raw meats and raw milk products: Incubate selenite cystine
and tetrathionate broth 18 ± 2 h at 35°.
(c) Post-enrichment. — Remove selective enrichment cul-
tures from incubation and mix by hand or with vortex mixer.
Transfer 1 mL tetrathionate culture to tube contg 10 mL GN
broth (prewarmed to 35°). Transfer I mL selenite cystine cul-
ture to sep. tube contg 10 mL GN broth. Incubate GN broths
12-18 h at 35° with the exception of raw meats and raw milk
products (see below). Return tetrathionate and selenite cystine
tubes to 35° for incubation up to total of 24 ± 2 h.
Raw meats and raw milk products: Incubate GN broths 6 h
at 35°. Return tetrathionate and selenite cystine tubes to 35°
for incubation up to total of 24 ± 2 h.
G. Filtration
(J) Connect manifold base to vac. pump thru trap. Add dis-
infectant soln to filtration flask. Two manifolds may be con-
nected in parallel to single vac. source. This configuration will
accommodate up to 24 food samples in 1 run plus requisite
pos. and triplicate neg. controls.
(2) Place manifold top(s) on manifold base(s). Fit manifold
top(s) with filter cup assemblies; 1 pos. control filter cup, 3
neg. control filter cups, and up to 24 pre-numbered sample
filter cups for each set of assays. Fit unused manifold positions
with No. 3 rubber stoppers (provided).
(3) Remove sample GN broths from 35° incubation. Vortex-
mix or otherwise mix each culture. For each sample, pipet I
mL from each of the 2 GN broths (one derived from tetra-
thionate, one from selenite cystine) into single filter cup. Rec-
ord sample numbers and filter numbers on data sheet.
(4) Mix pos. and neg. control solns. Pipet 2 mL pos. control
soln into pos. control filter cup. Pipet 2 mL neg. control soln
into each of the 3 neg. control filter cups.
Note: Pos. and neg. controls are shipped frozen on dry ice.
Thaw at room temp. (15—24°) before first use. Store controls
at 2-8° between uses,
(5) When all filter cups have been loaded with samples, open
vac. control valve on manifold base and turn on vac. pump.
Apply vac. (8-10 in. Hg) until all samples are filtered. Avoid
vac. of excessive pressure or prolonged duration. Close valve
on manifold base and turn off pump. Be sure vac. on manifold
base is released before proceeding.
490
Microbiological Methods
AOAC Official Methods of Analysis (1990)
(6) Add (squirt in) ca 1.5—2.0 mL denaturation soln (Soln
1) into each filter cup, completely covering surface of filters.
Wait 2 min, then apply vac. as before (see step 5) until soln
has filtered thru all of the cups. Turn off vac.
(7) Add (squirt in) ca 1 .5-2.0 mL neutralization soln (Soln
2) into each filter cup. Wait 2 min, then apply vac. Turn off
vac. after soln has filtered thru all of the cups.
(8) Add (squirt in) ca 1 .5-2.0 mL fixation soln (Soln 3) into
each filter cup. Wait 2 min, then apply vac. Turn off vac. after
soln has filtered thru aJJ of the cups.
(9) Snap off top part of each cup assembly. Be careful not
to discard membrane filters. With forceps, remove membrane
filters from cup assembly bases and place on sheet of absorb-
ent paper to dry. All other parts of cup assembly should be
treated as biohazardous waste and discarded. Manifold bases
and tops should be treated with disinfectant soln (do not au-
toclave).
H. DNA Hybridization Assay
(J) Using forceps, place filters (up to 24 sample filters plus
I pos. and 3 neg. control filters) into 50 mL polypropylene
centrf. tube, (i).
(2 ) Mix bottle of pre-hybridization soln (Soln 4) that has
been equilibrated to 65°. Pour Soln 4 into conical tube contg
filters to 25 mL mark. Store remaining Soln 4 at room temp.
With back-and -forth motion, gently shake tube until all filters
are completely immersed in soln and none are stuck to sides
of tube. Filters should stack in vertical array at bottom of tube.
Incubate tube for 30 min in 65° H 2 bath.
(3) Carefully drain pre-hybridization soln from tube and dis-
card soln. Use of funnel will prevent losing stack of filters into
collection vessel. Immediately mix bottle of hybridization soln
(Soln 5) that has been equilibrated to 65° and add 12 mL to
conical tube (measure accurately with pipet). Return remaining
Soln 5 to room temp, storage.
(4) Working behind Lucite shield, remove plastic vial contg
thawed 32 P-labeled Salmonella probe soln from Lucite con-
tainer. Mix probe soln by tapping on lower portion of plastic
vial. Using precision micropipet, add probe soln to conical
tube according to following schedule:
Age of Probe,
Days
Calendar
Day
(xL of
Probe Soln
1
1
2
3
4
5
6
7
8
Mon.
Tue.
Wed.
Thur.
Fri.
Sat.
Sun.
Mon.
Tue.
127
133
140
147
154
162
170
179
Day is indicated by date on vial. Day is normal shipping day; Day
1 is normal day of receipt, but this may vary. User should always refer to
age and calendar day for detn of probe soln vol. to use.
Dispose of pipet tip in container reserved for solid radio-
active waste. Return remaining probe soln to 2-8° storage in
its Lucite container.
(5 ) Tighten cap on conical tube. With back-and-forth mo-
tion, gently shake tube until all filters are completely im-
mersed in soln and none are stuck to sides of tube. Again,
filters should stack in vertical array at bottom of tube. Incubate
tube for 2 h in 65° H 2 bath.
(6) Working behind Lucite shield, carefully drain soln from
conical tube into container reserved for liq. radioactive waste,
using funnel to protect against losing stack of filters into waste
vessel. Drain off as much soln as possible. (Radiochemical
concn of waste is ca 1 .0 jmCi 32 P/mL in molecular form of
DNA.)
(7) Immediately add 25 mL wash soln (Soln 6) that has been
equilibrated to 65° by pouring soln into conical tube to 25 mL
mark. Return bottle of Soln 6 to 65° H 2 bath. Gently shake
tube for at least 10 s, or until all filters are free from sides of
tube and are stacked in vertical array at bottom of tube. In-
cubate conical tube for 5 min in 65° H 2 bath.
(8) Remove tube from H 2 bath and gently shake for at least
10 s, then carefully drain soln into liq. radioactive waste con-
tainer. Drain off as much soln as possible.
(9) Repeat steps 7 and 8 five addnl times for total of 6 washes.
Store Soln 6 at room temp, after use.
(10) Using forceps, remove filters from conical tube and
place them on sheet of absorbent paper. Discard conical tube
into solid radioactive waste container. Using forceps, sep. fil-
ters and let dry briefly (5-10 min).
(11) Turn on power to beta detector, (e), and let instrument
warm up ca 10 min. Counting time switch should be set to
0.5 min position.
To count each filter, center filter, using forceps, on lower
window of beta detector. Detector windows are delicate; be
careful not to damage them with forceps. Close detection unit
and press start switch. Radioactivity on filter will be counted
for 30 s, and result will be displayed as counts per minute
(cpm). Record result for each filter in cpm on data sheet. Fil-
ters should be saved until data analysis is complete. After-
wards, they should be discarded as solid radioactive waste.
Alternatively, filters can be counted using std scintillation
counter. Appropriate carrier vials and insert minivials are re-
quired so that filters can be positioned upright. Count each
filter for 30 s using settings capable of counting 32 P on dry
filters (do not use scintillation cocktail) with efficiency of 0.4-
0.5. Record data as cpm.
/. Data Analysis
(1) Det. av. of 3 neg. control filters. If this av. is >500
cpm, all filters must be re- washed (repeat steps 7 and 8 under
DNA Hybridization Assay) and re-counted. Accept data as fi-
nal after addnl wash, even if av. of 3 neg. control filters is
still >500 cpm.
(2 ) CPM of pos. control filter should be at least 5 times av.
of neg. control filters; otherwise assay is invalid.
(3) Add 500 cpm to av. of the 3 neg. control filters. This
sum is CUTOFF value.
Neg. criterion: Test sample is considered to be neg. (non-
reactive for presence of Salmonella) if its cpm is < CUTOFF
value.
Pos. criterion: Test sample is considered to be pos. (reactive
for presence of Salmonella) if its cpm is > CUTOFF value.
J. Confirmation of Positive DNA Hybridization Assays
Because a certain percentage of false pos. DNA hybridiza-
tion assays can be expected (3-4%), all samples found pos.
by DNA hybridization assay must be confirmed by culture
methods. HE, XLD, and BS plates should be streaked from
tetrathionate broth, selenite cystine broth, and GN broth tubes
as described in 967. 26B, and typical and suspicious colonies
should be identified as in 967. 26C, 967.27, 967.28.
Ref.: JAOAC 70, 527(1987).
989.13 Rflotile Salmonella in Foods
Immunodiffusion Screening Method
First Action 1989
Method is screening procedure for presence of motile Sal-
monella in all foods. It is not a confirmatory test because poly-
valent H (flagellar) antibodies used in test may cross-react with
AOAC Official Methods of Analysis (1990)
Salmonella
491
small percentage of non-Salmonella. Note: Method does not
detect nonmotile salmonellae.
If test is pos., enrichment broth from inoculation chamber
of test unit must be streaked onto selective/differential agar
media as in 967. 26B, or, if test is performed on raw or highly
contaminated product, enrichment broth must be transferred to
tetrathionate broth and incubated 18-24 h in 43 ± 0.5° H 2
bath, and then streaked onto selective/differential agar media.
Typical or suspicious colonies must be identified as in 967. 26C,
967.27, 967.28.
A. Principle
Detection of Salmonella is based on presence and obser-
vation of Salmonella immobilized in motility medium by poly-
valent H (flagellar) antibodies. Immobilization of motile Sal-
monella results in development of well defined band of cells
(immuno-band). Fig. 989.13 shows small disposable plastic
device (1-2 TEST unit) which has 2 chambers. Smaller in-
oculation chamber contains selective tetrathionate broth sup-
plemented with brilliant green and L-serine. Enriched sample
is inoculated into this chamber. Central motility chamber of
unit contains peptone-based, nonselective motility medium.
Motility chamber is sealed with gel-former plug. Tip of this
plug forms void in motility medium for addn of flagellar an-
tibody prepn. For shipping, opening between 2 chambers is
sealed with polyethylene chamber plug, which is removed and
discarded prior to addn of inoculum. Salmonella inoculated
into tetrathionate-brilliant green- serine broth move from this
medium into motility medium to react with flagellar antibod-
ies.
B. Method Performance
For all foods:
95% Confidence
Results
Percent
Range (Approx.)
Agreement 1
96.1
94.5-97.7
False neg.
1.7
0.5-3.0
(BAM/AOAC) 2
False neg.
3.6
1.8-5.5
(immunodiffusion) 3
1 Rate reflects no. of samples read identically between BAM/AOAC (Bac-
teriological Analytical Manual (1984) 6th ed. AOAC, Arlington, VA) culture
method and immunodiffusion method.
2 Rate reflects no. of samples found to be pos. by immunodiffusion method
but detd as neg. by BAM/AOAC culture method.
3 Rate reflects no. of samples found to be pos. by BAM/AOAC culture method
but detd as neg. by immunodiffusion method.
Of 17 laboratories, 8 had complete agreement between cul-
ture and immunodiffusion methods; 1 1 showed agreement on
>97% of samples; 14 showed agreement on ^95%; 16 showed
agreement on >93%.
C. Reagents
Items (a)-(c) are available as BioControl 1-2 TEST
(BioControl Systems, Inc., 19805 North Creek Pkwy, Bothell,
WA 9801 1). Store at refrigerator temp. (4-8°C; 39-46°F). Items
are stable 3 months (90 days) from date of receipt.
(a) 1-2 TEST unit.— See Fig. 989.13.
(b) Iodine-iodide soln. — 1 vial for 12 units.
(c) Antibody. — 1 vial for 12 units.
(d) Diagnostic reagents. — Necessary for cultural confir-
mation of presumptive pos. 1-2 TESTS. See 967. 25B.
D. Preparation of Sample
Most foods require only pre-enrichment of product in non-
inhibitory broth to initiate growth of salmonellae before in-
oculation into unit. Exceptions are given below. Methods used
for sample prepn may vary with product and should be per-
formed as in 967.26A and 975.54D.
WHITE CAP
GEL FORMER BASE
GEL FORMER TIP
GEL VOID
MOTILITY CHAMBER
CHAMBER PLUG^_
__\T
BLACK CAP
INOCULATION CHAMBER
FIG. 989.13— Test unit for immunodiffusion screening method
for motile Salmonella
Raw flesh foods or highly contaminated products . — No pre-
enrichment is required. Aseptically weigh 25 g sample into
sterile blender jar. Add 225 mL tetrationate broth without bril-
liant green dye. Blend 2 min. Securely cap jars and let stand
60 min at room temp. Mix well by shaking. Add 2.25 mL
0.1% soln of brilliant green dye. Aseptically transfer contents
of each jar to sterile 500 mL wide-mouth, screw-cap jar. Loosen
jar caps l / 4 turn and incubate 24 ± 2 h at 35°.
Flour-containing products (soy flour, wheat flour , dough,
pasta, cake mix, and processed animal by-products). — Trans-
fer 1 mL incubated pre-enrichment mixt. to tetrathionate broth
as in 967.26B(a). Incubate 24 ± 2 h at 35°.
E. General Instructions
Components and procedures of test kit have been stdzd for
use in 1-2 TEST procedures. Components or procedures other
than those supplied by BioControl Systems, Inc., may yield
unsatisfactory results, and should be pretested.
F. Immunodiffusion Detection
(a) Test unit preparation. — Each test unit has 2 chambers:
inoculation chamber and motility chamber (Fig. 989.13). Each
step of prepn sequence can be performed on individual unit or
multiple units as needed. Sample nos. can be recorded on lower
portion of motility chamber but must NOT interfere with read-
ing of results. Alternatively, sample nos. may be recorded on
flat surface of white cap. When cap is replaced, it must be
screwed on tightly.
(1 ) Position unit with black cap UP, and remove black cap.
Add 1 drop of iodine-iodide soln to inoculation chamber, and
replace black cap. Gently shake unit to mix and resuspend en-
richment ingredients.
(2) Position unit with white cap UP, and remove white cap.
Snip off tip of gel-former plug with scissors and discard tip.
Cut should be made at point where tip meets base of plug. If
tip of gel -former plug is not removed, antibody soln will be
displaced from gel void when white cap is replaced.
(3) Add 1 drop of antibody prepn to gel void in motility
chamber. Replace white cap. Antibody prepn should fill ca 2 / 3
of gel void. This can be detd by observing blue antibody soln
in gel void.
(4) Position unit with black cap UP, and remove black cap.
Remove chamber plug from inoculation chamber with sterile
forceps and discard plug. Do not replace black cap until unit
is inoculated. If chamber plug is not removed, bacteria will be
unable to move from inoculation chamber to motility chamber.
492
Microbiological Methods
AOAC Official Methods of Analysis (1990)
(b) Inoculation. — Prior to inoculation, be sure that enrich-
ment broth contg sample is well mixed. Use pipet to transfer
0.1 mL enriched sample into inoculation chamber. Replace
black cap.
(c) Incubation. — Place inoculated unit in incubator with white
cap UP. Incubate unit in shipper/incubation tray at 35° for
min. of 8 h.
(d) Reading positive results. — After 8 h incubation, unit may
be inspected for pos. results: With white cap UP, hold unit
next to strong light. Desktop fluorescent light is recommended
for reading test results. Carefully observe motility chamber gel
by rotating unit back and forth thru various angles in front of
light source.
Pos. test is indicated by presence of white band that is U-
shaped or meniscus-shaped. Band, which forms as motile Sal-
monella are immobilized by antibodies that have diffused into
gel, is seen in upper half of motility chamber gel.
Pos. test indicates that sample contains Salmonella. Pos. test
results should be confirmed by std culture methods outlined in
Confirmation of Positive Samples.
Pos. unit can be stored up to 1 week at refrigerator temp.
(4-8°).
(e) Reading negative results. — If no band is seen after ini-
tial 8 h incubation, reincubate units for min. of 6 h but not
more than 12 h. After this incubation period, read units as
described in (d), Reading positive results. Units that show no
band after this second incubation indicate neg. test results. Neg.
units that were incubated at least 14 h require no addnl in-
cubation. Neg. test results indicate that sample does not con-
tain levels of motile Salmonella detectable by immunodiffu-
sion test.
Neg. units show uniform turbidity thruout motility chamber
as result of movement of motile bacteria in gel. However, after
initial 8 h incubation, movement of bacteria thru gel may not
be complete.
G. Confirmation of Positive Sampies
Presence of band of cells indicates that Salmonella may be
present in sample. Perform cultural confirmation by using 3
mm loop to obtain inoculum from tetrathionate-brilliant green-
serine broth in inoculation chamber and streaking HE, XLD,
and BS plates.
For raw or highly contaminated products, transfer 0.1 mL
of the tetrathionate-brilliant green- serine broth to test tube contg
10 mL tetrathionate-brilliant green broth. Incubate 18-24 h in
43 ± 0.5° H 2 bath, and then streak into HE, XLD, and BS
plates. Identify typical or suspicious colonies from selective
plates as in 967.26C, 967.27, 967.28.
Ref.: JAOAC72, 303(1989).
VIBRIO
988.20 Vibrio cholerae in Oysters
Elevated Temperature Enrichment Method
First Action 1988
A. Principle
Recovery of V. cholerae is based on selection of typical col-
onies on isolation agar. Although V. cholerae grows well at
35°, many other species of competing bacteria also proliferate
in enrichment broth when incubated at 35°. Some species of
competing microflora mimic colonial appearance of V. chol-
erae on isolation medium. Subsequent selection of these mim-
icking colonies decreases probability of recovering any V.
cholerae colonies that may be present, and increases labor and
materials needed for analysis. Ability of almost all strains of
V. cholerae to grow at 42° distinguishes them from many other
bacterial species associated with oysters and results in higher
confirmation rate of suspects as V. cholerae.
B. Culture Media and Reagents
(a) AP broth. — Suspend 10.0 g peptone and 10.0 g NaCl
in 1 L H 2 and mix thoroly. Adjust pH so that value after
sterilization is 8.5 ± 0.2. Dispense portions into 500 mL flasks
so that final vol. after autoclaving 10 min at 121° is 225 mL.
(b) TCBS agar. — Suspend 5.0 g yeast extract, 10.0 g pro-
teose peptone No. 3, 10.0 g Na citrate, 10.0 g Na 2 S 2 3 , 8.0
g oxgall, 20.0 g sucrose, 10.0 g NaCl, 1.0 g ferric citrate,
0.04 g bromthymol blue, 0.04 g thymol blue, and 15 g agar
in 1 L H 2 and mix thoroly. Heat with frequent agitation until
medium just boils, 1-2 min. Cool in H 2 bath and pour 20
mL portions into 15 X 100 mm petri dishes. Let dry ca 2 h
with covers partially removed; then close plates. Final pH, 8.6
± 0.2. Do not autoclave. Do not use wet plates.
(c) TjNj agar. — Suspend 10.0 g tryptone or typticase, 10.0
g NaCl, and 20.0 g agar in 1 L H 2 and mix thoroly. Heat
with frequent agitation until medium boils. Dispense into 16
X 125 mm screw-cap tubes (if tubed medium is required). Au-
toclave 15 min at 121°, Slant tubes until cool or let medium
cool to 50° and pour into 15 x 100 mm petri dishes. Let dry
2 h with plates covered. Do not use wet plates. Final pH, 7.2
± 0.2.
(d) Tryptone broth. — Suspend 10.0 g tryptone or trypticase
in 1 L H 2 and mix thoroly. Dispense 5 mL portions into 16
X 125 mm or 16 X 150 mm test tubes. Autoclave 15 min at
121°. Final pH, 6.9 ± 0.2.
(e) Kligler iron agar (KIA). — Suspend 3.0 g beef extract,
3.0 g yeast extract, 15.0 g peptone 5.0 g- proteose peptone,
10.0 g lactose, 1.0 g dextrose, 0.2 g FeS0 4 , 5.0 g NaCl, 0.3
g Na 2 S 2 3 , 12.0 g agar, and 0.024 g phenol red in 1 L H 2 0,
mix thoroly, and heat with occasional agitation. Boil ca 1 min
until ingredients dissolve. Fill 13 x 100 mm screw-cap tubes
l /3 full and cap to maintain aerobic conditions during use. Au-
toclave 15 min at 121°. Before medium solidifies, place tubes
in slanted position so that deep butts (ca 3 cm) and adequate
slants (ca 5 cm) are formed on solidification. Final pH, 7.4 ±
0.2.
(f) Hugh-Leifson glucose broth (HLGB). — Suspend 2.0 g
peptone, 0.5 g yeast extract, 30.0 g NaCl, 10.0 g dextrose,
0.015 g bromcresol purple, and 3.0 g agar in 1 L H 2 0, mix
thoroly, and heat with agitation. Boil ca 1 min until ingredients
are dissolved. Final pH, 7.4 ± 0.2. Fill 13 x 100 mm screw-
cap test tubes V3 full and cap. Autoclave 15 min at 121°. After
inoculation cover with ca 1 mL sterile mineral oil to test for
fermentation of dextrose.
(g) Purple carbohydrate broth. — Suspend 10.0 g proteose
peptone No. 3, 1.0 g beef extract, 5.0 g NaCl, and 0.015 g
bromcresol purple in 1 L H 2 and heat with gentle agitation
until dissolved. Dissolve 10.0 g inositol or 10.0 g mannitol in
basal broth. Dispense 2.5 mL portions into 13 X 100 mm test
tubes. Autoclave 10 min at 121°. Final pH, 6.8 ± 0.2. j
(h) Decarboxylase test media (Moeller). — Suspend 5.0 g
peptone, 5.0 g beef extract, 0.5 g dextrose, 0.01 g bromcresol
purple, 0.005 g cresol red, and 0.005 g pyridoxal in 1 L H 2
and heat with gentle agitation until dissolved. Dissolve 10.0
g L-lysine-2HCl, 10.0 g L-arginineHCl, or 10.0 g [.-orni-
thine -2HQ in basal broth. Use 1 portion of basal medium,
without adding any amino acid, as control. Dispense 3-4 mL
portions into 13 x 100 mm screw-cap tubes. Cap loosely and
autoclave 10 min at 121°. Screw caps on tightly for storage.
After inoculation cover with ca 1 mL sterile mineral oil. Final
pH, 6.0 ± 0.2.
AOAC Official Methods of Analysis (1990)
Vibrio
493
C. Diagnostic Reagents
(a) Oxidase test soln. — Dissolve 1.0 g N,N,N' yN'-tctm-
methyl-/?-phenylenediamine-2HCl in 100 mL H 2 0. Store <7
days in dark glass bottle in refrigerator. Do not autoclave.
(b) String test soln. — Dissolve 0.5 g Na desoxycholate in
100 mL H 2 0. Store tightly capped in refrigerator. Do not au-
toclave.
(c) V. cholerae polyvalent (O) antiserum, — Contains ag-
glutinins for Inaba and Ogawa (O) antigens (Difco, or equiv.).
Rehydrate with 5.0 mL sterile physiological saline soln (e).
Store refrigerated.
(d) V. cholerae individual somatic (O) antisera. — For In-
aba and Ogawa (O) groups (Difco, or equiv.). Rehydrate and
store as described in (c).
(e) Sterile physiological saline soln. — Dissolve 8.5 g NaCl
in 1 L H 3 and autoclave 15 min at 121°.
(f) NaOH soln, IN. — Dissolve 42.11 g 95% reagent grade
NaOH in sterile H 2 and dil. to 1 L.
(g) HCl soln, IN.—DW. 89 mL HC1 to 1 L with sterile H 2 0.
(h) Sterile mineral oil. — Autoclave 500 mL mineral oil in
1 L flask for 30 min at 121°.
(i) Bromcresol purple soln, 0.2%. — Dissolve 0.2 g brom-
cresol purple in sterile H 2 and dil. to 100 mL.
D. Apparatus
(a) Incubator. — Air, 35 ± 2°.
(b) H 2 bath.— Covered, 42 ± 0.2°.
(c) High-speed blender. — 2 speed, with high-speed opera-
tion at 18 000-21 000 rpm, and 1 L glass or metal blender
jars with covers. Use 1 jar for each test sample.
(d) Sterile equipment. — (I) Flasks or jars, 500 mL capac-
ity. (2) Knives and spoons for opening and manipulating oys-
ters. (3) Petri dishes. 15 x 100 mm. (4) Pipets, 1.0 and 10.0
mL with 0.1 mL graduations. (5) Inoculating needles and loops,
ca 3 mm. (6) Culture tubes, 13 x 100 mm, 16 x 125 mm,
and tube racks. (7) Wooden applicator sticks.
(e) Balance. — 2000 ± 0.1 g capacity.
V. cholerae Recovery
E. Preparation of Test Sample
Aseptically remove oyster meats and liquor from ca 12 shell
stock oysters or 12 shucked oysters from container. Aseptic-
ally weigh ca 200 g oyster meat and liquor into sterile empty
blender jar. Blend at high speed 1 min. Aseptically weigh 25
g portions into 500 mL flasks contg 225 mL AP broth. Cover
flask with sterile Al foil. Swirl mixt. 25 times clockwise and
25 times counterclockwise to suspend oyster homogenate. In-
cubate 6-8 h at 42 ± 0.2° in H 2 bath.
F. Isolation
Gently remove flasks from H 2 bath. Streak 3 mm loopful
of surface or pellicle growth from incubated AP broth on TCBS
agar plate. Incubate plates 18-24 h at 35°. Typical V. cholerae
colonies on TCBS agar appear large, smooth, yellow, and
slightly flattened with opaque centers and translucent peri-
pheries. Colonies of V. mimicus, which is closely related to
V. cholerae, appear as smooth, green, slightly flattened col-
onies.
G. Treatment of Typical or Suspicious Colonies
Inoculation ofT } Nj agar. — Pick with needle 2-5 suspicious
colonies from TCBS agar plate. Streak to TjNj agar and in-
cubate 18-24 h at 35°.
Initial screening reactions. — Scrape agar surface with ster-
ile wooden applicator stick and touch to filter paper impreg-
nated with oxidase reagent.
Oxidase test. — V. cholerae cultures are oxidase pos. and
should produce dark purple spot within 1 min.
String test. — Emulsify oxidase pos. cultures in drop of 0.5%
Na desoxycholate by stirring with same wooden applicator stick
used previously. Within 1 min, V cholerae cultures form mu-
coid mass, which strings (string test) when stick is lifted 2-3
cm from slide. Treat oxidase and string test pos. cultures as
presumptive V. cholerae and submit them to further exami-
nation.
Inoculation ofKligler iron agar (KIA) and tryptone broth. —
Inoculate KIA slant with each suspect colony by streaking slant
and stabbing butt with inoculating needle. After inoculating
KIA with needle, do not obtain more inoculum from colony
and do not heat needle, but directly inoculate tryptone broth.
Incubate KIA and tryptone broth overnight at 35°. Cap tubes
lightly to maintain aerobic conditions while incubating slants
to prevent excessive H 2 S production.
KIA. — V. cholerae cultures typically have alk. (red) slant
and acid (yellow) butt, without H 2 S (blackening of agar) or
gas (cracking or lifting of agar). Do not eliminate KIA culture
as V. cholerae solely on basis of acid slant.
Tryptone broth. — V. cholerae cultures typically produce
growth in tryptone broth without added NaCl. Discard only
apparent non-V. cholerae cultures. Test retained presumptive
pos. KIA and tryptone cultures to det. if they are V. cholerae.
Biochem. reactions characteristic of V. cholerae are summa-
rized in Table 988.20.
V. cholerae Identification
H. Identification Tests
Pure 18-24 h T^N, agar cultures are required for inoculation
of biochem. media. Select isolated colony and transfer with
needle to each biochem. medium without obtaining more in-
oculum or heating needle.
Dextrose fermentation. — After inoculation, cover with ca 1
mL sterile mineral oil and incubate overnight at 35°. V. chol-
erae gives pos. test, shown by acid reaction (yellow). Discard
all cultures that give neg. test.
Acid production from mannitol and inositol. — Incubate at
35° and read daily up to 4 days. Pos. tests are shown by acid
production (yellow). V. cholerae gives pos. mannitol and neg.
inositol test. Do not eliminate culture as V. cholerae solely on
neg. mannitol test.
Decarboxylase broth. — After inoculation cover with ca 1
mL sterile mineral oil and incubate at 35°. Read daily up to 4
days. Pos. test is shown by purple alk. reaction thruout broth
(final color is slightly darker than original purple of medium).
Sometimes tubes that become yellow after 8-12 h incubation
change to purple later. Neg. test is permanently yellow thruout
broth and is seen with decarboxvlase control tube without added
Table 988.20
Biochemical Reactions of V. cholerae
Test or
V
cholerae
substrate
Positive ( + )
Negative ( - )
reaction
H 2 S (KIA)
blackening
no blackening
-
Gas (KIA)
lifting or
no lifting or
-
cracking
cracking
Tryptone
visible growth
no visible growth
+
HLGB
yellow
purple
+
Mannitol
yellow
purple
+
Inositol
yellow
purple
-
Decarboxylase I
Droth:
Lysine
purple
yellow
+-
Arginine
purple
yellow
-
Ornithine
purple
yellow
+
494
Microbiological Methods
AOAC Official Methods of Analysis (1990)
amino acid. If medium appears to be discolored (neither purple
nor yellow), add several drops of 0.2% bromcresol dye. V.
cholerae gives pos. (purple) reaction in lysine and ornithine
and neg. (yellow) reaction in arginine.
Serological Tests for V. cholerae
Reconstitute antisera with 5.0 mL sterile 0.85% saline and
refrigerate. Pretest all antisera with known test cultures to en-
sure reliability of results with unknown cultures. Caution:
Handle viable cultures carefully to prevent contaminating en-
vironment. Use pure 18-24 h T,N| cultures for all serological
tests. Perform serological test only on cultures that give biochem.
reactions typical of V. cholerae.
I. Polyvalent Somatic O Group 1 Slide or Plate Test
Use wax pencil to mark off 2 sections ca 1 X 2 cm on inside
of glass or plastic petri dish. Place 1 drop of 0.85% saline soln
to one section and 1 drop of V. cholerae polyvalent somatic
(O) antisera to other section. With sterile wooden applicator
stick or inoculating loop or needle, emulsify culture in saline
soln for one section and repeat for other section contg anti-
serum. Tilt mixt. in both sections back and forth 1 min and
observe against dark background. Any degree of agglutination
is pos. reaction.
Classify polyvalent somatic (O) group 1 test as:
Positive. — Agglutination in culture-saline-serum mixt.
Negative. — No agglutination in culture-saline-serum mixt.
Nonspecific. — Both mixts agglutinate.
J. Determination of Individual Somatic O Group 1 Serotypes
Test only somatic O group 1 pos. cultures in individual O
group 1 antisera. Perform serological somatic O group 1 test
on culture as above, by using Inaba and Ogawa antiserum in-
stead of V. cholerae polyvalent somatic O group 1 antiserum.
Classify individual somatic O group 1 test as:
Inaba positive, — Agglutination in culture-saline-Inaba anti-
serum mixt. and no agglutination in culture-saline or in cul-
ture-saline-Ogawa antiserum mixt.
Ogawa positive. — Agglutination in culture-saline-Ogawa
antiserum mixt. and no agglutination in culture -saline or in
culture-saline-Inaba antiserum mixt.
Hikojima positive. — Agglutination in both culture-saline-In-
aba antiserum mixt. and culture-saline-Ogawa antiserum mixt.
but no agglutination in culture-saline mixt.
Negative. — No agglutination in culture-saline-Inaba anti-
serum mixt., culture-saline-Ogawa antiserum mixt., or in cul-
ture-saline mixt. This pattern indicates faulty individual so-
matic O group 1 antisera or presence of non-O group 1 antisera
in polyvalent somatic O group 1 antiserum.
Nonspecific. — All mixts agglutinate.
Ref.: JAOAC71, 584(1988).
MISCELLANEOUS
975.56 Virus in Beef (Ground)
Microbiological Method
First Action 1975
Final Action 1989
A. Media and Reagents
(a) Diethylaminoethyl (DEAE) dextran sulfate soln. — Add
1 g DEAE dextran sulfate, 2 x 10 6 MW (Pharmacia Fine
Chemicals, Inc., 800 Centennial Ave, Piscataway, NJ 08854),
to H 2 0, dil. to 100 mL, mix on mag. stirrer, and filter thru
0.22 jxm filter.
(b) Magnesium chloride soln. — Add 50.75 g Mg0 2 .6H 2
to H 2 0, dil. to 100 mL, mix on mag. stirrer, and filter thru
0.22 pmi filter.
(c) Neutral red soln. — Add 1 g neutral red to 1 L H 2 0, mix
overnight on mag. stirrer, autoclave 15 min at 12.1°, and dis-
pense into 100 mL bottles for storage at 10°.
(d) Sodium bicarbonate soln.—ptt 8.0. Add 75 g NaHC0 3
to H 2 0, dil. to 1 L, and filter thru 0.22 fxm filter.
(e) Tissue culture. —Propagate Vero monkey kidney cell
cultures (ATCC CCL 81) in 6 oz (45 sq cm) prescription bot-
tles contg growth medium, (f). After cell sheets are confluent,
ca 7 days, decant medium, add 10 mL 0.02% Na 4 EDTA in
phosphate buffered saline soln, 975.54C(a), and shake. When
cells resuspend, ca 20 min, pour suspension into centrf. tube,
centrf. 15 min at 700 rpm in International PR-2, rotor 259,
and decant supernate. Add 146 mL growth medium to cell
pellet, mix, and distribute into 8 prescription bottles. Repeat
propagation until enough cultures are prepd to perform anal-
ysis.
(f) Growth medium. — To Leibovitz medium (L-15) (Grand
Island Biological Co. ,3175 Staley Rd, Grand Island, NY 14072)
add equal vol. Eagle's minimum essential medium (MEM) with
Hank's salt (Grand Island Biological Co.). Add 10% fetal bo-
vine serum (flow Laboratories). To final mixt. add 10 mL of
7.5% NaHC0 3 soln, (d). Medium will maintain cells 15 days
without having to be changed.
(g) Virus.— Poliovirus 1, Chat, attenuated (ATCC VR-192).
Passage in Vero cell cultures. Prep, virus pool and titer. Dil.
pool to provide 10-50 plaque forming units (pfu)/g.
(h) High antibiotic minimum essential medium (HA-
MEM). — Prep. MEM with nonessential amino acids in Hank's
salt soln contg in each L: 20 mL fetal bovine serum, 5.0 mL
MgCl 2 soln, (b), 10 mL DEAE dextran sulfate soln, (a), 4.643
g K penicillin G, 5.0 g streptomycin sulfate, 0.25 g tetracy-
cline. HCl, and 5.0 mg amphotericin B. Adjust to pH 8.5 with
IN NaOH for elution of virus and to prevent coagulation of
sample slurry.
(i) Agar medium. — Mix 400 mL "2x" MEM (filtered thru
0.22 jxm filter), 20 mL fetal bovine serum, 30 mL NaHC0 3
soln, (d), 15 mL neutral red soln, (c), 10 mL DEAE dextran
sulfate soln, (a), 10 mL MgCl 2 soln, (b), 2 mL amphotericin
B soln (10 |xg/mL), 2 mL tetracycline. HCl soln (50 u,g/mL),
5 mL K penicillin G soln (1435 units/mL), and 6 mL strep-
tomycin sulfate soln (1 mg/mL).
(j) Agar overlay medium. — Add 9.5 g Oxoid Ion agar No.
2 or 12 g Difco purified agar to H 2 0, dil. to 490 mL, mix on
mag. stirrer, autoclave 15 min at 121°, and temper in 47° H 2
bath. Add tempered agar to 500 mL agar medium, (i), and
temper in 43° H 2 bath. Add 10 mL canned sterile milk (Real-
fresh, Inc., PO Box 1551, Visalia, CA 93277) just before use.
B. Preparation of Sample
Place 100 g sample in plastic Whirl-Pac bag (Fisher Sci-
entific Co.) and add 200 mL HAMEM, (h). Shake vigorously
by hand, adjust pH of slurry to 8.5, and let stand 1 hr at room
temp, shaking vigorously 1 min every 20 min. Readjust pH to
8.5 and pour thru funnel contg 5 g Pyrex glass wool pretreated
with HAMEM. Let filter 1 hr (ca 180 mL filtrate is obtained)
and compress glass wool and slurry with wooden tongue de-
pressor to express remaining liq.
C. Assay
inoculate 1 mL filtrate into each of 10 bottles of Vero cell
monolayers, (e), rotating bottles to obtain even distribution of
inoculum. Incubate 1 hr at 36°. Return bottles to room temp.
AOAC Official Methods of Analysis (1990)
Viruses
495
Dispense 18 mL agar overlay medium, (j), into each bottle
against inside surface away from cell sheet. Cap bottles and
turn so overlay gently floods cell surface. Let solidify at room
temp. 30 min with bottles covered to exclude light. Turn bot-
tles so that overlay side is up, and incubate in dark at 36°.
Remove bottles daily from incubator, and count and mark
plaques until no new plaques appear in 48 hr. Discard after
14 days.
Plaque forming units (pfu)/100 g sample = (Av. plaque
count/bottle) X (total vol. filtrate/mL filtrate inoculated per
bottle).
Ref.: J AOAC 58, 576(1975).
985.43 Poliovirus 1 in Oysters
Microbiological Method
First Action 1985
Final Action 1989
A. Apparatus
(a) Tissue culture flasks. — Polystyrene, 1 50 sq. cm (Corn-
ing Glass Works, or equiv.).
(b) Specimen containers. — 220 mL, polyethylene, dispos-
able (Becton-Dickinson Labware, 2 Ridgewater Ln, Lincoln
Park, NJ 07035, or equiv.).
(c) Blender. — Waring, or equiv.
(d) Funnel.— FT 100 polypropylene (No. 4252-0100, Nalge
Co, 75 Panorama Creek Dr, PO Box 20365, Rochester, NY
14602, or equiv.).
(e) Centrifuge bottle. — 250 mL, linear, polyethylene (Nalge
No. 3121-0250, or equiv.).
(f) Refrigerated centrifuge. — Sorvall RC 5B (Ivan Sorvall,
Inc., Norwalk, CT 06852, or equiv.).
(g) Shaker. — Wrist action (Burrell Corp., or equiv.).
B. Media
(Use double-distd H 2 for prepn of media and reagents.)
(a) MEMH. — Eagle's min. essential medium with Hanks'
balanced salts (Gibco Laboratories, Life Technologies, Inc.,
3175 Staley Rd, PO Box 68, Grand Island, NY 14072-0122,
or equiv.).
(b) Plaque assay agar. ^ Add 12 g purified agar (Difco, or
equiv.) to 500 mL H 2 autoclave 15 min at 121°, and temper
fluid 30 min at 47°. ""
(c) Plaque assay medium. — Add 10 mL 50% MgCl 2 .6H 2 0,
10 mL 1% DEAE, 15 mL 7.5% NaHC0 3 , 15 mL 0.1% neut.
red, 30 mL 10% (w/v) nonfat dry milk autoclaved 10 min,
and 1 mL (50 mg) gentamicin sulfate to 420 mL double strength
MEMH. (All concns w/v with H 2 0). Bring to 36° in H 2 bath
and place in 36° incubator until use.
C. Reagents
(a) Freon IF. — (DuPont, Inc., or equiv.).
(b) Antifoam. — Antifoam C (Dow Corning, or equiv.)
(c) Nonfat dry milk. — Quality equiv. to Carnation Co., Los
Angeles, CA, product.
(d) Cat Floc.—\Q% (w/v) (Calgon Corp., PO Box 1346,
Ellwood, PA 15230, or equiv.).
(e) DEAE. — 1% (w/v) diethylaminoethyl dextran, MW —
5 x 10 5 (Pharmacia Fine Chemicals, Inc., 800 Centennial Ave,
Piscataway, NJ 08854 or equiv.).
D. Preparation of Celts
Buffalo African Green Monkey cell line (BGM). — (BGM cell
line is available from several commercial sources.). Incubate
cells at 36° in planting medium composed of equal vols of
Leibovitz L-15 medium and MEMH supplemented with 10%
fetal bovine serum plus 100 U penicillin G, 100 (xg strepto-
mycin sulfate, and 50 p,g gentamicin /mL. Treat confluent cul-
tures with 0.02% tetrasodium ethyl enediamine tetraacetate in
phosphate-buffered saline, split 1 to 10, and passage weekly
in tissue culture flasks.
E. Preparation of Virus
Poliovirus 7. —Chat strain (ATTCC VR-192). Propagate in
monolayers of BGM cells and harvest after observing 4 + cy-
topathic effect. Filter culture fluid contg virus thru 0.22 jxm
porosity membrane. Shake filtrate vigorously with equal vol.
Freon TF, and centrf. 30 min at 4000 X g at 10°. Refilter
supernate.
F. Sample Preparation
For each 100 g sample, select ca 10 std size oysters, shuck,
and pour liquor and meat into specimen container. Adjust liq-
uor and meat in container to 100 g.
Pour 100 g oyster sample into 1 L blender, and pi pet 1 mL
antifoam onto oysters. Rinse specimen container with 150 mL
37° H 2 0, and pour rinse into blender. Pipet 2.5 mL IN HC1
into mixt. and blend contents 20 s at 18 500 rpm. Adjust ho-
mogenate, using 1 s blending mixes, to pH 4.8 ±0.1 by add-
ing 1 N HC1 or 1 N NaOH as needed. Pour homogenate thru
polypropylene funnel into 250 mL linear polyethylene centrif.
bottle. Cap centrif. bottles tightly to prevent leakage during
various assay procedures. Centrf. 10 min at 5000 x g without
refrigeration in GSA rotor, using refrigerated centrif. Pour off
supernate and discard.
G. Elution of Virus from Oyster Tissue
Add following cold reagents (5°) to pellet (ca 40 g): 50 mL
MEMH without phenol red or NaHC0 1? 10 mL 10% w/v non-
fat dry milk, 2 mL 50% MgCl 2 .6H 2 0, 50 mL Freon TF, and
3 mL IN NaOH. Vigorously shake mixt. horizontally 5 min
on wrist-action shaker, adjust pH to 9.1 ± 0.1, and centrif.
mixt. 20 min at 5000 x g at 10°.
H. Concentration of Virus by Precipitation
Pipet supernate (ca 80 mL) into 250 mL centrf. bottle, tak-
ing care not to harvest Freon TF, which settles below super-
nate. Discard sediment and Freon TF. Add enough 37° FLO
to supernate to bring iiq. level to shoulder of top of bottle.
Form floe (15 min) by adjusting pH of Iiq. to 4.8 ± 0.1 with
IN HCl (ca 1 mL). Centrif. sample 10 min at 1500 x g (3000
rpm) without refrigeration. Pour off and discard supernate.
/. Elution of Viruses from Precipitate
Add 4 mL 0.2M Na 2 HP0 4 (ca 23°) and I mL freshly prepd
10% Cat floe to pellet (10 g) and suspend pellet by vortex-
mixing 30 s. Centrf. suspension (pH 7.5 ± 0.2) 20 min at
10 000 x g (8000 rpm) at 10°.
J. Plaque Assay of Eiuate
Pipet supernate (ca 10 mL) onto two 150 sq. cm BGM cell
monolayers previously rinsed with 50 mL MEMH adjusted with
7.5% NaHC0 3 to pH 7.0. Discard pellet. Incubate inoculated
cell monolayers 2 h at 36°. Tilt culture flasks at least twice
during incubation to redistribute eiuate over cell monolayers;
then overlay monolayers as described below.
K. Overlay Procedure
Pour tempered plaque agar into 36° medium, and mix by
inverting flask few times. Pour mixt. (60 mL) into cell mono-
layer flask onto side opposite cell sheet, and rotate flask so
that agar flows over monolayer once and then covers cells. Let
overlay agar solidify at room temp, (ca 23°). Cover flasks with
cloth to exclude light during solidification (ca 15 min). In-
496
Microbiological Methods
AOAC Official Methods of Analysis (1 990)
cubate 7 days at 36°, agar side up,
plaques and count as they appear.
Ref.: JAOAC68, 884(1985).
in dark incubator. Mark
973.68*
SOMATIC CELLS
Somatic Cells in Milk
Optical Somatic Cell Counting (OSCC) Method I
First Action 1973
Final Action 1980
Surplus 1989
See 46.152-46.160, 14th ed.
978.25*
Somatic Cells in Milk
Optical Somatic Cell Counting (OSCC) Method II
First Action 1978
Final Action 1980
Surplus 1989
See 46.161-46.170, 14th ed.
978.26 Somatic Cells in Milk
Optical Somatic Cell Counting (OSCC) Method ill
First Action 1978
Final Action 1979
A. Principle
Fresh or preserved milk samples are automatically sampled
at 40°, mixed with buffer and dye, and stirred. Portion of mixt.
is transferred to rotating disk which serves as object plane for
microscope, Xe arc lamp excites somatic cell nuclei-dye com-
plex to emit fluorescent light, and energy emitted by each nu-
cleus is measured as elec. pulse.
B. Apparatus
Optical somatic cell counter. — Fossomatic (manufactured
by Foss Food Technology Corp.), consisting of heating coils,
rotating table, stirrer, syringes for delivering buffer and dye,
rotating disk, microscope equipped to detect fluorescence, and
totalizing circuit and printer (see Fig. 978.26).
C. Reagents
(a) Ethidium bromide dye soln. — (/) Stock soln. — 0.1%.
Dissolve 1.00 g ethidium bromide (Aldrich Chemical Co., Inc.,
or equiv.) in 1 L H 2 by heating to 40-50° and mixing thoroly.
Stock soln is stable 60 days in light-proof, air-tight bottle. (2)
Working soln. — 0.002%. Dil. 20 mL dye stock soln to 1 L
with KH phthalate buffer soln, (c), and mix thoroly.
'(b) Rinsing liquid.— (1) Stock soln.—\% Triton X-100
(Rohm & Haas Co., or equiv.). Dissolve 10 mL Triton X-100
in 1 L H 2 0. Stock soln is stable 25 days in air-tight container.
(2) Working soln. — Add 10 mL stock soln to 25 mL NH 4 OH
(1 + 3), dil. to 10 L with H 2 0, and mix thoroly.
(c) Potassium hydrogen phthalate buffer soln. — 0.025M.
Dissolve 51.0 g KH phthalate and 13.75 g KOH in 10 L H 2
by heating to 50° and mixing thoroly. Add 10 mL 1% Triton
X-100, (b)(7), and again mix thoroly. Store <7 days in air-
tight container.
D. Analytical System
Two fl oz (60 mL) milk sample is heated to 40°, placed on
self- feeding rack, and stirred to ensure even cell distribution
just before 200 (jlL milk is withdrawn. Sample is combined
with 1.800 mL 60° buffer soln and 2.000 mL 60° dye soln.
Final mixt. is stirred continuously until 20 \xL is spread 10 u,m
deep on edge of rotating disk, 0.5 mm wide along effective
length of 3500 mm. Field is viewed with 15 x microscope ob-
jective. Cell-dye complex is excited by filtered blue light (400-
570 nm) from Xe lamp to emit red fluorescence, and filtered
FIG. 978.26— Optical somatic cell counter flow diagram (Fossomatic) (Method Ml)
AOAC Official Methods of Analysis (1 990)
Somatic Cells
497
fluorescence (590-700 nm) over background (removed by dis-
criminator) is sensed by photomultiplier. Each pulse is trans-
formed and reading of total cells /20 |xL is digitized on display
as well as on printer. Rinsing liq. is used to flush system be-
tween milk samples to ensure no carryover effect of sample.
Somatic cells/mL milk = No. pulses x 1000
E. Standardization
Perform direct somatic cell counts (DMSCC).on 3 std milk
samples within range 300,000-2,000,000 cells/mL as in 973.68
or 978.25. (Before analysis, obtain 3 subsamples of each std
to avoid excessive reheating.) To arrive at optimum discrim-
inator setting, compare stds over >5 discriminator settings
having increments of 0.25-0.5 between settings. Choose ini-
tial setting near previous operating point and additional set-
tings to provide > 1 set of readings above and > 1 below ap-
parent optimum. Optimum is setting at which deviations of
Fossomatic readings from those of stds are minimal, with 1 of
opposite sign from rest. Check instrument every 700-800 sam-
ples or after each 4 hr of operation against std milk samples
preserved with 0.05% K 2 Cr 2 7 .
Ref.: JAOAC 61, 779(1978).
980.33* Somatic Cells in Milk
Membrane Fi Iter-Deoxyribonucleic Acid
(MF-DNA) Method
First Action 1980
Final Action 1981
Surplus 1989
See 46.176-46.180, 14th ed.
SPECIAL REFERENCE
FDA Bacteriological Analytical Manual (BAM) 6th edition
(1984) AOAC, Suite 400, 2200 Wilson Bivd, Arlington, VA
22201-3301.
18. Drugs: Part I
Martin Farskefson, Associate Chapter Editor
Food and Drug Administration
960.53
Drugs
General Directions
A. Extraction with Lighter-Than-Water Solvents
Perform preliminary steps directed in method prior to extn.
Ext. aq. soln in separator with specified vols of solv. (ether,
pet ether, etc.) by shaking >1 min. Let sep. completely, swirl
to remove H 2 droplets, transfer lower aq. layer to second
separator, and decant solv. layer thru pledget of solv. -washed
cotton or glass wool in short-stem funnel inserted in neck of
third separator. Wash mouth of separator with fine stream of
solv. Repeatedly shake aq. soln with addnl portions of solvs
until substance sought is extd, using second and first separa-
tors alternately for shaking, collecting solvs by filtering into
third. If aq. soln is to be further examined, dry cotton pledget
in funnel by drawing air thru stem, and wash with 5 mL H 2
into main aq. ext.
B. Purified Diatomaceous (Siliceous) Earth for Partition
Chromatography
Celite 545, acid- washed (ManvilJe Filtration and Minerals),
or equiv., is usually suitable for column chromatgy. When in-
terfering materials are present, purify as follows: Place pad of
glass wool in base of chromatgc tube >100 mm diam. and add
siliceous earth to ht ca 5 times diam. Add vol. HC1 equal to
ca V3 vol. of earth, and let percolate. Wash with MeOH, using
small vols at first to rinse walls of tube, and then until wash-
ings are neut. to moistened indicator paper. Extrude into shal-
low dishes, heat on steam bath to remove MeOH, and dry at
105° until material is powdery and MeOH-free. Store in tightly
closed containers.
927.09
Sampling of Drugs
Final Action
Methods require analysis of sample portions stated. Use in-
dividual dosage form or fraction of composite, depending on
information needed. Sampling procedure used may affect
interpretation of results. Typical procedures for obtaining ran-
dom samples are described below.
A. Tablets and Pills
(a) Bulk lots. — Remove approx. equal numbers of units from
>10 locations spaced turnout mixed lot, collecting >100 units.
Calc. av. wt/unit, and reduce to fine powder that would com-
pletely pass 60 mesh seive. Mix thoroly.
(b) Containers. — Remove equal numbers of units from one
or more containers representing same lot to achieve >60 units.
Calc. av. wt/unit, and reduce to fine powder that would com-
pletely pass 60 mesh seive. Mix thoroly.
B. Capsules
Follow sampling plan in 927.09A(a) or (b). Weigh counted
capsules and det. gross wt /capsule. Open capsules and quant,
remove contents. If dry contents, reduce to fine powder that
would completely pass 60 mesh seive. Mix thoroly, As nec-
essary, clean capsules (cutting in 2 if necessary) and wash by
agitating with alternate portions of alcohol and ether, with fi-
nal rinse of ether. (Few drops of HO Ac mixed with alcohol
aids cleaning.) Remove ether, deduct wt cleaned, empty cap-
sules from gross wt, and calc av. net contents.
C. Ampuls
Before opening ampuls, dislodge any liq, in neck. Open suf-
ficient number of ampuls to provide required sample vol., and
transfer contents of each with dry hypodermic syringe to dry
stdzd cylinder graduated to contain. Alternatively, contents of
syringe may be discharged into dry, tared weighing vessel, and
vol.. calcd as wt, in g, of fluid delivered divided by density of
injection soln.
D. Vials
(a) Solns. — With successive use of clean, dry hypodermic
syringes of appropriate size, remove indicated number of doses
from vial, delivering each into same stdzd cylinder graduated
to contain. Alternatively, use gravimetric procedure described
in 927.09C (ampuls).
(b) Suspensions. — Follow procedure under (a), except shake
vial vigorously until product is homogeneous (but >15 s), then
remove sample immediately.
E. Ointments
Remove entire contents of sufficient number of weighed tubes
to tared, covered weighing vessel contg stirring rod. Mix pooled
contents to obtain composite, cover vessel, and det. wt of
composite (C). Weigh empty tubes and calc. total wt of oint-
ment removed (O). Use ratio O/C to correct wt of anal, sam-
ples subsequently taken from composite.
Refs.: Banes, "Principles of Regulatory Drug Analysis,"
AOAC, 2200 Wilson Blvd, Arlington, VA 22201
(1966).
Banes, "A Chemist's Guide to Regulatory Drug Anal-
ysis," AOAC, 2200 Wilson Blvd, Arlington, VA 22201
(1974).
JAOAC 10, 99(1927); 56, 508(1973); 67, 357(1984).
SOLVENTS
942.28* Acetone and Alcohols in Drugs
Qualitative Tests
Final Action
Surplus 1972
See 36.006-36.010, 12th ed.
973.69 Acetone and Alcohols in Drugs
Gas Chromatographic Method
First Action 1973
Final Action 1975
(Applicable to liq. prepns contg ethanol with isopropanol or
acetone or individual compds)
498
AOAC Official Methods of Analysis (1990)
Halogenated Drugs 499
A. Reagents and Apparatus
(a) Ethanol std stock soln. — (7) 2% (v/v). — Dil. 5.0 mL
absolute alcohol to 250 mL with H 2 0. (2) 0.2% (v/v).— Dil.
10.0 mL soln (7) to 100 mL with H 2 0.
(b) Isopropanol std stock soln. — 2% (v/v). Dil. 5.0 mL
isopropanol to 250 mL with H 2 0.
(c) Acetone std stock soln. — 2% (v/v). Dil. 5.0 mL acetone
to 250 mL with H 2 0.
(d) Acetonitrile internal std stock soln. — 2% (v/v). Dil. 5.0
mL CH 3 CN to 250 mL with H 2 0.
(e) Gas chromato graph. — With 1 .8 m (6') x 4 mm id glass
column, packed with 80-100 mesh Porapak Q (Waters As-
sociates, Inc.) and H flame ionization detector. Approx. op-
erating conditions: temps (°) — column 135, detector 155, in-
jection port 165; N carrier gas flow rate 120 mL/min. CH 3 CN
peak should elute in 5 min. Adjust H and air flow rates and
electrometer sensitivity so that 5 |xL 0.2% EtOH std soln gives
50-70% scale deflection.
B. Preparation of GC Column
Carefully plug column exit with small pad of glass wool.
Apply vac. to exit and slowly add packing material thru inlet,
tapping very gently to pack firmly. Pack to within 1 cm of
area heated by injection port. Plug with glass wool and con-
dition overnight at 235° with slow N stream.
C. Preparation of Sample
(a) Ethanol. — Prep, soln contg ca 2% (v/v) EtOH by step-
wise diln with H 2 0. Proceed as in 973. 69D.
(b) Isopropanol. — Prep, soln contg ca 2% (v/v) isopro-
panol by stepwise diln with H 2 0. Proceed as in 973. 69D.
(c) Acetone. — Prep, soln contg ca 2% (v/v) acetone by
stepwise diln with H 2 0. Proceed as in 973. 69D.
If acetone concn is unknown, prep. 50% diln of product
with H 2 0, prep, acetone std soln, and inject sample and std
as in 973. 69D. To det. amt acetone, adjust product and std
dilns to give comparable peak hts; % internal std added to the
2 solns should be equal to % acetone present in std soln.
D. Determination
Pipet 10 mL sample soln into 100 mL vol. flask. Pipet 10
mL each std stock soln needed into sep. 100 mL vol. flask.
Pipet 10 mL internal std stock soln into each flask and dil. to
vol. with H 2 0.
Inject 5 |xL sample and std solns, each in duplicate, using
10 )jlL syringe. Approx. retention times of peaks relative to
CH 3 CN internal std peak are as follows: EtOH, 0.76; acetone,
1.32; isopropanol, 1.40.
Calc. % EtOH, acetone, or isopropanol in sample as
% C = C x (/////') x (/'//) x /
where C and C" — % component in sample and std, resp., H
= av. sample peak ht or area in sample chromatogram , H' =
av. std peak ht or area in std chromatogram, / and /' = resp.
values for internal std, and/ - sample diln factor,
Ref.: J AOAC 56, 684(1973).
CAS-67-64-1 (acetone)
CAS-64-17-5 (ethyl alcohol)
CAS-67-63-0 (isopropyl alcohol)
928.10* Ether in Drugs
Dichromate Oxidation Method
Final Action
Surplus 1965
(Not applicable in presence of essential oils)
See 32.370-32.374, 10th ed.
953.18* Propylene Glycol in Drugs
Periodate Oxidation Method
Final Action
Surplus 1975
See 36.016-36.017, 12th ed.
929.10* Chloroform
or Carbon Tetrachloride in Drugs
Volhard Titrimetric Method
Final Action
Surplus 1970
See 36.355-36.357, 11th ed.
934.08* Tetrachloroethylene in Drugs
Total Chloride Method
Final Action
Surplus 1970
See 36.434-36.435, 11th ed.
939.15* Trichloroethylene in Drugs
Total Chloride Method
Final Action 1965
Surplus 1970
See 36.437-36.439, 11th ed.
963.30* Chlorinated Hydrocarbons
in Drugs
Infrared Spectrophotometric Method
First Action 1963
Surplus 1982
See 36.018-36.022, 14th ed.
HALOGENATED DRUGS
936.17* Chlorobutanol in Drugs
Gravimetric Method
Final Action
Surplus 1972
See 36.023-36.024, 12th ed.
967.29 Chlorobutanol in Drugs
Gas Chromatographic and Infrared Methods
First Action 1967
Final Action 1968
(Caution: See safety notes on flammable solvents, toxic sol-
vents, carbon disulfide, and carbon tetrachloride.)
A. Reagents
(a) Diatomaceous earth. — See 960. 53B.
(b) Chlorobutanol. — USP. Store over soln satd with both
sugar and salt; product contains 1 / 2 mole H 2 of hydration.
(c) Glass wool. — Fine; washed with CS 2 and dried.
500
Drugs: Part I
AOAC Official Methods of Analysis (1990)
(d) Dichlorodimethylsllane . — Dissolve 5 mL in 100 mL
toluene. (Caution: Dichlorodimethylsllane is toxic. Avoid
contact with skin or eyes. Use effective fume removal device.)
B. Apparatus
(a) Chromatographic tube. — 23 X 400 mm with drip tip
small enough to fit into 10 mL vol. flask, and with close-
fitting tamping rod.
(b) Infrared spectrophotometer. — With matched 1 mm path
length, liq. -filled NaCl cells.
(c) Gas chromato graph. — With 1.8 m (6') x 4 mm glass
column, packed with Carbowax 6000 on 100-110 mesh An-
akrom ABS, H flame ionization detector, and strip chart re-
corder.
C. Preparation of GC Column
Carefully wash inside of column and small amt of glass wool
with d ic hi orodi methyls i lane soln, rinse with MeOH, and dry.
Slowly sprinkle ca 25 g Anakrom ABS into 400 mL beaker
almost filled with CC1 4 . Remove fine particles remaining at
surface with vac. line and trap. Decant solv., oven-dry sup-
port, and transfer 20.0 g to 500 mL filter flask fitted with trap
and stopper. Dissolve 5.0 g polyethylene glycol (Carbowax
6000) in 100 mL toluene, warming if necessary. Add Car-
bowax soln to flask and apply vac. 5 min, swirling occasion-
ally. Return to atm. pressure and let stand 5 min. Transfer
slurry with rapid swirling to buchner fitted with coarse paper.
Maintain reduced pressure on funnel 5 min; then dry coat sup-
port by spreading on smooth surface. Air dry 1 hr. Oven-dry
addnl hr at 90°.
Carefully plug column exit with small pad fine glass wool
and thru-hole septum. Apply vac. to exit port and slowly add
coated support thru injection port, tapping very gently to pack
firmly. Pack to within 1 cm of area heated by flash heater.
Plug with fine glass wool and condition ca 3 days at 200° with
slow N stream.
D. Preparation of Standard Solutions
Dissolve ca 0.5 g chlorobutanol. V2H2O, accurately weighed,
in 1 mL alcohol and transfer to 100 mL vol. flask with 8 mL
alcohol. Dil. to vol. with H 2 0. Using 5 mL aliquots, prep,
duplicate diat. earth columns with trap layers as in 967. 29E.
Prep, and elute columns individually. Calc. mg anhyd. chlo-
robutanol /mL CS 2 = C hyd X 0.9517. (Chlorobutanol is ap-
preciably volatile at room temp.; expose to atm. as little as
possible.)
E. GC Determination
Weigh 3 g diat. earth, add 2 mL IN HO, and mix until
uniform. Transfer to chromatgc tube plugged with small pad
glass wool and tamp moderately tight.
Calc. vol. sample contg ca 25 mg chlorobutanol. Weigh diat.
earth equal to J g/mL sample. Pipet sample into diat. earth,
and mix (ca 1 min) until uniform. Transfer quant, to same
column, dry washing with small amt of dry diat. earth, and
tamp firmly. Pack as few portions as possible, each portion
<5 g diat. earth. Rinse beaker with small portions CS 2 and
transfer to column until sample portion is wet with CS 2 . Let
each portion sink into column before adding next. Add 20 mL
CS 2 to column and collect eluate in 10 mL vol. flask. Rinse
column tip with few drops CS 2 (pipet) when 8-9 mL collects.
Continue to collect eluate to vol., stopper flask, and mix. This
should yield proper concn for either GC or IR detns. (Tightly
stoppered solns of chlorobutanol in CS 2 may be stored over-
night.)
Inject, as below, 6 u,L sample soln and est. concn from std
curve.
F. GC Standard Curve
Operating conditions: temps (°) — column 135, detector 215,
flash heater 230; N flow rate, ca 35 mL/min to elute chlo-
robutanol in ca 6 min; and H flow rate, 30 mL/min.
Adjust electrometer sensitivity so that 12 fxg chlorobutanol
gives ca 50% deflection. Inject 4, 5, 6, 7, 8, and 9 u,L of each
std eluate from 10 |ulL syringe. Read vol. in syringe before and
after injection; take difference as vol. injected. Plot net (A) %
deflection against jig anhyd. chlorobutanol injected.
G. Preparation of Infrared Standard
Record spectrum of each std eluate from 9 to 15 |am, using
quant, instrument settings and CS 2 in ref. beam. Det. av. A
of max. at 12.5-12.6 |xm, using baseline technics with min.
ca 9.5 and ca 14.2 |xm as base. (Av4 is linear from 0.5 to 4.5
mg chlorobutanol /mL CS 2 .)
H. Infrared Determination
Det. A of sample soln at 12.5-12.6 fxm, as above.
mg Chlorobutanol in sample aliquot
= (A A sample eluate/AA std eluate)
X mg chlorobutanol in 10 mL CS 2 std
Ref.: J AOAC 50, 669(1967).
CAS-57-15-8 (chlorobutanol)
931.11 Iodoform Drug Substance
Gravimetric Method
Final Action
A. Reagents
(a) Ammonium thiocyanate std soln. — 0.05JV. Stdze against
0AN AgN0 3 , using equal vol. alcohol and 3 mL FeNH 4 (S0 4 ) 2
soln as indicator.
(b) Ferric ammonium sulfate indicator. — Dissolve 8 g
FeN'H 4 (S0 4 )2.12H 2 in 100 mL H 2 0.
B. Determination
Accurately weigh ca 0.25 g CHI 3 and transfer quant, to 200
mL erlenmeyer. Add 40 mL alcohol, swirl gently until CHI 3
dissolves, filter if necessary, and immediately add 40 mL 0. 17V
AgN0 3 and 10 mL HN0 3 . Swirl gently ca 5 min, let stand at
room temp. 2-3 hr, and then swirl occasionally as aid in floc-
culating the Agl. Titr. excess AgN0 3 with 0.05JV NH 4 SCN,
using 3 mL of the FeNH 4 (S0 4 ) 2 indicator. 1 mL 0. IN AgN0 3
= 0.01312 g CHI 3 . Or: Proceed as in 932.17, last par. begin-
ning "Collect Agl on weighed gooch, ..."
Ref.: JAOAC 14, 370(1931).
CAS-75-47-8 (iodoform)
932.17
iodoform in Ointments
Gravimetric Method
Final Action
Transfer ca 2.5 g sample to tared 50 mL beaker and weigh.
Add 5 mL CHCl 3 , stir gently with glass rod, and transfer bulk
of undissolved ointment and CHCI 3 soln to 250 mL g-s flask.
Add 5 mL CHC1 3 to ointment remaining in beaker and stir until
dissolved. Add soln to flask and finally wash beaker 3 times,
using <5 mL CHC1 3 each time, and add washings to flask.
Or: weigh sample in small, tared glass capsule, drop capsule
with contents into 250 mL g-s flask, and add <20 mL CHC1 3 .
(Use glass capsule only in volumetric detn.) Swirl gently until
all ointment dissolves. Add 40 mL 0. IN ale. AgN0 3 and swirl
AOAC Official Methods of Analysis (1 990)
Inorganic Drugs
501
to wash down any CHI 3 that adheres to sides of flask. Slowly
add 10 mL HNO^ and let stand at room temp, ca 18 hr. Titr.
excess of 0. IN ale. AgN0 3 with 0.05/V NH 4 SCN, 931.11 A(a),
using 3 mL FeNR^SO^ indicator, 931.11A(b), vigorously
shaking mixt. near end of titrn. 1 mLO. W AgN0 3 = 0.01312
gCHI 3 .
For gravimetric detn use ordinary erlenmeyer instead of
g-s flask. Weigh ointment base into 100 mL beaker and add
CHC1 3 . After ointment base dissolves, filter thru gooch, using
suction. Wash beaker and crucible once with alcohol. Wash
crucible several times with CHC1 3 without suction. Collect fil-
trate in erlenmeyer and add 40 mL 0. IN AgN0 3 and 10 mL
HNO3 in small portions. Let mixt. stand 18 hr. Collect Agl
on weighed gooch, using suction. Wash with H 2 and then
with alcohol. Finally, wash repeatedly with CHC1 3 without
suction. Drv gooch and contents at ca 125° to const wt. 1 g
Agl = 0.5590 g CHI3.
Ref : JAOAC 15, 434(1932).
CAS-75-47-8 (iodoform)
932.18
Iodoform on Gauze
Final Action
Weigh, in tared g-s weighing bottle, sample of CH1 3 gauze
contg ca 1 g CHI3. (CHI3 gauze is usually moist and loses wt
rapidly when exposed to air.) Transfer to 150 mL beaker, add
ca 75 mL alcohol, and stir until CH1 3 dissolves. Filter into 200
mL vol. flask, draining ale. soln by pressing on gauze. Wash
with four or five 25 mL portions alcohol, filter washings, and
finally dil. to vol. with alcohol. Pipet 40 mL aliquot into 200
mL erlenmeyer and immediately add 40 mL 0. 17V AgN0 3 and
10 mL HNO3. Proceed as in 932.17, first par., beginning "...
let stand at room temp, ca 18 hr."
Ref.: JAOAC 15, 441(1932).
CAS-75-47-8 (iodoform)
INORGANIC DRUGS
927.10* Arsenic in Iron-Arsenic Tablets
Distillation Method
Final Action
Surplus 1980
See 36.034-36.036, 13th ed.
928.11* Arsenic in Iron Methylarsenate
Distillation Method
Final Action
Surplus 1980
See 36.037, 13th ed.
tings: scan rate, 2 mv/sec; scan direction, " — "; range, 1.5 v;
initial potential, -0.15 v; modulation amplitude, 50 mv; op-
eration mode, differential pulse; current range, 0.05 ma, or as
needed; output offset, as required; display direction, " + "; drop
time, 1 sec; low pass filter, off; selector, off; pushbutton, ini-
tial; recorder: X-axis, 0.1 v/in., Y-axis, 1 v/in.
(b) Cells. — Std cell bottom with satd calomel ref. electrode
(SCE), C rod counter electrode, and dropping Hg indicating
electrode.
(c) Pipet. — 100 |xL Bppendorf pipet, or equiv.
B. Reagents
(a) Supporting electrolyte
. — \M HCl. Add 82 mL HCl to
flask, dil. to vol. with H^O, and
ca 500 mL H 2 in 1 L vol
mix.
(b) Arsenic std soln. — 2.000 mg As/mL. Dissolve 0.2640
g As 2 3 , dried 1 hr at 105°, in ca 25 mL \N NaOH in 100
mL vol. flask, acidify to litmus with \M HCl, and dil. to vol.
with H 2 0.
C. Digestion
Transfer to 100 mL borosilicate beaker accurately measured
vol. of injection, dild if necessary, and contg ca 29 mg Na
cacodylate (ca 10 mg As), and add 1.0 g .Mg(N0 3 )2.6H 2 and
1 mL HNO3. Heat on hot plate at low heat until H 2 is evapd;
then at high heat to dry residue. Complete digestion by placing
beaker in furnace at 450° until no brown fumes are evolved
(30 min). Remove from furnace and let cool to room temp.
Perform blank similarly.
D. Reduction of Arsenic
Add to residue 20 mL 5M HCl (2 H- 3) and swirl to dissolve,
warming on steam bath, if necessary; then add 5 mL 40% HBr
and 0.3 g H2NNH2.H2SO4. Cover beaker with watch glass and
place on steam bath 30 min. Cool to room temp., transfer with
H 2 to 100 mL vol. flask, dil. to vol. with H 2 0, and mix.
E. Determination
Add to polarographic cell 20 mL supporting electrolyte and
bubble N thru soln 5 min; then direct gas to sweep over soln.
Switch selector to "Cell", and allow sufficient time for re-
corder pen to come to rest. Depress "Scan" pushbutton and
record polarogram from —0.15 to —0.9 v. Pipet 2 mL of sam-
ple soln into cell, bubble N thru soln 1 min, direct gas to sweep
over soln, and record polarogram as before, using same in-
strumental settings. Repeat operations on 2 addnl 100 |jlL ali-
quots of std soln. Polarograph blank similarly. Plot std addn
curve as follows: mg std added, 0, 0.2, and 0.4, on X-axis
and first As peak lit which appears at ca —0.37 v against SCE
on Y-axis. Extrapolate linear plot to X-axis to obtain mg As
in aliquot. Correct for blank, if necessary. Stds need not be
polarographed beyond ca —0.60 v, since anal, peak is at ca
-0.37 v against SCE.
Ref.: JAOAC 60, 1015(1977).
CAS-7440-38-2 (arsenic)
977.28 Arsenic in Cacodylate Injections
Differential Pulse Polarographic Method
First Action 1977
Final Action 1979
A. Apparatus
(a) Polarograph. — Capable of effectively scanning —0.15
to —0.9 v in differential pulse mode. Typical instrument set-
926.16* Arsenic in Sodium Cacodylate
Titrimetric Method
Final Action
Surplus 1977
See 36.044, 12th ed.
502
Drugs: Part I
AOAC Official Methods of Analysis (1990)
932.19 Bismuth Compounds in Drugs
Gravimetric Method
Final Action 1965
(Applicable in absence of Pb. Caution: See safety notes on
distillation, nitric acid, and hydrogen sulfide.)
Thoroly mix sample and weigh 0.5 g into 500 mL Kjeldahl
flask. Ignite gently over small flame, using wire gauze under
flask, and increase heat towards end. Let cool, add 15-20 mL
HN0 3 , evap. to dryness, and ignite as before until yellow or
orange Bi 2 3 is formed. Cool residue and dissolve in 10-15
mL warm HNO3, using few mL 3% H 2 2 if residue does not
dissolve readily. Boil off excess H 2 2 and wash into 400 mL
beaker with H 2 0, rinsing flask well. Oil. to ca 200 mL, make
just neut. to litmus with NH 4 OH, and add 5 mL HC1. Ppt with
H 2 S completely.
Transfer ppt to filter paper and wash once with HO (5 +
200) and then several times with H 2 0. Dissolve ppt of Bi 2 S 3
on filter with hot HN0 3 (1 + 2). Small residue of S (and HgS
if Hg salts are present) usually remains. Neutze filtrate with
NH4OH (2 + 3) and ppt with 25 mL 20% (NH 4 ) 2 C0 3 soln.
Cone, to ca 150 mL (by boiling, if desired) and let stand on
steam bath 1-2 hr. Collect ppt in previously ignited, weighed
gooch, wash with small amt of H 2 0, dry, ignite in furnace at
ca 550°, and weigh as Bi 2 3 .
Ref.: J AOAC 15, 422(1932).
972.46 Bismuth Compounds in Drugs
Polarographic Method
First Action 1972
Final Action 1974
A. Apparatus
(a) Polarogmph. — Any voltammetric or polarographic in-
strument with necessary accessories (cells, electrodes, Hg,
capillaries) capable of scanning to — 1.0 v and measuring
0.040 |xa/mm.
(b) Cells, — Microcell with satd calomel or Hg pool ref.
electrode.
B. Reagents
(a) Supporting electrolyte. — 1M HCl. Add 171 mL HC1 to
ca 1 L H 2 in 2 L vol. flask and mix. Cool to room temp.,
dil. to vol. with H 2 0, and mix.
(b) Gelatin maximum suppressor. — 1 mg/mL. Accurately
weigh 100 mg gelatin into small beaker and dissolve in small
amt of H 2 on steam bath. Transfer quant, to 100 mL vol.
flask and dil. to vol. with H 2 0. Prep, fresh daily.
(c) Bismuth std solns. — (I) Stock soln. — 1 mg/mL. Trans-
fer 122.2 mg Bi subcarbonate, equiv. to 100 mg Bi (mg Bi
subcarbonatex0.8182 (factor derived from primary std) - mg
Bi), to 100 mL vol. flask with supporting electrolyte, (a). Dil.
to vol. with same soln. (2) Working soln. — 0.2 mg/mL. Pipet
20.0 mL stock soln into 100 mL vol. flask, add 1 .0 mL gelatin
max. suppressor, (b), and dil. to vol. with supporting electro-
lyte, (a). Mix thoroly.
C. Preparation of Sample
(a) Tablets. — Det. av. wt/tablet. Grind to pass No. 60 sieve.
Quant, transfer amt tablet material contg 10 mg Bi, accurately
weighed, to 50 mL vol. flask with aid of \M HCl, (a). Add
0.5 mL gelatin max. suppressor, (b), and dil. to vol. with \M
HCl. Shake thoroly or use sonic vibrator. Filter thru rapid pa-
per just before polarographic detn.
(b) Magma, emulsions, and injectables . — Mix thoroly to
disperse suspension. Immediately transfer aliquot contg ca 100
mg Bi to 100 mL vol. flask. Rinse inside of pipet with \M
HCl, (a), and dil. to vol. with same soln. Pipet 10 mL into
50 mL vol. flask, add 0.5 mL gelatin max. suppressor, (b),
and dil. to vol. with \M HCL Mix thoroly.
(c) Powder. —Transfer entire contents of vial, or amt ma-
terial equiv. to 200 mg Bi for bulk powders or capsules, to
200 mL vol. flask. Wash vial into flask and dil. sample to vol.
with \M HCl, (a). Proceed as for magma, beginning "Pipet
10 mL . . ."
D. Determination
Transfer soln to cell and bubble N thru soln 10 min. Record
polarogram from to — 1.0 v against satd calomel ref. elec-
trode. Measure ht of diffusion current (/ d ) at half-wave poten-
tial (E l /2), and det. Bi concn by comparing wave hts of sample
soln with those of std soln polarographed immediately before
and after samples. Do all detns at same current sensitivity and
consecutively.
E l / 2 value is qual. identification of Bi.
Calc. concn of Bi as follows:
(a) Tablets.— mg Bi /tablet - 50 x (///') x C x (W/W);
(b) Magma, emulsions, and injectables. — mg Bi/mL = 500
x (///') x (C/V);
(c) Powder.— mg Bi/mg sample = 1000 x (///') x (C/
W);
where / and /' = diffusion currents of sample and std, resp.;
C = mg Bi/mL working soln, (c)(2); W and W = av. tablet
wt and wt sample taken (mg), resp.; and V = mL liq. prepn
taken.
E. Measurement of Diffusion Current
Measure diffusion current (/ d at E^/o) . Draw lines tangent to
tops of residual and limiting currents. Draw third line tangent
to vertical slope. Measure its length, mark off half-way point
at / d /2; then drop perpendicular thru this point and thru ab-
scissa (applied voltage). Diffusion current is perpendicular
portion of this line cutting thru limiting current and residual
current tangent lines.
Ref.: JAOAC 55, 155(1972).
967.30 Calcium and Magnesium in Drugs
Titrimetric Method
First Action 1967
Final Action 1968
(Applicable to pharmaceuticals and vitamin-mineral prepns)
A. Reagents
Use H 2 redistd from glass (preferable) or deionized H 2 0.
(a) Calcium carbonate, — Primary std grade, dried 2 hr at
285°.
(b) Hydroxy naphthol blue. — Ca indicator (Mallinckrodt
Chemical Works No. 5630 in dispenser bottle ready for use,
or equiv.). Store in dark and replace after 1 year.
(c) Calmagite. — Ca + Mg indicator (Mallinckrodt No. 4283
in dispenser bottle ready for use, or equiv.). Store in dark and
replace after 1 year.
(d) Di sodium dihydrogen ethylenediamine tetraacetate
(EDTA) std soln.-0.0lM. Dissolve 3.72 g Na 2 H 2 EDTA.2H 2
(99 + % purity) in H 2 in 1 L vol. flask, dil. to vol., and mix.
Accurately weigh enough CaC0 3 (ca 40 mg) to give ca 40 mL
titrn with 0.01M EDTA and transfer to 400 mL beaker. Add
50 mL H 2 and enough HCl (1 + 3) to dissolve the CaC0 3 .
Dil. to ca 150 mL with H 2 and add 15 mL IN NaOH, dis-
regarding any ppt or turbidity. Add ca 200 mg hydroxy naph-
AOAC Official Methods of Analysis (1990)
Inorganic Drugs
503
thol blue indicator and titr. from pink to deep blue end point,
using mag. stirrer. Add last few ml EDTA soln drop wise.
Molarity EDTA soln - mg CaC0 3 /(mL EDTA x 100.09).
(e) Buffer soln.— pH 10. Dissolve 67.5 g NH 4 C1 in 200 raL
H 2 0, add 570 mL NH 4 OH, and dil. to 1 L.
(f) Potassium hydroxide-potassium cyanide soln, — Dis-
solve 280 g KOH and 66 g KCN in I L H 2 0.
(g) Potassium cyanide soln. — 2%. Dissolve 2 g KCN in 100
mL H 2 0.
B. Apparatus
(a) Titration stand. — Fluorescent illuminated.
(b) Ion exchange column. — Approx. 20 X 600 mm, fitted
with coarse porosity fritted glass disk and Teflon stopcock.
Place 30-40 g moist Amberlite IR-4B resin (anion exchange
resin with high phosphate capacity) from fresh bottle in 600
mL beaker and exhaust with three 250 mL portions 5% Na 2 C0 3
or NaOH. Wash with H 2 until excess base is removed. Treat
resin with three 250 mL portions 5% HCI (3 + 22), mixing
thoroly after each treatment. Rinse with H 2 until color is re-
moved, and transfer with H 2 to column. Column is ready for
use after draining H 2 to top of resin column. (Exchange ca-
pacity for phosphate is ca 1500 mg; therefore, number of ali-
quots can be passed thru column before regeneration is nec-
essary. Rinse column with ca 250 mL H 2 before each use
until eluate is colorless.)
C. Preparation of Sample
Transfer 2 g well mixed sample to 100 mL Pt or porcelain
dish. Ash at <525° until apparently C-free (gray to brown).
Cool, add 20 mL H 2 0, stir with stirring rod, and add 10 mL
HCI cautiously under watch glass. Rinse off watch glass into
dish and evap. to dryness on steam bath. Add 50 mL HCI (1
+ 9), heat on steam bath 15 min, and filter thru quant, paper
into 200 mL vol. flask. Wash paper and dish thoroly with hot
H 2 0. Cool filtrate, dil. to vol., and mix.
D. Determination
Transfer 50 mL aliquot prepd sample to 250 mL beaker and
adjust to pH 3.5 with 10% KOH soln added dropwise, using
pH meter and mag. stirrer. Pass sample thru resin column (col-
umn is in CI form), collecting effluent in 250 mL vol. flask
and adjusting flow rate to 2-3 mL/min. Wash column with
two 50 mL portions H 2 0, passing first portion thru at same
rate as sample soln and second at 6-7 mL/min. Finally, pass
enough H 2 freely thru column to dil. to vol. Mix thoroly.
Pipet two 100 mL aliquots into 400 mL beakers.
Titration J (calcium + magnesium).— Adjust first aliquot to
pH 10 (using pH meter and mag. stirrer) with pH 10 buffer
soln, (ca 5 mL). Add 2 mL 2% KCN soln, and 200 mg Cal-
magite indicator, and titr. immediately with 0.01M EDTA soln
thru red to deep blue end point, using mag. stirrer.
Titration 2 (calcium). — Adjust second aliquot to pH 12.5-
13.0 (using pH meter and mag. stirrer) with KOH-KCN soln
(ca 10 mL). Add 0.100 g ascorbic acid and 200-300 mg hy-
droxy naphthol blue indicator. Titr. immediately with 0.01 M
EDTA soln thru pink to deep blue end point, using mag. stir-
rer.
%Ca = Fxfx 0.4008 x 10 x 100/mg sample
%Mg = (X-y)xFX 0.2431 x 10 x 100/mg sample
where X and Y - mL EDTA soln from titrns I and 2, and F
- molarity EDTA soln/0.01.
Refs.: JAOAC 49, 287(1966); 50, 663, 787(1967).
CAS-7440-70-2 (calcium)
CAS-7439-95-4 (magnesium)
932.20 Calcium Gluconate in Drugs
Colorimetric Method
Final Action
(Applicable to prepns whose aq. solns are neut. and which do
not contain salts of other optically active hydroxy acids. Cau-
tion: See safety notes on uranyl acetate and toxic dusts.)
Weigh two 0.5 g portions Ca gluconate or two 1 g portions
powd tablets contg <50% of the salt. If chocolate or fatty base
is present, wash samples several times on hardened filter with
absolute ether, then warm residue until ether is driven off.
Transfer each portion to sep. 25 mL vol. flasks, add 15 mL
H 2 0, and warm until Ca salt dissolves. (Samples contg cocoa
will have undissolved residue.) Cool mixt. to room temp.
To one flask (No. 1) add 3.5 g finely pulverized uranyl ace-
tate, stopper, and shake mech. 1 hr. (If agitation is not vig-
orous enough, >1 hr of shaking may be required.) Let other
flask (No. 2) stand. If sample contains chocolate, add little
alumina cream, 925.46B(b), to each flask. Cool to room temp.,
dil. flask No. 1 to vol. with uranyl acetate soln (10 g shaken
with 95 mL H 2 until satd and then filtered), and flask No.
2 with H 2 0. Filter, and polarize each soln in 200 mm tube,
using 50 mm tube contg 1 .8% K 2 Cr 2 7 soln as light filter. If
soln is too dark to read in 200 mm tube, make reading in 100
mm tube and multiply result by 2. If X = rotation in °S of
Soln No. 2 and Y — rotation of Soln No. 1 , with 1 g sample:
% Ca(C 6 H,,0 7 ) 2 = 4.34 x (Y - X); and with 0.5 g sample:
% Ca(C 6 H n 7 ) 2 = 8.52 X (Y - X).
Refs.: JAOAC 15, 456, 461(1932); 16, 379(1933); 17,
425(1934),
CAS-299-28-5 (calcium gluconate)
977.29 Calcium, Potassium, and Sodium
in Electrolyte Replenishers
Atomic Absorption Spectrophotometric-
Flame Photometric Method
First Action 1977
Final Action 1979
(Applicable to Ringer's and Lactated Ringer's Injections)
A. Reagents
(Use H 2 of > 0.5 megohm resistivity for all rinsing and diln.
Use borosilicate volumetric glassware, including pipets, meet-
ing NIST tolerances. Use 5:4 mL single transfer pipets for all
dilns, except that Class A 5 mL Mohr pipets may be used to
complete intermediate and final dilns. Clean all glassware with
HN0 3 (1+3) until washings show no Na at 589 nm when
compared with H 2 used in Na detn.)
(a) Buffer soln.— 0.25M EDTA + 25 mg La 2 3 /mL. (Re-
quired in AA detn of Ca and K only.) Transfer 73.1 g EDTA
into 1 L erlenmeyer. Add ca 100 mL H 2 0, and shake cau-
tiously with just enough NH 4 OH to dissolve. Transfer 25.0 g
low-Ca La 2 3 to 500 mL erlenmeyer, add 25 mL FLO, and
very cautiously dissolve with 70% HCIO4. Cool both solns.
Pour HCIO4 soln into EDTA soln, and wash mixt. into 1 L
vol. flask. Dissolve completely and adjust to pH slightly alk.
to Me orange, using NH 4 OH or 70% HC10 4 , as needed. Dil.
to vol. with H 2 0, mix, and store in clean, dry polyethylene
or Teflon bottle.
(b) Calcium std soln. — 10 mg/mL. Dry CaC0 3 (low in al-
kalis) >:2 hr at 285° (Caution: Higher temp, may convert CaC0 3 ,
in part, to CaO), cool in desiccator, and transfer 24.975 g to
504
Drugs: Part I
AOAC Official Methods of Analysis (1990)
1 L vol. flask. Add ca 150 mL H 2 and acidify cautiously
with 45 mL HCI from freshly opened bottle. Cool to room
temp., dil. to vol. with H 2 0, and mix. Store in clean, dry
polyethylene or Teflon bottle.
(c) Potassium std sola. — 1 mg/mL. Dry KC1 >2 hr at 500-
600°, cool in desiccator, and transfer 1.9070 g to 1 L vol.
flask. Dissolve and dil. to vol. with H 2 0, mix, and store in
clean, dry polyethylene or Teflon bottle.
(d) Sodium std soln. — 10 mg/mL. Dry NaCJ >2 hr at 500-
600°, cool in desiccator, and transfer 25.420 g to 1 L vol.
flask. Dissolve and dil. to vol. with H 2 0, mix, and store in
clean, dry polyethylene or Teflon bottle.
(e) Mixed cation std soln. —(5 |xg Ca + 10 fxg K + 200
|xg Na)/m.L. Pipet 10 mL Ca std soln, (b), into 200 mL vol.
flask, dil. to vol. with H 2 0, and mix. Pipet 10 mL this dild
soln into 1 L vol. flask, pipet in 10 mL K std soln, (c), and
20 mL Na std soln, (d), dil. to vol. with H 2 0, and mix. Store
in clean, dry polyethylene or Teflon bottle. Stable >1 month.
(Proportionate vols may be prepd.)
B. Instrument Suitability
(Caution: See safety notes on A AS and flame photometers.)
Use spectrophtr in AA or flame emission mode. Keep A
readings between 0.100 and 0.820 unit, or emission readings
between 20 and 95% T by adjusting sample dilns, if necessary.
Prep, sufficient std solns to bracket sample detns. Readings
of all stds must be on linear portion of std curve. Linearity is
detd by running intermediate test std which must agree to within
1 % of reading indicated by straight line relationship between
the bracketing std points.
Spectrophtr must pass following precision test: Read, se-
quentially, low std, sample, and high std. Repeat twice and
average readings of each soln. Results are acceptable if each
individual reading differs from av. value for particular soln by
<1.4%.
C. Determination
(a) Calcium. — (7) For absorption. — Pipet 5 mL sample and
4 mL buffer soln into 100 mL vol. flask, dil. to vol. with H 2 0,
and mix. Prep, "median range std" by dilg mixed cation std
soln, including 4.00 mL buffer soln in each 100 mL std soln.
Analyze sample and std solns at 422.7 nm, and est. no. and
concn of stds needed for final detn as required in 977. 29B,
par. 2. (2) For emission. — Proceed as in (7), omitting buffer
soln.
(b) Potassium. — (7) For absorption. — Pipet 5 mL sample
to 100 mL vol. flask, dil. to vol. with H 2 0, and mix. Pipet
10 mL this soln into 50 mL vol. flask, pipet in 2 mL buffer
soln, dil. to vol. with H 2 0, and mix. Prep, "median range std"
by dilg mixed cation std soln, including 2.00 mL buffer soln
in each 50 mL final soln. Analyze sample and std solns at
766.5 nm, and est. no. and concn of stds needed for final assay
as required in 977. 29B, par. 2. (2) For emission. — Proceed
as in (7), omitting buffer soln.
(c) Sodium. — Pipet 5 mL sample into 100 mL vol. flask,
dil. to vol. with H 2 0, and mix. Pipet 4 mL this soln into 500
mL vol. flask, dil. to vol. with H 2 0, and mix. Prep, "median
range std" by dilg mixed cation std soln. Analyze sample and
std solns at 589.0 nm, and est. no. and concn of stds needed
for final assay as required in 977. 29B, par. 2.
(d) Calculations. — Det. concn of each cation from std curve.
Calc. concn of each cation as mg CI salt/ 100 mL sample.
Ref.: J AOAC 60, 929(1977).
CAS-7440-70-2 (calcium)
CAS-7440-09-7 (potassium)
CAS-7440-23-5 (sodium)
949.14* Calcium, Phosphorus,
and Iron in Vitamin Preparations
Titrimetric Method
Final Action
Surplus 1977
See 36.058-36.059, 13th ed.
938.1 3* Effervescent Potassium
Bromide with Caffeine in Drugs
Volhard Titration
Final Action
Surplus 1975
See 36.057-36.058, 12th ed.
941.19
Elixir of Five Bromides
Final Action
A. Preparation of Dilution
Transfer 50 mL sample to 1 L vol. flask, dil. to vol., and
mix. Measure aliquots of this dil n at original temp, of sample.
B. Determinations
(a) Ammonium bromide .—Place 200 mL aliquot of diln in
Kjeldahl flask; add small piece of paraffin and excess 10%
NaOH soln (ca 5 mL). Distil NH 3 into excess std acid (40 mL
O.liV usually is enough). Titr. excess acid with 0.1N NaOH,
using Me red. 1 mL 0AN acid - 0.00979 g NH 4 Br.
(b) Calcium bromide. — Pipet 100 mL aliquot of diln into
casserole or Pt dish and evap. to dryness. Ignite at dull red (ca
525°) until org. matter is thoroly charred. Cool, add 5 mL HC1
(1 + 3) to dissolve Ca salts, filter, and wash well with hot
H 2 0. Return filter and unoxidized C to casserole or dish and
ignite at 600° until residue is white. Treat residue with 5 mL
HCI (1 + 3), filter, and wash with hot H 2 0, combining fil-
trates.
Det. Ca as in 910.01, and reserve filtrate for detn of Na,
K, and Li. If 0.1 AT KMn0 4 is used, 1 mL = 0.0100 g CaBr 2 .
(c) Lithium bromide. — Dil. filtrate and washings from Ca
detn to 200 mL and mix. Evap. 100 mL aliquot to dryness and
drive off all NH 4 salts by heating to faint red (ca 525°) in Pt
dish. Treat residue with little H?0, filter into Pt dish, add few
mL HCI, and evap. to dryness.
Complete conversion of alkali bromides to chlorides by
treating residue with C1-H 2 and evapg to dryness. Repeat addn
and evapn of C1-H 2 twice more, or until there is no apparent
darkening of soln due to liberation of Br.
Dissolve mixed chlorides in min. amt of cold H 2 (ca 1.5
mL is more than enough for 0.5 g salts), in tall 200 mL beaker.
Add 1 drop HCI, and then gradually add 20 mL absolute al-
cohol, dropping alcohol into center of beaker (not on sides)
while rotating soln. (NaCl and KC1 should be pptd in perfectly
uniform granular condition.) In similar manner add 60 mL ether
(sp gr 0.716-0.717 at 25°) and let mixt. stand ca 5 min or
until ppt is well agglomerated and supernate is almost clear,
rotating mixt. occasionally. Filter with suction thru weighed
gooch into erlenmeyer, using bell jar arrangement, washing
beaker thoroly with mixt. of alcohol and ether (1 4- 5), and
collecting all ppt on gooch with aid of policeman. Thoroly
wash ppt on gooch, set crucible aside, and rinse funnel with
alcohol-ether mixt. to wash any adhering Li soln into flask
contg filtrate. Evap. filtrate to dryness on steam bath, using
air current.
AOAC Official Methods of Analysis (1
Inorganic Drugs
505
Treat residue with 10 mL absolute alcohol, warming if nec-
essary, so that practically all residue dissolves. If slight film
remains on bottom and sides of flask, remove with policeman.
Then, while rotating soln in flask, add 50 mL ether (sp gr
0.716-0.717 at 25°), followed by 1 drop HCI. Let stand 30
min, rotating soln frequently. When fine ppt has agglomerated
(only very small amt is usually pptd), filter into tall beaker
with suction thru gooch contg first ppt. Wash combined ppts
with the ether- alcohol mixt. , taking same precautions as in first
pptn. Air dry gooch and contents; then dry in oven, ignite gently,
cool, and weigh to obtain combined wt NaCl and KC1. Re-
serve crucible and contents for K detn.
Evap., on steam bath, ether-alcohol filtrate and washings
contg the Li. Dissolve residue in little H 2 0, add slight excess
of H 2 S0 4 (1 + 1), and transfer to weighed porcelain or Pt dish.
Evap. as far as possible on steam bath and then gently ignite
residue over flame. (By placing dish on triangle over asbestos
gauze and using low flame, soln can be evapd without spat-
tering.) Finally ignite carefully over full flame, cool, and weigh.
If charring has occurred, repeat ignition with H 2 S0 4 . Calc. to
LiBr, using factor 1.5800.
(d) Sodium bromide. — Remove combined KC1 and NaCl
from gooch by washing with hot H 2 0, dil. to 50 mL, and use
5 mL aliquot for detn of Na. Proceed as in 941.03, beginning
"... add 100 mL Mg uranyl acetate soln . . ." Calc. to
NaBr, using factor 0.0688.
(e) Potassium bromide. — To 25 mL aliquot of soln of KG
and NaCl, add enough Pt soln (0.105 g H 2 PtCl 6 /mL) to con-
vert KC1 and NaCl to K 2 PtCl 6 and Na 2 PtCl 6 , and evap. to dry-
ness. Treat residue with 80% alcohol by vol., filter, and wash
until excess of H 2 PtCl 6 and Na 2 PtCl 6 is removed. Dry filter
and ppt, dissolve residue in hot H 2 0, and transfer to weighed
Pt dish. Evap. on steam bath, dry 30 min in oven at 100°,
cool, and weigh as K 2 PtCl 6 . Calc. to KBr, using factor 0.4898.
(f ) Total bromine.— Transfer 20 mL of diln to 500 mL flask.
Add 100 mL H 2 0, 2 mL HN0 3 , and excess of 0.LV AgNO.
(usually 30 mL). Titr. excess AgN0 3 with 0.1 .N NH 4 SCN, us-
ing Fe alum indicator. 1 mL 0AN AgNO^ = 0.00799 g Br.
Refs.: JAOAC 24, 842(1941); 25, 847(1942).
CAS- 12 124-97-9 (ammonium bromide)
CAS-7789-41-5 (calcium bromide)
CAS-7550-35-8 (lithium bromide)
CAS-7758-02-3 (potassium bromide)
CAS-7647-15-6 (sodium bromide)
941.20* Elixir of Three Bromides
Final Action
Surplus 1975
See 36.061-36.062, 12th ed.
984.37 Fluoride in Drug Tablets
and Solutions
Fluoride-Selective Electrode Method
First Action 1984
Final Action 1986
A. Principle
F in tablets and solns is detd by F- selective electrode, using
total ionic strength adjustment buffer (TISAB) to eliminate
complexation of F with polyvalent cations and direct poten-
tiometry to measure concn of F ions in soln. (Caution: Store
all samples and stds in plastic containers because F reacts with
glass.)
B. Reagents
(a) Total ionic strength adjustment buffer (TISAB). — TISAB
II (Orion Research Inc. Cat. No. 94-09-09); alternatively, in
ca 500 mL H 2 in 1 L beaker, dissolve 57 mL HO Ac, 58 g
NaCl, and 4 g CDTA (cyclohexylene dinitrilo tetraacetic acid
or l^-diaminocyclohexane-A^/V.A^Af' -tetraacetic acid). Cool
and adjust pH to 5.0-5.5 with 5M NaOH. Cool to room temp.,
transfer to 1 L vol. flask, and dil. to vol. with H 2 0.
(b) Fluoride std solns.— (1) 10~ 2 M.— Transfer 209.9 mg
accurately weighed Ref. Std NaF, previously dried 4 h at 150°,
to 500 mL vol. flask, dissolve, and dil, to vol. with H 2 0. (2)
J0~ 3 M.— Pipet 25.0 mL of 10~ 2 M soln to 250 mL vol. flask.
Dil. to vol. with H 2 0. (3) J0~~ 4 M.— Pipet 25.0 mL 10~ 3 M soln
to 250 mL vol. flask. Dil. to vol. with H 2 0.
C. Apparatus
(Equiv. app. may be substituted.)
(a) Meter. — Expanded-scale mV pH meter or selective ion
meter.
(b) F -selective electrode. — F-combination electrode filled
with internal filling soln.
(c) Magnetic stirrer. — Suitable mag. stirrer with asbestos
pad placed on top to reduce heat transfer. Use teflon -coated
stir bar.
D. Preparation of Samples
(a) Tablets. — Det. no. of tablets equiv. to 10 mg F and
completely disperse in 500 mL vol. flask contg ca 400 mL
H 2 by heating on steam bath and shaking intermittently. Cool
to room temp, and dil. to vol. with H 2 0.
(b) Solutions. — Pipet aliquot of soln contg 1-2 mg F into
100 mL vol. flask, dil. to vol. with H 2 0, and mix.
Do not let solns remain in glass >1 h.
E. Determination
Pipet 20.0 mL of each std and sample prepn into sep. plastic
beakers. Pipet 20.0 mL TISAB into each beaker. Use poten-
tiometer equipped with F-selective electrode. Immerse elec-
trode in 10~ 3 M std soln and stir at const, rate with mag. stirrer.
Take mV measurement when potential has stabilized within
±0.1 mV. Repeat with 10" 2 M and 10~ 4 M std solns and with
sample prepn. Using 2-cycle semi-log graph paper, prep, std
curve plotting molar F std concn on log axis vs mV potential
on linear axis. Det. molar F concn (C ; ) in sample prepn from
std curve. Prep. sep. mV measurements for each std soln and
std curve for each sample detn.
F. Calculations
Tablets:
mg F/tablet = C s x
(19 mg F/mL)x 500 mL/no. of tablets
= 9500 CJ no. of tablets
Solns:
mg F/aliquot = C, X (19 mg F/mL) X 100 mL
= 1900 Q
where C { — molar concn in sample prepn as detd from std
curve.
Ref.: JAOAC 67, 682(1984).
935.66* Mypophosphites In Sirups
Final Action
Surplus 1975
(Applicable in absence of phosphates; if phosphates are
present, make suitable correction.)
506
Drugs: Part I
AOAC Official Methods of Analysis (1990)
A. Method I
See 36.063, 12th ed.
0. Method II
(Not applicable in presence of other reducing agents or phe-
nolic compds)
See 36.064-36.065, 12th ed.
932.21
Iodine in Drugs
Titrimetric Method
Final Action
now be clear yellow.) Titr. with 0AN AgN0 3 , approaching
end point drop wise and swirling frequently. (AgN0 3 soln causes
turbidity due to formation of colloidal Agl and development
of reddish-brown color similar to that observed in over-titrd
Volhard detn. End point, which is produced by J drop AgN0 3
soln, is characterized by flocculation of colloidal Agl and
complete disappearance of reddish brown tinge, leaving almost
clear, pale yellow supernate.) mL 0.\N AgN0 3 — mL 0AN
Na 2 S 2 3 , (a) = mL consumed by iodide originally present. 1
mL 0.17V AgN0 3 = 0.0166 g KI.
Ref.: JAOAC 24, 833(1941).
CAS-7553-56-2 (iodine)
CAS-6881-11-0 (potassium iodide)
Transfer sample contg <0.1 g iodide (0.05 g is ample) to
crucible, preferably Ni. If sample contains only slight amt of
org. material, add 1 g starch. Add 2-3 g solid KOH. If sample
is solid, add 10-15 mL alcohol before adding KOH. Alkali
must be thoroly mixed with sample to prevent loss of I in fur-
nace (either stir, leaving stirring rod in crucible, or heat and
swirl on steam bath until KOH is in soln). Dry and char tho-
roly. (Use as low temp, as possible to prevent loss of 1; <525°.)
Ext charred mass with hot H 2 0, filter into erlenmeyer, and
wash well with hot H 2 0.
Neutze filtrate with H 2 S0 4 (1 + 1), make alk. again with
4% NaOH soln, and add 1 mL excess. Heat to bp and slowly
add satd KMn0 4 soln until KMn0 4 color remains after several
min of boiling. Then add ca 0.5 mL excess, continue boiling
ca 5 min, and let cool. Add enough KMn0 4 to completely
oxidize all iodide to iodate so that KMn0 4 color, not brown
Mn0 2 color, is present at end of boiling period. Add few mL
alcohol and place on steam bath. (KMn0 4 color should be
bleached; if it is not, add little more alcohol.) When ppt has
settled, filter, and wash with hot 1% NH 4 Cl soln. If filtrate is
not clear, digest on steam bath until the Mn0 2 can be retained
on filter. After cooling, add 1-2 g KI, acidify with HCI, and
titr. with 0AN Na 2 S 2 3 . 1 mL 0.17V Na 2 S 2 3 = 0.00277 g KI,
0.00250 g Nal, or 0.00212 g 1.
Refs.: JAOAC 15, 419(1932); 32, 555(1949).
CAS-7553-56-2 (iodine)
941.21
Iodine in Ointments
Titrimetric Method
Final Action
(a) Free iodine. — Weigh (to 1 mg) ca 2 g ointment, and
transfer to 250 mL I flask. Melt on H 2 bath (<70°), add 30
mL CHC1 3 , mix well, and then add 30 mL H 2 0. (All of base
should be dissolved in CHCI3 before H 2 is added.) Titr. with
0AN Na 2 S 2 3 , using starch indicator (mix ca 2 g finely powd.
potato starch with cold H 2 to thin paste; add ca 200 mL boil-
ing H 2 0, stirring const., and immediately discontinue heating;
add ca 1 mL Hg, shake, and let soln stand over the Hg). Ap-
proach end point dropwise, shaking flask vigorously to ensure
that all I has been extd from CHC1 3 layer. 1 mL 0. 17V Na 2 S 2 3
- 0.01269 g 1.
(b) Potassium iodide. — Pour liqs from free 1 detn, (a), into
500 mL I flask, rinsing flask with 200 mL H 2 0, added in sev-
eral portions. (It is desirable to maintain this vol. within rather
narrow limits.) Add 0.5 mL 0.2% ale. p-ethoxychrysoidin in-
dicator and 1 -4 drops 0, 1 N NaOH (to neutze). (Aq. layer should
977.30 Iron in Drugs
Spectrophotometric Method
First Action 1977
Final Action 1979
(Applicable to drugs listed in Table 977.30. Rinse all glass-
ware with deionized H 2 before use to avoid Fe contamination
from tap H 2 0.)
A Principle
Sample is dissolved in dil. HCI or H 2 and dild to concn
of 3 mg/100 mL. Fe 12 is detd by complexing with a,a'-di-
pyridyl at pH 4.5 and measuring A at 523 nm. Total Fe is detd
by reducing Fe +3 to Fe ' 2 with ascorbic acid and complexing
with dipyridyl. Fe +3 is detd by difference.
B. Reagents
(a) Dipyridyl soln. — Dissolve 0.1 g a,ot'-dipyridyl in H 2
and dil. to 100 mL. Soln is stable up to 4 months if stored in
cool, dark place.
(b) Iron std solns. — (J) Stock soln. — 0.3 mg Fe/mL. Ac-
curately weigh ca 0.3 g std Fe powder (99.999%), dissolve in
100 mL H 2 and 20 mL HCI by heating on steam bath, if
necessary, dil. to I L with H 2 0, and mix. (Complete soln may
require as Jong as 5 hr heating.) (2) Working soln. — 0.03 mg/
mL. Dil. 50.0 mL stock soln to 500 mL with H 2 0.
(c) Acetate buffer soln. — pH 4.5. Dissolve 273 g
Na0Ac.3H 2 in H 2 0, add 240 mL HOAc, and dil. to 2 L
with H 2 0.
C. Preparation of Sample
(Complete immediately to avoid oxidn of Fe 12 to Fe H \)
(a) Powders. — Accurately weigh sample contg ca 60 mg
Fe, transfer to 200 mL vol. flask, and dissolve and dil. to vol.
with initial solv. specified in Table 977.30. Dil. 10. mL ali-
quot to 100.0 mL with specified diln solv.
(b) Tablets. — Accurately weigh portion powd tablets or in-
dividual tablets contg ca 60 mg Fe into 200 mL vol. flask, add
100 mL H 2 and 4 mL HCI, heat on steam bath 30 min (Fe
gluconate and FeS0 4 do not require heat; place 5-10 min in
ultrasonic bath), cool to room temp., and dil. to vol. with H 2 0.
Filter thru Whatman No. 1 paper, or equiv. Dil. 10.00 mL
filtrate to 100.0 mL with H 2 0.
(c) Elixirs, sirups, and injections. — Pipet sample (use "to
contain" pipet and rinse, if sample is viscous) contg ca 60 mg
Fe into 200 mL vol. flask, dil. to vol. with H 2 0, and mix.
Pipet 10 mL into 100 mL vol. flask, add 2 mL HCI, and dil.
to vol. with HoO.
AOAC Official Methods of Analysis (1990)
Inorganic Drugs 507
Table 977.30 Conditions for Analysis of Various Iron Prepa-
rations
Preparation
Initial
Solv.
Color
Diln Develop.
Solv. Temp.
Ferrous ammonium sulfate, powder
Ferrous sulfate, powder, tablets
Ferrous gluconate, powder, tablets
Ferrous fumarate, powder, tablets
Ferric ammonium citrate, powder
Ferric glycerophosphate, powder
Iron cacodylate, powder, injection 3
Iron peptonate, powder
Soluble ferric pyrophosphate, powder
Ferrous sulfate, elixir
Iron sorbitex, injection (1.00 mL sample)
Iron dextran, injection {1.00 mL sample)
(1)
H 2
Room
(1)
H 2
Room
(1)
H 2
Room
(1)
H 2
Heat
(1)
H 2
Heat
(1)
H 2
Room
(1)
H 2
Heat
H 2
(2)
Heat
H ?
(2)
Room
H 2
(2)
Room
H 2
(2)
Heat
H 2
(2)
Heat
Solv. (1) = 100 mL H 2 contg 4 mL HCI, dil. to vol. with H 2
Sotv. (2) - 2 mL HCI, dil. to vol. with H 2
a For iron cacodylate injection, use sample contg 5-6 mg Fe and use initial
diln directly for detn.
D. Determination
(a) Ferrous iron.— Pi pet duplicate 10 mL aliquots sample
soln (1 as sample blank) and 10 mL working std soln, contg
ca 300 jxg Fe, into sep. 100 mL vol. flasks. Transfer 10 mL
reagent blank soln, prepd by dilg 4 mL HCI to 2 L with FLO,
into fourth 100 mL vol. flask. To all solns, add 15 mL buffer
soln and ca 20 mL FLO, and mix. To 1 sample soln, std, and
reagent blank, add 5 mL dipyridyl soln. To std soln, add 20-
25 mg USP ascorbic acid powder. Dil. all solns to vol. with
H 2 0, and mix. Let stand 3 hr.
Record A of working std soln, sample soln, and sample blank
soln (no addn of dipyridyl soln) from 700 to 500 nm against
reagent blank soln, setting spectrophtr at A at 523 nm against
reagent blank soln. Use A at max., ca 523 nm.
% Fe in powder = [(A - A )/A'] x C x (200/UO
where A, A , and A' refer to sample, sample blank, and std,
resp.; C = concn working std soln in jjig/mL; and W = mg
sample.
mg Fe/tablet = [(A -
where T = av. mg /tablet.
A () )/A'] X C x (2/VV) X T
mg Fe in original aliquot elixir, sirup, or injection
taken for assay = [(A - A Q )/A F ] x C
mg Fe compd = (mg Fe x MW)/55.85
x 2
where MW - molecular wt of Fe compd.
(b) Total iron. — Proceed as in (a), adding ascorbic acid to
sample, sample blank, and std solns. Develop color 3 hr at
room temp, or 1 hr, without delay, on steam bath as specified
in Table 977.30, heating before dilg solns to vol. Det. A as
in (a), and calc. % total Fe or mg/dose or aliquot.
(c) Ferric iron. — Fe' 3 - total Fe - Fe f 2 , all expressed in
same units.
Ref.: JAOAC60, 1350(1977).
CAS- 1332-98-5 (ferric ammonium citrate)
CAS- 130 J -70-8 (ferric glycerophosphate)
CAS- 10058-44-3 (ferric pyrophosphate)
CAS- 10045-89-3 (ferrous ammonium sulfate)
CAS- 141-01-5 (ferrous fumarate)
CAS-229-29-6 (ferrous gluconate)
CAS-7782-63-0 (ferrous sulfate)
CAS-9004-66-4 (iron dextran)
CAS- 1 338- 16-5 (iron sorbitex)
978.27 Ferrous Sulfate in Drugs
Semiautomated Method
First Action 1978
Final Action 1980
A. Principle
FeS0 4 in tablets, capsules, or liqs is dissolved in 2% H 2 S0 4 ,
mixed with o-phenan thro line in acetate buffer to form stable
Fe +2 complex, and A is measured in flowcell at 502 nm.
B. Apparatus
(a) Automatic analyzer. — With following modules (Techni-
con Instruments Corp.): Sampler II with 30/hr (3:1) cam; 2
proportioning pumps (I or II); manifold; colorimeter I, with 15
mm tubular flowcell and matched 502 nm filters, or spec-
trophtr; compatible recorder (see Fig. 978.27), or equiv.
(b) Shaker. — Wrist action.
(c) Ultrasonic generator. — 150 watt.
C. Reagents
(Use deaerated, deionized FLO thruout.)
(a) o-Phenanthroline reagent. — 35 mg/100 mL. Dissolve
350 mg c-phenanthroline.H 2 in 500 mL FLO, dil. to 1 L with
FLO, and add 10 drops wetting soln, (c).
'(b) Sulfuric acid.— 2% (w/v). Dil. 1 1.4 mL H 2 S0 4 to 1 L
with H 2 0.
(c) Acetate buffer. — pH 4.6-4.7. Dissolve 136.08 g NaOAc
in mixt. of 57 mL HOAc and 500 mL FLO. Dil. to 2 L with
FLO, add 20 drops of wetting soln, (d), and mix well.
(d) Wetting soln. — 30% soln (w/v) polyoxyethylene lauryl
ether in H 2 (Brij-35, Technicon No. T21-01 10).
(e) Iron std soln. — 60 mg Fe/100 mL. Accurately weigh
ca 60 mg Fe wire and transfer to 100 mL vol. flask. Add 10
mL H 2 and 1.3 mL FLS0 4 , heat on steam bath until dis-
solved, and dil. to vol. with FLO.
D. Preparation of Sample
Place individual tablet or capsule, liq. aliquot, or weighed
composite in accurately measured vol. 2% H 2 S0 4 to give Fe
concn of 0.6 mg/mL. Use ultrasonic generator to disintegrate
solid dosage formulations. After complete disintegration, ag-
itate 15 min on mech. shaker. Let settle 2 hr.
E. Analytical System
Sample is withdrawn and dild with air- segmented stream of
H 2 in double mixer, resampled, and mixed with acetate buffer.
o-Phenanthroline reagent is added and, after mixing in double
mixer, soln is debubbled and passed thru 15 mm flowcell, where
A is measured at 502 nm.
F. Start-Up and Shut-Down Operations
Place all lines in resp. solns and pump until steady baseline
is obtained (ca 15 min). To shut down system, place all lines
in FLO and pump 10 min. Remove lines from H 2 reservoir
and pump system dry. If irregular bubble pattern occurs during
sample run, pump soln contg 10 drops wetting soln/L H 2
thru system ca 5 min before finally flushing with FLO for shut-
down.
G. Determination
Fill sample cups in following order: 3 cups std soln, 5 cups
sample soln, 1 cup std soln, 5 cups sample soln, etc., ending
with 3 cups std soln. (First 2 cups of std soln are used to equil-
ibrate system, but are not included in calcns.) Start sampler.
After last cup has been sampled, let system operate until steady
baseline is obtained. Draw tangent to initial and final base-
lines. Subtract baseline to det. net A and A' for each sample
508 Drugs: Part I
AOAC Official Methods of Analysis (1990)
TO PUMP A -4-
AO INLET
TO WEIR BOX
2X MIXER
I UPPER
'level
®.
WASTE -4-
-©-
2.50 T FROM FLOWCELL
®.
-*-e-
2.00 T WATER
©
©
©
H3
-«-fe*
0.42 T AIR
©
4 Gfl
1.60 T WATER
-Md-
®.
0.32 T RE3AMPLE
WASTE <4-
"©-
©
\
UOTjM A-0
WASTE
FROM PUMP
A
PUMP B
TO
PUMP B
WASTE
2X Mixor 1X Mixer
AO
V
H3
^@- i.eojr buffer
®-
WA8TE
-«-«-
0.32 T RE SAMP L E
WASTE -4-
® r—
Q 10 ° T » pT
(AMPLE ^^"
4D1 AO
©
^ Q 1.40 T COLOR REAQEWT
©
COLORIMETER RECORDER
15 mm Flowcell
502 nm
PROPORTIONING
PUMP
PUMP A
FIG. 978.27— Flow diagrams for semiautomated analysis for ferrous sulfate
and std peak, resp. Discard values for first 2 and last std peaks
and calc. av. std A' .
mg Fe in portion taken = (A/A') X C x D
where C ~ concn of std in mg/mL and D = diln factor.
Ref.: J AOAC 61, 968(1978).
CAS-7720-78-7 (ferrous sulfate)
983.27 Mercury in Mercury-Containing Drugs
Atomic Absorption Spectrophotometric Method
First Action 1983
Final Action 1985
A. Principle
Samples are digested in H 2 0~HC1-HN0 3 , and Hg is detd
by AAS using air-C 2 H 2 flame or flameless technic (low Hg
levels).
AOAC Official Methods of Analysis (1990)
Inorganic Drugs
509
B. Apparatus
Rinse all glassware before use with HN0 3 (1 + 1) followed
by H 2 0. For low Hg levels, decontaminate boiling flasks be-
fore use as follows: Add 5 mL H 2 0-HC1-HN0 3 (4 + 3 + 1),
place on steam bath 20 min, and rinse with H 2 0.
(a) Atomic absorption spectrophotometer. — Equipped with
air-C 2 H 2 flame, or equipped with Hg hollow cathode lamp and
gas flow-thru cell (Fig. 986. J5B), 25 (id) x 1 15 nm with quartz
windows cemented in place. Operating conditions: wavelength
253.7 nm, slit width 160 |jLm, lamp current 3 mA, and sen-
sitivity scale 2.5.
(b) Diaphragm pump. — Neptune Dyna-Pump, orequiv. Coat
diaphragm and internal parts of pump with acrylic-type plastic
spray. Use 16 gage Teflon tubing for all connections.
(c) Gas inlet adapter. — 24/40 I (Kontes Glass Co. No. K-
181000).
(d) Digestion flask. — 250 mL flat-bottom boiling flask with
24/40 1 joint.
C. Reagents
(a) Reducing soln. — Mix 50 mL H 2 S0 4 with ca 300 mL
H 2 0. Cool to room temp, and dissolve 15 g NaCl, 15 g hy-
droxylamine sulfate, and 25 g SnCl 2 in soln. Dil. to 500 mL.
(b) Diluting soln. — To I L vol. flask contg 300-500 mL
H 2 0, add 58 mL HN0 3 and 67 mL H 2 S0 4 . Dil. to vol. with
H 2 0.
(c) Magnesium perchlorate. — Drying agent placed in filter
flask (Fig. 986. 15B). Replace as needed. (Caution: Mg(C10 4 ) 2
is explosive when in contact with org. substances.)
(d) Mercury stock soln. — 1000 |xg/mL. Dissolve 0.1354 g
HgCl 2 in 100.0 mL H 2 0.
(e) Digestion soln.— H 2 0-HC1-HN0 3 (4 + 3 + 1). Pre-
pare immediately before use.
(f ) K 2 Cr 2 Q 7 soln.— 5%, aq.
D. Sample Preparation
(a) Ointments. — Mix sample thoroly and accurately weigh
portion contg ca 5 mg Hg into 50 mL beaker. Add 5 mL H 2 0-
HCI-HNO3 (4 + 3+1). Cover with watch glass and heat on
steam bath 30 min. Cool to room temp., swirl beaker to co-
agulate fat, and decant soln and three 10 mL H 2 rinses into
50 mL vol. flask. Add 2 mL 5% K 2 Cr 2 7 , dil. to vol., and
mix. Prep, reagent blank, beginning "Add 5 mL H 2 0-H0-
HNO3 - . .".
(b) Tinctures. — Pipet aliquot contg ca 5 mg Hg into 50 mL
vol. flask. Place on steam bath, and evap. almost to dryness
in current of air. Add 5 mL H 2 0-HC1-HN0 3 (4 + 3 + 1), and
heat on steam bath 30 min. Blow air into flask 2-3 min, while
swirling contents, to expel N oxides. Cool to room temp., add
ca 30 mL H 2 and 2 mL K 2 Cr 2 7 soln, dil. to vol. with H 2 0,
and mix. Prep, reagent blank, beginning "Add 5 mL H 2 0~
HCI-HNO3 . . .".
(c) Injectables. — Pipet aliquot contg ca 5 mg Hg into 50
mL vol. flask, add 5 mL H 2 0-HC1-HN0 3 (4 + 3 + 1), and
proceed as in (b), beginning tt . . . and heat on steam bath
(d) Preservatives and solns (or samples contg low levels of
Hg). — Pipet duplicate aliquots contg 0.5 |xg Hg (0.1 mL Ep-
pendorf pipet, or equiv., dilg sample if necessary), into sep.
decontaminated 250 mL boiling flasks, add 5 mL H 2 0-HC1-
HNO3 (4 + 3 + 1) to each flask, and heat on steam bath 30
min. Cool to room temp., and add 95 mL dilg soln, (b). Prep.
2 reagent blanks, beginning "Add 5 mL H 2 0-HC1-HN0 3
E Standard Preparation
(a) and 100 (ig Hg/mL std solns (for samples a, b, and
c). — Pipet and 5 mL 1000 |xg/mL Hg stock soln into 50
mL vol. flasks, add 5 mL H 2 0-HC1-HN0 3 (4 + 3 + 1), ca
30 mL H 2 0, and 2 mL K 2 Cr 2 7 soln, dil. to vol. with H 2 0,
and mix.
(b) 0.5 fJLg Hg std soln (for sample d). —Dilute 1000 jxg/
mL Hg stock soln to 5 u,g/mL. Pipet duplicate 0.1 mL ali-
quots of this soln (Eppendorf pipet or equiv.) into sep. de-
contaminated 250 mL boiling flasks, (d). Add 5 mL H 2 0-
HCI-HNO3 (4 + 3 + 1), and heat on steam bath 30 min. Cool
to room temp., add 95 mL dilg solution, (b), and mix.
F. Determination
(a) Samples a, b, and c. — Operate atomic absorption spec-
trophtr with air-C 2 H 2 flame according to manufacturer's spec-
ifications. Zero instrument with |mg/mL Hg std soln, and
measure A of 100 u.g/mL Hg std soln, blank soln, and sample
solns, using 4x scale expansion.
(b) Sample d. — Adjust output of pump to ca 2 L air/min
by regulating speed of pump with variable transformer. Con-
nect app. as in Fig. 986. 15B, except for gas inlet adapter.
With pump working and spectrophr zeroed, add 20 mL re-
ducing soln to dild aliquot. Immediately connect gas inlet adapter
and aerate ca 3 min. (Adjust aeration time to obtain max. A.)
Record A, disconnect pressure on "out" side of pump, and
open vent on filter flask to flush system. Analyze in following
sequence: reagent blank, 0.5 |xg Hg std soln, sample solns,
and 0.5 fig/mL std soln.
G. Calculations
(a) Flame AAS:
mg Hg/g or mL - (A - A B ) x C f(A' x W x 20)
(b) Flameless AAS:
mg Hg/g or mL
- (A ~A B )/(A' - A B )X (C'/V) X F x 1/1000
where A, A B , and A' = absorbance of sample, blank, and std
solns, resp.; C = concn of std soln (jxg/mL, flame AAS; u.g,
flameless AAS); W = wt (g) or vol. (mL) of sample taken; V
= vol. sample (mL) added to 250 mL boiling flask; F = diln
factor if sample was dild.
Ref.: JAOAC66, 1203(1983).
CAS-7439-97-6 (mercury)
957.19
Mercury in Drugs
Gravimetric Method
Final Action 1965
(Applicable to Hg in phenylmercuric chloride, Hgl 2 , nitronv
ersol, HgO ointment, and calomel tablets.)
A Reagents
(a) Strychnine sulfate soln. — Approx. 0.0 1M; 4.3 g/500
mL.
(b) Valsers reagent .—Dissolve 10 g KI in H 2 and dil. to
100 mL. Sat. with Hgl 2 (ca 14 g) and filter.
B. Apparatus
(a) Digestion flask. — Acetylation or r-b, 100 mL, fitted to
H 2 0-cooled straight-tube condenser with ¥ joint.
(b) Gooch crucibles. — Fitted with 21 mm filter paper disks,
covered with thin layer of asbestos and dried at 105°. Use to
filter and weigh ppt of strychnine. HI'. Hg.T 2 .
510
Drugs: Part I
AOAC Official Methods of Analysis (1990)
C. Preparation of Samples
(Caution: See safety notes on bromine.)
Accurately weigh (avoid use of metal containers) or measure
sample contg 20-100 mg Hg (optimal ca 50 mg) and treat as
follows:
(a) Solns of organic mercurials. — Transfer sample to beaker
and evap. just to dryness with low heat (60-70°) and air cur-
rent. Dissolve residue in ca 5 mL 10% NaOH soln and transfer
to digestion flask. Rinse beaker with four 3-4 mL portions
H 2 and add rinsings to digestion flask. Add excess liq. Br to
soln and connect flask to condenser. Boil 4-5 min and add 3
mL HCI thru top of condenser. Continue to heat soln until Br
collects in condenser tube. Remove heat and cool until Br re-
turns to soln in digestion flask.
Alternately heat and cool until Br has almost completely dis-
sipated. (After 3 intervals of heating, flow of H 2 thru con-
denser may be discontinued to aid in removing Br.) Let flask
cool, and rinse inside of condenser with ca 5 mL H 2 0. Dis-
connect flask and rinse tip of condenser with small stream of
H 2 from wash bottle. Filter thru 9 cm paper into 150 mL
beaker, and rinse flask and filter with four 5 mL portions H 2 0.
(b) Ointments .—Transfer sample to digestion flask and add
5 mL HCI (1 + 3) followed by 5 mL satd Br-H z O. Add small
pieces of porcelain, SiC, or few glass beads to prevent bump-
ing. Connect flask to condenser and fit flask over hole cut in
asbestos board so that bottom extends just below undersurface
of board. Heat over low flame, maintaining slow and contin-
uous boiling ca 10 min, and then cool to room temp. Discon-
nect flask and decant aq. portion thru 9 cm paper into 150 mL
beaker. Take precautions to retain all ointment base in flask.
Rinse neck of flask into filter with few drops of H 2 from
wash bottle. Add 1 mL HCI (1 +3), 1 mL satd Br-H 2 0, and
8 mL H 2 to flask and reflux. Again cool contents of flask
and decant aq. phase thru filter.
Repeat refluxing and decanting with two 10 mL portions
H 2 and finally rinse condenser tube into flask with ca 5 mL
H 2 0. Disconnect flask, rinse condenser tip, and decant rins-
ings thru filter. Rinse filter with 2 small portions H 2 from
wash bottle.
Test for complete removal of Hg by adding 5 mL H 2 and
2 drops HCI (1 +3) to digestion flask and refluxing as before.
Pass this soln thru original filter into 50 mL beaker. To filtrate
add 1 drop 10% KI soln and 1 drop strychnine sulfate soln.
No turbidity should be produced. If extn is incomplete, repeat
refluxings with H 2 until all Hg is removed. Reserve all test
solns showing presence of Hg to add to major portion after
pptn of Hg.
(c) Calomel tablets. — Det. av. wt/tablet. Grind to fine
powder and transfer accurately weighed portion to digestion
flask. Add 10 mL satd Br-H 2 and 5 mL HCI (1 + 3). Con-
nect flask to reflux condenser and gently boil contents until
most of Br vapors collect in condenser. Discontinue heating
until Br returns to soln in flask. Repeat alternate heating and
cooling until Br vapors are dissipated. Cool flask and contents
to room temp, and rinse condenser tube with ca 10 mL H 2 0.
Disconnect flask and rinse condenser tip into flask. Filter soln
thru gooch into 150 mL beaker. Rinse flask with three 5 mL
portions H 2 and pass rinsings thru crucible, and finally rinse
crucible with fine stream of H 2 0.
(d) Tablets containing purgative drugs. — If tablets contain
purgative drugs, add 10 m.L alcohol to weighed sample in flask.
Heat on steam bath with gentle agitation until alcohol begins
to boil. Remove flask, cool under tap, and filter supernate thru
gooch fitted with asbestos mat. Retain as much of insol. res-
idue in flask as possible. Rinse flask and contents with three
10 mL portions alcohol and two 5 mL portions H 2 0, and de-
cant thru crucible as above. Remove asbestos mat with fine
wire or needle and transfer to flask. Rinse crucible with 10
mL satd Br-H 2 and 5 mL HCI (1 + 3), and add rinsings to
flask. Connect flask to condenser, and treat as in (c).
D. Determination
Add 10 mL 10% KI soln to filtrate, and if necessary, evap.
on steam bath under air current to ca 50 mL. If soln has not
previously been acidified, add 3 mL HCI (1 + 3). Add 1%
NaHS0 3 soln until I color is discharged, and keep soln free
from I color by addn of NaHS0 3 soln until final filtration is
made. Add strychnine sulfate soln slowly from buret or pipet
until ppt coagulates and settles rapidly. (Strychnine sulfate soln
may be added as rapidly as it will flow from buret if theoretical
amt is used, based on 1 mL soln for each 4 mg Hg expected
to be present.) Avoid undue excess of strychnine because of
slight solubility of its hydriodide.
Let ppt settle and test for complete pptn by adding 2-3 drops
strychnine sulfate soln to clear supernate. If pptn is incom-
plete, indicated by cloudiness around the drops, add strychnine
sulfate soln in 1 mL increments until pptn is complete. Let ppt
remain in beaker with occasional stirring 0.5-1 hr.
Decant supernate thru weighed gooch, 957.19B(b). Wash
ppt into crucible with fine stream of H 2 0. Completely transfer
ppt to crucible, and wash residue and crucible with three 5 mL
portions H 2 0. Scrub beaker thoroly with policeman. Transfer
crucible and holder to another small suction flask and wash
residue with 2-3 mL H 2 0. Test filtrate for complete removal
of strychnine by addn of Valser's reagent. If necessary, con-
tinue washing ppt with small portions H 2 until last washings
give no more than faint opalescence upon addn of Valser's
reagent. Always test main filtrate by addn of ca 1 mL strych-
nine sulfate soln to assure complete pptn of Hg. If pptn was
incomplete, repeat detn. Dry crucible 1 hr at 105°, cool in
desiccator, and weigh. Calc. % Hg compd in sample on basis
of MW of 916.74 for ppt of strychnine. HI. Hgl 2 .
Ref.: J AOAC 40, 819(1957).
CAS-7546-30-7 (calomel)
CAS-7774-29-0 (mercuric iodide)
CAS-7439-97-6 (mercury)
CAS- 133-58-4 (nitromersol)
CAS- 100-56-1 (phenylmercuric chloride)
934.09
Merbromin in Drugs
4. Tests for Purity
—Procedure
(a) Acidify portion of merbromin soln with 10% H 2 S0 4 and
filter off ppt. Filtrate is only slightly yellow.
(b) Pass H 2 S (Caution: See safety notes on hydrogen sul-
fide.) into portion of filtrate. No ppt or coloring occurs.
(c) Add few mL 10% HN0 3 to another portion of filtrate
and add AgN0 3 soln. No ppt forms.
B. Total Solids in Solution
—Final Action
Pipet 10 mL merbromin soln into tared, extra-wide-form
weighing bottle and evap. to dryness on steam bath. Let dry
overnight in open bottle in desiccator contg H 2 S0 4 . Weigh.
C. Determination of Mercury
—Final Action
(Caution: See safety notes on wet oxidation, sulfuric acid,
flammable solvents, toxic solvents, carbon disulfide, carbon
tetrachloride, hydrogen sulfide, permanganates, and asbestos.)
AOAC Official Methods of Analysis (1990)
Inorganic Drugs
511
Pipet 10 mL ca 2% merbromin soln into 500 mL tall beaker
and evap. to dryness on steam bath (or accurately weigh ca
0.2 g powder). Dissolve residue in 4 mL H 2 and slowly add,
with const mixing, 10 mL H 2 S0 4 . Incline beaker and cau-
tiously add small portions finely powd KMn0 4 , mixing after
each addn, until deep purple color shows that considerable ex-
cess has been added. Let stand 30 min, mixing occasionally.
Mixt. should still be purple.
Add 100 mL H 2 and mix thoroly. Add small portions finely
powd oxalic acid, mixing after each addn, until soln is clear.
Filter thru small filter into 400 mL beaker, wash original beaker
and filter until filtrate measures ca 200 mL, and pass H 2 S thru
soln 20 min. Warm on steam bath until ppt of HgS settles
quickly after stirring, and again pass H 2 S thru warm soln 5
min. Immediately filter soln into weighed gooch; thoroly wash
ppt on filter with H 2 0, 3 times with alcohol, and then with 4
or 5 portions CC1 4 or CS 2 , letting liq. run thru crucible without
suction; finally wash with ether. Dry ppt to const wt at 100°
and weigh as HgS. HgS x 0.8622 - Hg.
Qual. test dried ppt for Hg and other heavy metals. If slow
filtration occurs during washing with H 2 0, let ppt drain, and
wash once with alcohol; then continue as directed.
Refs.: J AOAC 17, 75, 432(1934).
CAS- 129- 16-8 (merbromin)
931.12
Calomel in Ointments
Titrimetric Method
Final Action
Accurately weigh ca I g ointment, transfer to 250 mL g-s
erlenmeyer, and treat with ca 50 mL CHCl 3 . When base is
dissolved, decant thru dry, closely packed asbestos mat in
Caldwell crucible {Caution: See safety notes on asbestos.), us-
ing light suction. Wash flask and contents several times with
20-30 mL portions CHCl 3 , decanting thru crucible. Let any
residual CHCl 3 in flask evap., and transfer asbestos mat and
contents to flask, wiping sides of crucible and mouth of flask
with damp piece of filter paper and adding it to flask. Add 2.5
g KI and 30 mL std 0.17V I, 939.13 (stdzd against Na 2 S 2 3 ),
stopper, and mix well. Let stand ca 1.5 hr or until soln of
calomel is complete, agitating frequently and fairly vigor-
ously. Titr. with 0. l/V Na 2 S 2 3 , 942.27B, adding 1 or 2 mL
excess and using starch indicator, (mix ca 2 g finely powd.
potato starch with cold H 2 to thin paste, add ca 200 mL boil-
ing H 2 with stirring, and discontinue heating; add ca 1 to 6
Hg, shake, and let soln stand over Hg). When all traces of 1
disappear, back-titr. with std I soln to blue color. 1 mL 0.1/V
I - 0.02360 g Hg 2 Cl 2 .
Ref.: J AOAC 14, 312(1931).
CAS-7546-30-7 (calomel)
asbestos mat placed on plate of Caldwell crucible (Caution:
see safety notes on asbestos.). Wash once with H 2 by de-
cantation and then successively with alcohol and ether. Trans-
fer removable plate holding mat and insol. material to original
flask, washing into flask any insol. material adhering to sides
of crucible. Add 2.5 g KI, 10 mL H 2 0, and then 30 mL std
0.17V I soln, 939.13A. Complete detn as in 931.12.
Refs.: JAOAC 10, 367(1927); 11, 343(1928); 12, 280(1929).
CAS-7546-30-7 (calomel)
929.11 Mercurous Iodide in Tablets
Titrimetric Method
Final Action
Accurately weigh well mixed powd sample contg 0. 1 9-0. 26
g (3-4 grains) Hg 2 l2. Transfer sample to 200 mL g-s flask,
and proceed as in 927.11, omitting addn of H 2 after the KI.
1 mL 0.UV I - 0.03275 g Hg 2 I 2 .
Note: Some com. tablets are difficult to filter thru asbestos
mat without loss of Hg 2 I 2 . Placing few drops of alumina cream
on mat before filtration is started (wash free from NH 3 ), sat-
isfactorily prevents loss, tho it retards filtration. Alumina
cream. — Prep, cold satd soln of alum in H 2 0. Add NH 4 OH
with constant stirring until soln is alk. to litmus, let ppt settle,
and wash by decantation with H 2 until wash H 2 gives only
slight test for sulfates with BaCl 2 soln. Pour off excess H 2
and store residual cream in g-s bottle.
Ref.: JAOAC 12, 280(1929).
CAS-7783-30-4 (mercurous iodide)
935.67 Mercury in Mercurial Ointments
Titrimetric Method
Final Action
(Caution: See safety notes on distillation and nitric acid.)
After mixing ointment thoroly with glass rod, avoiding con-
tact with metals, weigh 1 g sample into erlenmeyer. Add 20
mL H 2 and 20 mL HN0 3 , and heat gently over small flame
until red fumes cease to evolve. Cool, and decant aq. soln
from ointment base into separator. Wash ointment base with
50 mL boiling H 2 0, cool, and decant into separator. Repeat
washing until all Hg is removed.
Shake combined solns in separator with 50 mL ether. Trans-
fer aq. soln to erlenmeyer. Wash ether soln with three 10 mL
portions H 2 until Hg is removed, adding washings to flask.
Add 3 mL FeNH 4 (S0 4 ) 2 soln, 931.11A(b), and titr. with 0. l/V
NH 4 SCN. 1 mL 0.LV NH 4 SCN - 0.01003 g Hg.
Ref.: JAOAC 18, 520(1935).
CAS-7439-97-6 (mercury)
927.11
Calomel in Tablets
Titrimetric Method
Final Action
Count and weigh representative number of tablets. Powder
tablets and accurately weigh well mixed sample contg 0.19™
0.26 g (3-4 grains) Hg 2 CI 2 . Transfer to 200 mL g-s erlen-
meyer, add ca 50 mL H 2 0, acidify with HOAc, and after sol.
fillers dissolve, decant with aid of suction thru tightly packed
957.20* Mercury in Ointments
of Mercuric Nitrate
Titrimetric Method
Final Action
Surplus 1977
See 36.090, 13th ed.
512
Drugs: Part I
AOAC Official Methods of Analysis (1990)
955.51* Nitrites in Tablets
Hydrazine Method
Final Action
Surplus 1975
(Applicable in presence or absence of nitrates or chlorides)
See 36.080-36.081, 12th ed.
945.69
Meperidine in Drugs
Final Action
925.58
A. Total Silver
Silver Protein in Drugs
Titrimetric Method
Final Action
(Caution: See safety notes on distillation and nitric acid.)
Place 1 g sample, accurately weighed, in 500 mL Kjeldahl
flask; add 15 mL H 2 S0 4 and then 10 mL HN0 3 . Place on
steam bath few min, with occasional rotation, to ensure ho-
mogeneous mixt., and boil to white fumes. Add more HN0 3 ,
boil again to clear colorless soln, and cool. Add 100 mL H 2
and boil until free of N oxides. Cool, dil. to 300 mL, add 5
mL HNO, and 5 mL FeNH 4 (S0 4 ) 2 soln, 931.11A(b), and titr.
with QAN NH 4 SCN. 1 mL 0.W NH 4 SCN = 0.01079 g Ag.
B. lonizable Silver Compounds
Weigh strip of com. dialyzing tubing 55 mm wide and ca
30 cm Jong, wet with H 2 until uniformly pliable, shake free
of adhering H 2 0, and partially dry by rolling in clean paper
towel. Reweigh while still moist and place in 250 mL beaker.
(Sheets of dialyzing parchment paper may be used in place of
tubing. Over one end of glass tube 10 cm long and ca 2.5 cm
od, fold and secure with rubber band sq piece of parchment
paper in form of sack large enough to hold sample soln. Di-
alyzing material should be kept in humid container to prevent
breaking when handled.)
Weigh I g sample, dissolve in 15 mL H 2 0, and transfer to
dialyzing tube. Calc, and add enough H 2 to beaker to make
100 mL (this ensures 20 mL in dialyzing tube and 80 mL in
beaker). Adjust tubing to form "U" in beaker, cover with watch
glass, and keep cool and in dark 24 hr.
(a) Qualitative test. — Test few mL clear, colorless soln from
beaker for Ag ions by addn of few drops HO (1 + 3) and
trace of HND 3 .
(b) Determination. — If Ag ions are present, remove 50 mL
clear, colorless soln (representing 0.5 g sample) from beaker,
dil. to 100 mL, and add 2 mL FeNH 4 (S0 4 ) 2 soln, 931.11A(b),
and 2 mL colorless HN0 3 . Titr. with 0.017V NH 4 SCN and calc.
to % by wt ionizable Ag. 1 mL 0.017V NH 4 SCN = 0.001079
g Ag.
Refs.: JAOAC 8, 551(1925); 9, 312(1926); 10, 374(1927).
CAS-7440-22-4 (silver)
ANTIHISTAMINES
974.39* Selected Drug Combinations
Ion Exchange Chromatography
First Action 1974
Final Action 1978
Surplus 1989
(Applicable to 14 antihistamines, antitussive agents, expec-
torants, and sedatives, alone or combined)
See 36.108-36.114, 14th ed.
A. Distillation Method*
—Surplus 1970
See 36.093-36.095, 11th ed.
B. Extraction Method
Accurately weigh portion of powd sample contg ca 0.1 g
meperidine, and macerate 2 hr with 10 mL H 2 and 1 mL \N
H 2 S0 4 . Decant liq. thru small filter into separator. Macerate
residue 20 min with 5 mL FLO, filter thru same filter, and
wash residue and filter with small portions of H 2 0.
Sat. soln with NaCl; then add 5 mL \N NaOH and ext with
25 mL and six 20 mL portions ether as in 960. 53A. Wash
combined ether exts with two 5 mL portions H 2 0; ext this H 2
with 10 mL ether and add this ether to main ether ext. Ext
ether soln first with 20.0 mL 0.02/V H 2 S0 4 , and then succes-
sively with 10 and 5 mL H 2 0. Combine H 2 S0 4 and H 2 exts
in beaker and warm on H 2 bath until no ether odor is de-
tected. Cool soln, and titr. excess acid with 0.027V NaOH, us-
ing Me red. 1 mL 0.02W H 7 S0 4 - 0.005676 g meperi-
dine.HCl, C 15 H 21 2 N.HC1.
Refs.: JAOAC 28, 711(1945); 31, 540(1948).
CAS-50-13-5 (meperidine hydrochloride)
978.28 Mephentermine Sulfate in Drugs
Spectrophotometric Method
First Action 1978
Final Action 1981
A. Principle
Mephentermine sulfate is sepd on ion exchange column, eluted
with alcoholic HC1, and measured by UV spectrophotometry.
B. Apparatus
(a) Chromatographic tube. — Glass, 150 x 12 (id) mm, fit-
ted with replaceable coarse fritted glass disk, Teflon stopcock,
and Buna-N "O" ring seal (Kontes Glass Co., No. K-422280,
or equiv.).
(b) Ion exchange resin. — See 974.39C(d).
C. Reagents
(a) Alcoholic hydrochloric acid. — (/) 1.57V. — Mix 1 part
HC1 with 7 parts alcohol-H 2 (1 + 1). (2) 6/V.— Mix 1 part
HC1 with 1 part alcohol-H 2 6 (1 + 1).
(b) Mephentermine sulfate std soln. — 0.5 mg/mL. Accu-
rately weigh ca 25. mg mephentermine sulfate, previously dried
1 hr at 105°, into 50 mL vol. flask. Dissolve and dil. to vol.
with \.5N alcoholic HCi, (a)(7), and mix.
D. Preparation of Column
Prep, slurry of 2 g resin, (b), with 20-25 mL alcohol-H 2
(1 + 1), and transfer to tube, (a), with stopcock closed and
plug of glass wool under fritted disk. Let resin settle by grav-
ity; then top with small pledget of glass wool. (Column need
not be tamped.) Drain solv., wash column with three 10-20
mL portions aicohol-H 2 (1 + 1) followed by 20 mL H 2 0,
and discard all washings. Prevent column from drying out be-
fore use by maintaining head of 2-3 mL alcohol-H 2 (1 + 1)
or H 2 0.
When using column for first time and upon completion of
sepns, wash thoroly with 15-20 mL 6N alcoholic HCI, (a)(2),
to recondition resin. With stopcock closed, stir resin to obtain
slurry, let settle, and drain. Repeat twice. Finally, wash resin
with alcohol-H 2 (1 + 1) until excess acid is removed and
AOAC Official Methods of Analysis (1990)
Antihistamines 513
then with three 10-20 mL portions H 2 0. Store under H 2 when
not in use-
Perform blank detn on new column, beginning in 978. 28E
with "Elute mephentermine with three 5 mL portions . . ."If
UV spectrum has A >0.05 at 258 nm, recondition column again
or prep, new column, using different bottle of resin.
E. Preparation of Sample
(a) Tablets. — Grind tablets to pass No. 80 sieve. Accu-
rately weigh amt powder contg ca 25 mg mephentermine sul-
fate into 50 mL beaker. Add 10 mL H 2 0, heat on steam bath
2-3 min, cool, and filter thru 9 cm Whatman No. 541 paper
into prepd column, 978. 28D. Column flow rate should be ca
2-3 drops/sec. Thoroly wash filter paper with three 5—10 mL
portions H 2 0, letting each portion pass into column before addn
of next. Wash column with 25 mL alcohol-H 2 (1 + 1). Dis-
card all washings. Elute mephentermine with three 5 mL por-
tions 1.57V alcoholic HO, then with 30 mL 1.5N alcoholic
HCL Let each portion just enter column before adding next,
introducing eluant down sides of column so as not to disturb
resin. Collect eluate in 50 mL vol. flask, rinse liq. from tip
of column into flask, and dil. to vol.
(b) Elixir.— Add sample vol. contg ca 25 mg mephenter-
mine sulfate to prepd column, 978. 28D, and proceed as in (a),
beginning "Elute mephentermine with three 5 mL portions ..."
If sample contains parabens, wash column with 25 mL alcohol
before eluting.
F. Determination
Scan sample and std solns from 220-300 nm against 1.5N
alcohol-HCl as ref. blank. Draw baseline connecting min. A
at ca 254 and 262 nm, and use max. A at 258 nm to calc.
potency of mephentermine sulfate:
mg Mephentermine sulfate/tablet
- (A/A f )x (X/g sample) x W
where A and A f refer to sample and std, resp.; X — av. g/
tablet; and W - mg std in 50.0 mL 1.5/V alcoholic HCl; or
mg Mephentermine sulfate /mL
- (A /A') x (F/mL sample) x W
where F — diln factor.
Ref.: J AOAC 61, 60(1978).
CAS- 1212-72-2 (mephentermine sulfate)
959.15 Methapyrilene in Expectorants
Spectrophotometric Method
Final Action 1965
(See also 97439.)
A. Reagent
Methapyrilene hydrochloride std soln, — 0.015 mg metha-
pyrilene. HCl/mL. Transfer 60 mg methapyrilene. HCl, accu-
rately weighed, to 200 mL vol. flask. Dissolve in ca 0. \N
H 2 S0 4 and dil. to vol. with ca 0.1/V H 2 S0 4 . Transfer 5 mL
aliquot to 100 mL vol. flask and dil. to vol. with ca 0. \N
H 2 S0 4 .
&. Determination
Pipet 10 mL sample into separator, make alk. with NH 4 OH,
and ext with four 20 mL portions CHC1 3 . Combine CHC1 3 exts
in 100 mL vol. flask and dil. to vol. with CHC1 3 . Transfer
aliquot contg 1-3 mg methapyrilene to small beaker and evap.
just to dryness on steam bath with air current. Dissolve residue
in ca 0. ]N H 2 S0 4 , transfer to 100 mL vol. flask, and dil. to
vol. with ca 0. IN H 2 S0 4 . Det. A of this soln and A r of std
against ca 0.17V H 2 S0 4 blank at 315 nm.
mg Methapyrilene. HCl/ 100 mL sample
= (A X 1.5 x 100 x 10)/(A' x vol. aliquot).
Ref.: J AOAC 42, 466(1959).
CAS- 135-23-9 (methapyrilene hydrochloride)
959.16 Pyriiamine in Cough Sirup
Spectrophotometric Method
Final Action 1965
(See also 974.39.)
A, Reagent
Pyriiamine std soln. — 0.015 mg pyriiamine maleate/mL.
Transfer 150 mg pyriiamine maleate to 500 mL vol. flask,
dissolve in ca 0.1 Af H 2 S0 4 , and dil. to vol. with ca 0. IN H 2 S0 4 .
Transfer 5 mL aliquot to 100 mL vol. flask and dil. to vol.
with ca0.1vVH 2 SO 4 .
B. Determination
Proceed as for detn of methapyrilene, 959. 15B, but measure
A at 314 nm.
mg Pyriiamine maleate/100 mL sample
= (Ax 100 x 1.5 X 10) /)A' X mL aliquot)
where A and A' refer to sample and std, resp.
Ref.: JAOAC 42, 466(1959).
CAS-91-84-9 (pyriiamine)
CAS-59-33-6 (pyriiamine maleate)
981.24 Chlorpheniramine Maleate
in Drug Tablets
Semiautomated Method
First Action 1981
Final Action 1982
A. Principle
Method is automated version of general USP XIX assay for
salts of org. nitrogenous bases. Sample is made basic, extd
with isooctane, resampled, re-extd with acid, and measured at
265 nm.
B. Apparatus
(a) Automatic analyzer. — AutoAnalyzer with following
modules: Sampler II with cam or timing clock set at 30/hr
with sample to wash ratio of 2:1, proportioning pump III, and
manifold; see Figure 981.24 (Technicon Instrument Co.).
(b) Spectrophotometer . — Zeiss spectrophotometer, Model
PM2 DL (Carl Zeiss West Germany, PO Box 1369/1380, D-
7082, Oberkochen, West Germany), or equiv., fitted with Helma
10 mm, 18-180 \xL flowcell (Helma Cells, PO Box 544, Ja-
maica, NY 11424), or equiv.
(c) Recorder. — Texas Instrument Servo /R iter II (Texas In-
struments, Inc., 24500 Hwy 290, PO Box 144, Sypress, TX
77429), or equiv.
(d) Shaker. — Model BT, wrist action (Burrell Corp.), or
equiv.
(e) Ultrasonic generator. — Model II, 150 watt (Heat Sys-
tems-Ultrasonic Inc., 38 E Mall, Plainview, NY 11803), or
equiv.
514
Drugs: Part I
AOAC Official Methods of Analysis (1990)
glass carrier tube
use silicone tQ Welr Box
30 : a/1
See TOP VIEW for position of
bracketed portion of manifold
RECORDERS
265 nm
10 mm fc
V
uv
SPECTRO-
PHOTOMETER
^^^ 2x mixer
*_. I 2.4 mm I.D.
2.02 S resample from C3 ( short
^ length of small bore
V 1*06 S Solvaflex tubing )
0.034" I, p. polyethylene
PROPORTIONING P-JMP
* short length of small bore Teflon or polyethylene
Tv^on, S - Solvaflex,
FIG. 981.24 — Schematic of semiautomated analysis of chlorpheniramine tablets
C. Reagents
(a) Hydrochloric acid soln.— AN. Dil. 34 mL HCl to
4 L with H 2 0.
(b) Sodium hydroxide soln. — 0.5/V. Dil. 55 mL 50% w/w
NaOH to 2 L with H 2 0.
(c) Chlorpheniramine maleate std soln. — 0.08 mg/mL.
Accurately weigh ca 80 mg USP Ref. Std Chlorpheniramine
Maleate and transfer to 100 mL vol. flask. Dissolve and dil.
to vol. with 0. IN HCl. Transfer 10.0 mL aliquot to 100 mL
vol. flask and dil. to vol. with 0.1/V HCl. Soln is stable >2
weeks.
D. Preparation of Sample
Disintegrate individual tablets or disperse weighed compos-
ite in accurately measured vol. 0AN HCl to give concn of 0.08
mg/mL based on amt of drug claimed. Use ultrasonic gen-
erator or shake mech. ca 30 min to assure tablet disintegration.
Let particulates settle completely, overnight if necessary, be-
fore sampling. Use clear portion of sample soln for analysis.
B. Analytical System
Isooctane and sample soln are brought together on manifold,
0.5N NaOH is added, and stream is segmented with air. Phases
are passed thru mixing coil and org. phase is sepd, resampled,
and brought together with 0AN HCl. Stream is segmented with
air and passed thru mixing coil, and aq. phase is drawn thru
flowcell where A is measured at 265 nm.
F. Start Up
Place all aq. reagent lines in respective reagents and pump
5 min; then pump isooctane until steady baseline is obtained.
G. Shut Down
Remove isooctane line first; 5 min later, remove other lines.
Pump system dry.
H. Determination
Fill sample cups in following order: 3 cups std soln, 5 cups
sample soln, 1 cup std soln, etc. Place 2 cups std soln at end
of each run. (Extra cups of std solns at start and end of sam-
pling pattern will eliminate carryover effect in transitions from
wash soln to std soln and vice versa. Two extra cups at start
and 1 extra cup at end should suffice, but det. number needed
for equilibrium by experiment. System should give uniform
response for ^2 std cups before sampling pattern is started.)
Start Sampler II. After last cup has been sampled, let system
operate until steady baseline is obtained. Draw tangent line to
initial and final baselines. Subtract baseline to determine net
A and A' for each sample and std peak, resp. Discard values
for first 2 and last std peak. All std peaks can be averaged or
stds flanking sample pattern can be used.
mg chlorpheniramine maleate taken = (A/A f ) x C x D
where C = concn of std in mg/mL, and D = diln factor for
sample. If ground composite is used, av. tablet wt and sample
wt must be used in formula.
If peak shape or steady state slope creep upward exces-
sively, check for isooctane in flowcell. Remove all lines and
pump dry. Pump alcohol thru isooctane line 5 min; then pump
dry. Start up system as above.
Ref.: JAOAC62, 1197(1979).
CAS- 11 3-92-8 (chlorpheniramine maleate)
AOAC Official Methods of Analysis (1990)
Alkanolamines
515
958.10 Antihistamines in Drugs
in Presence of Aspirin, Phenacetin,
and Caffeine
Spectrophotometry Method
Final Action 1965
(Applicable to thonzylamine.HCl, pheniramine maleate, and
chlorpheniramine maleate in combination with APC)
A. Preparation of Standard Solutions
Prep. sep. std solns of thonzylamine.HCl, pheniramine mal-
eate, and chlorpheniramine maleate by dissolving 250 mg an-
tihistamine salt, accurately weighed, in 50.0 mL H 2 0. Pipet
5 mL of each soln into sep. 100 mL vol. flasks and diL to
vol. with ca 0.1 A? H 2 S0 4 . Transfer 10 mL of each acid soln
to sep. 100 mL vol. flasks and diL to vol. with ca 0. IN H 2 S0 4 .
(Concn = 2.5 mg/100 mL.) Det. A' of thonzylamine.HCl at
314 nm, pheniramine maleate at 265 nm, and chlorphenira-
mine maleate at 264 nm.
B. Determination
Place accurately weighed powd sample contg ca 10 mg an-
tihistamine in 125 mL separator. Add 15 mL H 2 and ca 0.5
mL H 2 S0 4 (1 + 1). Ext with CHCl 3 , using 30*", 20, 20, and
20 mL portions. Re-ext by passing CHC1 3 exts successively
thru 2 separators, each contg 10 mL ca 0.17V H 2 S0 4 , shaking
vigorously each time. Discard CHC1 3 and combine aq. solns.
Make combined solns alk. with 10% NaOH and ext with
30, 20, 20, and 20 mL portions CHC1 3 - Again pass CHC1 3
exts successively thru 2 separators, each contg 20 mL ca 0. IN
H 2 S0 4 , shaking vigorously each time. Discard CHC1 3 , com-
bine acid aq. solns, and diJ. to vol. with ca 0.1/V H 2 S0 4 in
100 mL vol. flask. Transfer 25 mL aliquot to 100 mL vol.
flask and dil. to vol. with ca 0AN H 2 S0 4 . Det. A at wave-
length of max. absorption against ca 0AN acid as ref.
% Thonzylamine.HCl
- (A x 2.5 x 4 x 100)/C4' x mg sample)
% Pheniramine or chlorpheniramine maleate
- 1.018 X (A X 2.5 X 4 x 100)/(A' X mg sample)
where 1 .018 corrects for absorbance of maleate moiety of std.
Ref.: J AOAC 41, 495(1958).
CAS-1 13-92-8 (chlorpheniramine maleate)
CAS- 132-20-7 (pheniramine maleate)
CAS-63-56-9 (thonzylamine hydrochloride)
Pseudoephedrine HCI
and Triprolidine HCI or Chlorpheniramine Maleate
in Drug Combinations
See 981.26.
ALKANOLAMINES
956.08 Norepinephrine
in Drug Preparations of Epinephrine
Spectrophotometric Method
Final Action
A. Apparatus
(a) Chromatographic tubes. — Fuse 6 cm length of 5-6 mm
tubing to piece of 25 mm tubing ca 25 cm long (25 X 200
mm test tube may be used). Constrict stem slightly ca 2 cm
below seal. Com. tubes with dimensions ±10% are satisfac-
tory. Pack wad of Pyrex glass wool in base as support.
(b) Tamping rod, — Flatten end of glass rod to circular head
with clearance of ca 1 mm in tube (a). Or use disk of stainless
steel, Al, etc., of diam. ca 1 mm less than id of column, (a),
attached to 30-45 cm (12-18") rod.
(c) Hypodermic syringe. — 1 mL without needle, graduated
in 0.01 mL.
B. Reagents
(a) Diatomaceous earth.- — See 960. 53B.
(b) Glass woo/.— Pyrex No. 3950.
(c) Benzene. — Distil reagent grade benzene in all -glass app.
Shake distillate with H 2 2-3 min and filter benzene layer thru
paper. Use this H 2 0-satd sol v. unless dry benzene is specified.
{Caution: See safety notes on distillation, flammable solvents,
toxic solvents, and benzene.)
(d) Concentrated phosphate buffer. — p'H 6. DiL 50.0 mL
0.2M KH 2 P0 4 soln, 941.17B(b), and 5.64 mL 0.2M NaOH,
941.17B(d), to 100 mL with H 2 0.
(e) Iodine -potassium iodide soln. — Dissolve 2 g I and 6 g
KI in H 2 0, and dil. to 100 mL.
(f ) Norepinephrine std soln . — . 1 00 mg norepinephrine base/
mL. Dissolve 19.9 mg /-norepinephrine (levarterenol) bitar-
trate. H 2 in exactly 100 mL H 2 0. Discard after 8 hr.
C. Preparation of Sample
(a) Aqueous solns of epinephrine. HCI containing bisulfite
and chlorobutanol. — If soln is 0.1% with respect to "total epi-
nephrine" (epinephrine + norepinephrine), pipet 30 mL sam-
ple into 125 mL separator provided with tightly fitting stopper
and stopcock. If soln is more coned, use sample contg 30 mg
"total epinephrine," and dil. to 30 mL with H 2 0.
(b) Suspensions of epinephrine in oil. — Mix suspension by
gentle swirling and agitation; add to separator accurately mea-
sured vol. contg ca 30 mg epinephrine and 25 mL pet ether,
and swirl until oily base dissolves. Add 10 mL 0.05/V H 2 S0 4
and ext epinephrine by shaking 1 min. Drain aq. layer into
125 mL separator, and wash pet ether layer with two 10 mL
portions H 2 0. Add washes to acid ext, wash combined aq.
layers with two 10 mL portions CC1 4 , and discard CCI 4 . Rinse
stopper and mouth of separator with few drops H 2 and let
rinsings drain into separator. Proceed as in 956.08D, begin-
ning "Add 2.10 g NaHC0 3 ..."
(c) Ointments of epinephrine bitartrate {petrolatum base). —
Transfer to separator accurately weighed sample contg ca 60
mg epinephrine bitartrate. Add 25 mL benzene and swirl until
ointment base dissolves. Proceed as in (b), beginning "Add
10 mL 0.05N H 2 S0 4 ..." except if bisulfite is present, it
must be removed with I, as below, before proceeding with
acetylation.
D. Acetylation
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Add 25 mL CC1 4 and shake vigorously to ext chlorobutanol.
After layers sep. completely, drain and discard solv., and re-
peat extn with two 25 mL portions CC1 4 . After each extn, drain
as much solv. as possible. Rinse stopper and mouth of sepa-
rator with few drops of H 2 0, and let rinsings drain into sep-
arator. Add 4 drops starch indicator, 949.15A(b); then, while
swirling, destroy NaHS0 3 by adding l-Kl soln, (e), dropwise
until soln remains blue. Immediately discharge blue color by
adding 0. IN Na 2 S 2 3 dropwise. Add 2. 10 g Na'HC0 3 (prevent
it from contacting wet mouth of separator) and swirl few sec
516
Drugs: Part I
AOAC Official Methods of Analysis (1990)
to dissolve most of NaHC0 3 . Immediately, using hypodermic
syringe, rapidly inject into separator exactly 1 mL Ac 2 (pre-
vent reagent from contacting mouth of funnel). Stopper sep-
arator at once and shake vigorously until evolution of C0 2 stops
(ca 7-8 min). Release pressure as necessary by momentarily
inverting separator and cautiously opening stopcock.
Let mixt. stand 5 min; then ext with six 30 mL portions
CHC1 3 . Filter each ext thru C.HCI3- washed compact pledget of
absorbent cotton into beaker, and evap. combined exts to small
vol. or to dryness on steam bath under air current. Quant,
transfer residue with small portions CHC1 3 to tared 50 mL beaker
and continue evapn until sol v. is removed. Dry 30 min at 105°,
let cool in desiccator, and weigh. Wt mixed amorphous tri-
acetyl derivatives of epinephrine and norepinephrine X 0.5923
= E — "total epinephrine."
£ Chromatographic Separation of Acetyiation Product
(Caution: See safety notes on distillation, flammable solvents,
toxic solvents, benzene, and chloroform.)
Place wad of glass wool in chromatgc tube and compress it
tightly at juncture of tube and stem, using packing rod.
Place 10 g diat. earth and ca 175 mL benzene in 250 mL
beaker. While stirring vigorously and continuously, add 7.0
mL H 2 0, drop wise, to produce uniform solid phase. Transfer
to chromatgc tube ca V10 of the solid, under benzene, and com-
press it firmly and evenly with packing rod. While keeping
column of benzene above solid in tube, add remainder of solid
in beaker in ca 5 equal portions and compress each portion
firmly and evenly before adding next. Properly prepd column
is ca 65 mm high and permits flow of ca 2-4 mL benzene/
min under head of 8 cm sol v. With wad of absorbent cotton
affixed to stiff wire, remove any solid adhering to tube above
column. Keep layer of benzene above column until used.
To beaker contg mixt. of triacetyl derivatives, add exactly
6 mL dry benzene. Warm gently and dissolve residue com-
pletely by stirring and swirling. Cover beaker with watch glass
to retard evapn, and cool to room temp.
Remove supernate benzene from tube by careful aspiration,
pipet onto top of column accurately measured aliquot of soln
of derivative equiv. to 20-25 mg total epinephrine, and im-
mediately place graduate under tube. As soon as last of ben-
zene soln is absorbed by column, rinse down wall of tube with
three 2 mL portions benzene, delivered conveniently from pi-
pet. Let each rinse be completely absorbed before adding next
portion. Then carefully add benzene into tube to ht of ca 8 cm
above top of column, and maintain level of the benzene with
suitable constant level device. After 160 mL effluent (contg
triacetylepinephrine) collects, thoroly rinse tip of tube with
CHCI3 and discard effluent and rinsings, or reserve for qual.
tests.
Remove layer of benzene above column by aspiration, place
clean receiver under tube, and let CHC1 3 pass thru column un-
til 100 mL effluent (contg triacetylnorepinephrine) collects.
Evap. effluent to dryness and transfer to 50 mL beaker, con-
fining residue near bottom of beaker.
F. Determination of Norepinephrine
Add exactly 10 mL 0.50N HC1 to residue of triacetyl-
norepinephrine, warm gently, and dissolve by stirring and rub-
bing with rubber policeman. Pour soln into g-s test tube (15
X 150 mm is convenient), place tube in boiling H 2 bath, and
stopper loosely. After 5 min, stopper tightly and maintain 30
addnl min at 100°. Remove tube and cool to room temp., lift-
ing stopper slightly from time to time to keep vac. from form-
ing-
Thoroly mix contents and transfer .1 mL aliquot to another
g-s test tube. Neutze acid by adding exactly 42 mg NaHC0 3 .
(Ensure that all NaHC0 3 is delivered to bottom of tube, and
that none adheres to wall above acid layer.) After efferves-
cence stops, add 1.5 mL H 2 and 2.5 mL buffer, (d). Mix by
swirling, and add 4 drops 0.17V I. Swirl, and after exactly 3
min (timed by stopwatch) from addn of 1, add 6 drops 0.17V
Na 2 S 2 3 , stopper, and mix thoroly. Measure A at 520 nm, 3
±0.5 min after addn of Na 2 S 2 3 soln, in 1 cm cells against
the pH 6 buffer blank in Beckman Model DU spectrophtr, or
equiv.
Transfer 1.5 mL aliquot std soln to g-s test tube. Add 1 mL
H 2 and 2.5 mL pH 6 buffer, (d), and swirl. Develop color
and measure A' at 520 nm as above.
Calc. amt norepinephrine in sample originally taken for
analysis, and from this value and E, 956. 08D, calc. % nor-
epinephrine in "total epinephrine."
Ref.: J AOAC 39, 639(1956).
CAS-5 1-43-4 (epinephrine)
CAS-5 1-42-3 (epinephrine bitartrate)
CAS-55-31-2 (epinephrine hydrochloride)
CAS-51-41-2 (norepinephrine)
965,42* Phenylephrine Hydrochloride
in Drugs
Acetyiation Method
First Action 1965
Surplus 1970
See 36.108-36.111, 11th ed.
969.49 Phenylephrine Hydrochloride
in Drugs
Colorimetric Method
First Action 1969
Final Action 1970
(Not applicable in presence of tetracycline, acetaminophen,
salicylamide, phenolic compds, and Zn salts)
A. Reagents
Prep, (a), (b), and (d)(2) fresh on day of use.
(a) 4-Aminoantipyrine hydrochloride soln. — (Eastman Ko-
dak, No. 6535) 3% in H 2 0.
(b) Potassium f err icy anide soln. — 4% K 3 Fe(CN) 6 in H 2 0.
(c) Sodium borate soln.— 2% Na 2 B 4 O 7 J0H 2 O in H 2 0.
(d) Phenylephrine hydrochloride std solns. — (/) Stock
soln, — Approx. 2.5 mg/mL. Weigh ca 125 mg phenyleph-
rine. HC1 to nearest 0.1 mg into 50 mL vol. flask and dil. to
vol. with H 2 0. Soln is stable several months under refriger-
ation. (2) Working std soln. — Approx. 0.25 mg/mL. Dil. 5
mL stock soln to 50 mL with H 2 0.
B. Preparation of Samples
(a) Tablets and capsules. — Det. av. wt/unit. Grind tablets
to powder and mix, or mix contents of capsules. Weigh por-
tion contg ca 12.5 mg phenylephrine. HC1 into 50 mL vol. flask,
add ca 30 mL H 2 0, and shake vigorously. Dil. to vol., shake
again, and filter if soln is not clear.
(b) Powders for oral suspensions, oral suspensions, sirups,
solns , etc. — Reconstitute powders for oral suspension as di-
rected on label or use solns, sirups, and oral suspensions as
is. Transfer aliquot contg ca 12.5 mg phenylephrine, HO into
50 mL vol. flask and proceed as in (a).
AOAC Official Methods of Analysis (1990)
Alkanolamines 517
C. Determination
(Reaction is time dependent; assay samples one at a time.)
Transfer 2 mL aliquot sample soln to 50 mL voL flask (omit
sample for reagent blank), add 1.0 mL 4% K 3 Fe(CN) 6 soln,
and swirl. Dil. to ca 48 mL with Na 2 B 2 7 soln and add 1.0
mL aminoantipyrine soln. Immediately dil. to vol. with Na 2 B 2 7
soln and shake vigorously. Immediately det. A of soln at 490
nm against reagent blank, in matched 1 cm cells.
Calc. sample concn, S — CFA/A', where C = mg std/mL,
F — diln factor, and A and A' refer to sample and std, resp.
Report mg phenylephrine. HC1 /tablet, capsule, or vol. liq.
dose.
Refs.: J. Pharm. Sci. 52, 802(1963). JAOAC 52, 500(1969).
CAS-6 1-76-7 (phenylephrine hydrochloride)
971.37 Phenylephrine Hydrochloride
in Drugs
Automated Method
First Action 1971
Final Action 1973
A. Principle
Oxidn products of phenylephrine with K 3 Fe(CN) 6 form col-
ored complex with 4-aminoantipyrine in borate soln. Method
is automated version of 969.49 and is not applicable in pres-
ence of tetracycline, acetaminophen, salicylamide, phenolic
compds, and Zn salts.
B. Apparatus
Automatic analyzer. — AutoAnalyzer with following mod-
ules (available from Technicon Corp.): (/) Sampler 11. — With
50/hr (2:1) cam. (2) Proportioning pump. (3) Colorimeter. —
With 15 mm tubular flowce.ll and matched 490 nm filters. (4)
Recorder. (5) Manifold.
Assemble app. as shown in Fig. 971. 37A. Make sample and
resample pump tubes as short as possible by cutting each end
6 mm from color-coded shoulders. Sample line is 20 cm. Re-
duce vol. at point of debubbling and flowcell by constricting
lower arm of C-5 debubbler with Tygon tubing to give push
fit with 0.015 x 2.5" polyethylene tubing leading to flowcell
as shown in Fig. 971.37B.
Prewash system with H 2 before placing reagent lines in
their appropriate reagent container. When tubes are pumping
satisfactorily and system is equilibrated (ca 15 min), adjust
colorimeter and recorder to produce steady baseline.
C. Reagents
See 969.49A(a), (b), (c), (d)(7) (2.5 mg/mL), and in addn:
Wetting agent. — Add 0.5 mL polyoxyethylene lauryl ether
(Brij 35) to 1 L H 2 0.
D. Preparation of Sample
Proceed as in 969. 49B, except weigh portion contg ca 125
mg in (a) and transfer aliquot (dild if necessary) contg 125 mg
in (b).
E. Stream Flow
See Fig. 971. 37A. Sample solns are withdrawn from sample
cups, segmented with air, and dild in manifold. Stream is de-
bubbled and resampled. Resultant stream is buffered with air-
segmented stream of borate soln. K 3 Fe(CN) 6 is added and mixed,
and color reagent is added and mixed. Stream is debubbled
and A is measured at 490 nm.
F. Determination
Fill 2 mL sample cups with prepd solns and aspirate at 2:1
sample-to- wash ratio, picking up sample with 0.016" stainless
steel probe. Include 2 std solns (2.5 mg/mL) at beginning and
end of each run of 5 samples in duplicate (10 detns). Draw
H9O wash
I UPPER
Ilevel
LOWER , TUBING SIZE (inches)
LEVEL 6
sample
-a>
0.056
0.015
"WW — £TT
105-0086
H 2 Q with Brij
resample
"WW"
105-0086
I sodium borate
"WW H-3 I "*" air
~® ~ 0.Q81
""O^ 0.073
-©
^® 0.025
"® 0.110
0.081
105-0086
®r
(?)
potassium ferr icya nide ^ 0.015
mae ^-
105-0086
4-aminoantipyrine HCf ^) 0.015
*— ^ ©
WASTE.
®
-®"
PROPORTlONtNG
PUMP
COLORIMETER RECORDER
15 mm TUBULAR f/fc
490 nm filters
WASTE
I
s
C-3
FIG. 971 .37 A — Flow diagram for phenylephrine hydrochloride
518 Drugs: Part I
AOAC Official Methods of Analysis (1990)
Tygon 1 /8" id x 1 /4" od
Waste -»--«--•-
XMWwHWJ —
^s
N6 Nipple
Pump Tube .
0.045"
Polyethylene
5-1/2", 0.015x0.043"
Withdrawal Tube to Flow Cell
FIG. 971 .37B— Assembly of debubbler
line between baseline at beginning and end of run, if neces-
sary. Subtract baseline A from max. A to obtain net A (A A)
for each peak. Calc. mg phenylephrine. HCl /unit dose as in
969. 49C from A A, using av. of duplicate detns.
Ref.: JAOAC54, 596, 600(1971).
CAS-6 1-76-7 (phenylephrine hydrochloride)
970.78 Phenylephrine Hydrochloride
In Drugs
Ion-Pair Column Partition Method
First Action 1970
Final Action 1971
(Not applicable in presence of phenolic nitrogenous bases)
A. Apparatus and Reagents
(a) Recording spectrophotometer. — With matched I cm cells.
(b) Chromatographic tubes. — Fuse 6 cm length of 5-6 mm
tubing to piece of 25 mm tubing ca 25 cm long (25 x 200
mm test tube may be used). Constrict stem slightly ca 2 cm
below seal. Com. tubes with dimensions ±10% are satisfac-
tory. Pack wad of Pyrex glass wool in base as support.
(c) Phosphate buffer.— pH 5.80 ± 0.05. Mix 1 vol. \M
K 2 HP0 4 (174 g/L) and 4 vols IM KH 2 P0 4 (136 g/L) and
adjust pH with either component.
(d) Phosphate-citrate buffer, — pH 5.10 ± 0.05. Mix 2 vols
\M K 2 HP0 4 and 1 vol. \M citric acid (192 gQH 8 7 or 210
g C 6 H 8 7 .H 2 0/L) and adjust pH with either component.
(e) Diatomaceous earth. — See 960. 53B.
(f) Bis-(2-ethylhexyl) hydrogen phosphate {DEHP) soln. —
Reagent grade. 2.4% v/v in H 2 0-satd ether. Prep, fresh daily.
(g) Sulfuric acid. — 0.17V, ether-satd. Prep, fresh daily.
(h) Chloroform and ether. — H 2 0-satd. Prep, fresh daily and
use thruout detn.
(i) Phenylephrine hydrochloride std solns . — (7 ) Stock soln . —
1 mg/mL. Accurately weigh ca 100 mg USP Phenyl-
ephrine. HCI Ref. Std in 100 mL vol. flask and dil. to vol.
with H 2 0. (2) Working soln. — 0.04 mg/mL. Dil. 2 mL stock
soln to 50 mL with 0.1N NaOH and use to obtain spectrum
between 400 and 200 nm (or as far as instrument permits) along
with sample detn.
B. Preparation of Samples
(a) Samples containing about J mg phenylephrine .HCl / mL
sirup. — Pipet 4.0 mL pH 5.8 buffer into 10 mL vol. flask.
Carefully add sirup to vol. Do not wet flask above mark.
(b) Samples containing more than J mg phenyl-
ephrine. HCl I mL s/rwp.— Dil. to 1 mg/mL and proceed as in
(a).
(c) Tablets. — Weigh ground sample contg ca 2 mg phenyl-
ephrine into 50 mL beaker. If components of tablets are H 2 0-
soL, add 2 mL H 2 0, warm slightly to dissolve, and add 1 mL
pH 5.8 buffer. If some components are not H 2 0-sol. (e.g.,
acetaminophen), add 1 mL dimethylsulfoxide, warm to dis-
solve, and then add 2 mL pH 5.8 buffer. For tablets contg
antacids (e.g., Mg(OH) 2 and Al(OH) 3 ), heat powd sample contg
ca 2 mg phenylephrine with 5 mL alcohol and 1 mL HCl to
dissolve alk. material; add 10 mL n-BuOH and evap. to dry-
ness. Dissolve residue in 1 mL dimethylsulfoxide and add 2
mL pH 5.8 buffer.
(d) Capsules. — Take portion of contents contg ca 2 mg
phenylephrine and proceed as in (c). Grind sample if neces-
sary.
C. Determination
Pack small glass wool plug in base of chrornatgc tube as
support. Transfer mixt. of 1 g diat. earth with 0.8 mL pH 5.1
buffer to tube and tamp to uniform mass. Mix 4 g diat. earth
with 3.0 mL aliquot prepd sample and carefully transfer di-
rectly above pH 5.1 layer, tamping gently. Dry-wash beaker
with 1 g diat. earth, add to column, and tamp. Cover with
small glass wool pad. Pass 75 mL CHCI 3 thru column fol-
lowed by 125 mL ether, and discard eluates. Place 125 mL
separator contg ca 20 mL 0..I./V H 2 S0 4 as receiver under col-
umn. Elute column with 50 mL DEHP-ether soln and then
with 25 mL ether, collecting in same separator. Shake sepa-
rator and transfer aq. phase to 50 mL vol. flask contg 6 mL
IN NaOH. Re-ext ether with 15 mL 0.1/V H 2 S0 4 . Combine
exts and dil. to vol. with H 2 0. Obtain spectrum between 200
and 400 nm on same day as elution.
D. Calculations
Det. corrected A (A A) of both std and sample as follows:
Construct baseline representing background A extension ob-
tained from 400 to ca 250 nm. (Constructed baseline A value
at wavelength of max. A, ca 290 nm, is designated as A B .)
Subtract A B from total A max observed at wavelength peak.
For std, calc. a = A A 1 /be, where b ~ cell pathlength (1
cm), and c = concn in g/L.
For samples, calc. c - (A A x F)/ab, where F = diln fac-
tor.
Report mg phenylephrine. HCl /tablet, capsule, or vol. liq.
dose.
AOAC Official Methods of Analysis (1990)
Alkanolamines
519
Ref.: JAOAC53, 120(1970).
CAS-6 1-76-7 (phenylephrine hydrochloride)
958.1 2 Phenylpropanolamine
Hydrochloride in Drugs
Extraction Method
Final Action
958.11
A. Apparatus
See 967.31A.
Phenylpropanolamine
Hydrochloride in Drugs
Spectrophotometry Method
Final Action 1965
B. Reagents
(a) Chloroform.— A at 258.5
blank, <0.200.
(b) Diatomaceous earth
nm, measured against H 2
.—See 960.53B
C. Preparation of Column
Fix pledget of glass wool in stem of chromatgc tube above
constriction. Clamp tube vertically. In small beaker mix 3 g
diat. earth and 2 mL H 2 0. Transfer to tube with metal spatula
and press down evenly with packing rod.
D. Determination
(a) Capsules and tablets. — To 150 mL beaker transfer ac-
curately weighed amt of powd sample contg ca 50 mg phenyl-
propanolamine. HC1. Add 5 mL NH 4 OH (1 + 4) and mix by
gentle swirling. Add 5 g diat. earth and mix with metal spat-
ula. Transfer to tube without loss thru powder funnel, in 4 or
more portions, pressing down each portion evenly with pack-
ing rod. When removing funnel from tube each time, tap it
lightly in tube to remove loosely adhering particles; then hang
it in beaker of such size that it does not touch bottom. After
using packing rod, scrape off most of adhering material into
tube with spatula, and tap rod and spatula over mouth of tube.
When laying down implements, place them in position such
that their ends do not touch anything. Finally use smooth, in-
tact rubber policeman to sweep material from beaker and fun-
nel into tube. Rub beaker, spatula, and packing rod with three
ca 1 g portions diat. earth, sweeping each portion thru funnel
into tube, using rubber policeman. Press down each portion
with packing rod.
Place 100 mL vol. flask in receiving position. Wash down
inside of tube with CHC1 3 , adding enough (ca 20 mL) to mois-
ten column and produce only few drops of eluate. Elute with
95 mL CHCI 3 , wash tip of tube with little CHC1 3 , and dil. to
vol. with CHCI3. Measure A at 258.5 nm, 2-5 min after pour-
ing into silica cell, against portion of same CHC1 3 used for
elution.
To 150 mL beaker transfer ca 50 mg pure phenylpropan-
olamine.HCl, accurately weighed. Proceed as with sample,
beginning "Add 5 mL NH4OH (1 + 4) . . ." Det. A of sample
and std eluates at ca same time, on same setting of wavelength
dial. Use same cell for both eluates, and same cell for both
blanks. Calc. phenylpropanolamine. HC1 content.
(b) Aqueous solns. — Prep, column as in958.11C. Into 150
mL beaker pipet vol. sample contg ca 50 mg phenylpropan-
olamine. MCI, or pipet 10 mL, whichever is less. Add 1 mL
NH4O.H and mix by gentle swirling. Add number of g diat.
earth equal to total number mL of liq. and mix with metal
spatula. Proceed as in (a), beginning "Transfer to tube without
loss . . .
Ref.: JAOAC 41, 499(1958).
CAS- 154-4 1-6 (phenylpropanolamine hydrochloride)
Proceed as in 957.21C-D.
Refs.: JAOAC 41, 509(1958); 58, 852(1975).
973.70 Phenylalkanolamine Salts
Including Phenylpropanolamine Hydrochloride and
Ephedrine Sulfate in Elixirs and Sirups
Spectrophotometric Method
First Action 1973
Final Action 1974
(Applicable to individual phenylalkanolamines when only one
is present, except for phenylephrine, which does not interfere
and which is not detd by this method.)
A. Principie
Phenylalkanolamine is eluted with CH 2 Cl 2 from weakly ba-
sic diat. earth column, retained on weakly acidic column, and
converted to benzaldehyde by on-column periodate reaction.
Benzaldehyde is detd by UV spectrometry and is proportional
to amt alkanolamine salt in sample.
B. Reagents and Apparatus
(a) Phosphate-chloride soln. — Dissolve 5 g KH 2 P0 4 and
7.5 g KCl in 100 mL H 2 0.
(b) Sodium metaperiodate soln, — Dissolve 2 g NaI0 4 in 20
mL H 2 0. Store in dark.
(c) Water-saturated methylene chloride. — Sat. ca 400 mL
spectral grade CH 2 Cl 2 by shaking 1 min with equal vol. H 2 0.
Use thruout.
(d) Diatomaceous earth. — See 960. 53B.
(e) Phenylalkanolamine salt std soln. — 0.4 mg/mL. Ac-
curately weigh ca 100 mg phenylalkanolamine salt and dis-
solve and dil. to 250 mL with H 2 0.
(f ) Recording spectrophotometer. — With matched 1 cm cells.
(g) Chromatographic tubes. — Fuse 6 cm length of 5-6 mm
tubing to piece of 25 mm tubing ca 25 cm long (25 X 200
mm test tube may be used). Constrict stem slightly ca 2 cm
below seal. Com. tubes with dimensions ±10% are satisfac-
tory. Pack wad of Pyrex glass wool in base as support.
(h) Tamping rod. — Flatten end of glass rod to circular head
with clearance of ca 1 mm in tube (a). Or use disk of stainless
steel, Al, etc., of diam. ca 1 mm less than id of column, (a),
attached to 30-45 cm (12-18") rod.
C. Preparation of Sample and Chromatographic Columns
Sample. — Accurately dil. sample with H 2 to final concn
of ca 0.4 mg/mL.
Column /.—Add 2.0 mL dild sample to 300 mg K 2 HP0 4
in 150 mL beaker. Swirl to dissolve. Add 3 g diat. earth, mix,
transfer quant, to column, and tamp. Dry- wash beaker with 1
g diat. earth, add wash to column, and tamp. Cover with small
glass wool plug. If acidic compds such as acetaminophen or
theophylline are present, underlay sample-diat. earth mixt. with
mixt. of 3 g diat. earth and 2 mL 10% NaOH.
Column II. — Mix 3 g diat. earth and 2 mL phosphate-chlo-
ride soln, and transfer to column. Tamp and cover with small
glass wool pad.
Column HI. — Mix 0.5 mL H 2 and 1 g diat. earth, transfer
520
Drugs: Part I
AOAC Official Methods of Analysis (1990)
to column, and tamp. Mix 3 g diat. earth and 2 mL NaI0 4
soln, transfer to column, tamp, and cover with glass wool pad.
D. Preparation of Standard
Prep. sep. column /// as above. Mix 2.0 mL phenyl alkan-
olamine salt std soln and 3 g diat. earth, transfer quant, to
column, and tamp. Dry-wash beaker with 1 g diat. earth, transfer
wash to column, tamp, and cover with glass wool pad. Place
100 mL vol. flask under column. Wet column with 10 mL
CH 2 C1 2 . With pipet, evenly distribute 1.0 mL NH 4 OH onto
surface of column packing. Elute column with four 25 mL
portions CH 2 C1 2 ; let each portion sink entirely into surface.
Rinse tip of column into flask and dil. eluate to vol. with CH 2 C1 2 .
Elute with addnl 25 mL CH 2 C1 2 and collect eluate for use as
blank.
E. Determination
Mount columns so that eluate from / flows onto //. Elute
combined columns with four 25 mL portions CH 2 C1 2 ; let each
portion sink entirely into surface of both columns. Rinse tip
of Column / into // with CH 2 C1 2 and discard /. Elute Column
// with addnl 25 mL CH 2 C1 2 . Discard all eluates.
Mount Column // above /// and place 100 mL vol. flask
under ///. With pipet, evenly distribute 1.0 mL NH 4 OH onto
surface of Column // packing. Elute combined columns with
four 25 mL portions CH 2 C1 2 ; let each portion sink entirely into
surface of each column. Rinse tip of Column // into /// with
ca 1 mL CH. 2 C1 2 . Discard Column 11 and continue to elute
Column /// until 100 mL eluate is collected.
Scan spectra of sample and std eluates from 350 to 230 nm,
against column blank eluate. If liq. is cloudy, let soln clear
(ca 1 min) before detg A. Calc. net A for sample and std solns,
A A and A/4', resp., at min. A, ca 267 nm, and max. A, ca
246 nm.
mg Phenylalkanolamine salt/mL = {A A/ A A') X C x F
where C = mg std/mL and F = diln factor.
Refs.: JAOAC 56, 100(1973); 58, 852(1975).
CAS- 134-72-5 (ephedrine sulfate)
CAS- 1 54-4 1-6 (phenylpropanolamine hydrochloride)
PHENETHYLAMINES
954.1 3* Amphetamine Drugs
Final Action 1965
Surplus 1973
A. Titrimetric Method
See 38.116, 12th ed.
B. Confirmatory Gravimetric Determination
See 38.117, 12th ed.
954.1 4* Amphetamine Drugs
Stereochemical Composition
Final Action
Surplus 1977
See 38.127-38.128, 13th ed.
972.47 Amphetamine in Drugs
Gas Chromatographic Method
First Action 1972
Final Action 1973
A. Apparatus
(a) Gas chromatograph. — Equipped with flame ionization
detector. Operate at sensitivity such that proline derivative from
10 |xg d-amphetamine sulfate gives peak 70-90% full scale.
Adjust injection zone and detector temps to 230°.
(b) Column.— Glass, 2 m x 4 mm id, packed with 1% Car-
bowax 20M on 80-100 or 100-120 mesh Gas-Chrom Q (Ap-
plied Science). Condition column 24 hr at 210° before use.
Operate at 185° and adjust N carrier gas flow rate so that pro-
line derivative of ^-amphetamine is eluted in ca 15 min (ca 60
mL/min).
(c) Chromatographic tube. — 22 x 300 mm, without stop-
cock.
B. Reagents
(a) Diatomaceous earth. —See 960. 53B.
(b) Proline reagent. — (Prep, reagent at <30°.) To 1.0 g /-
proline (Sigma Chemical Co.) in 125 mL g-s conical flask,
add 5 g trifluoroacetic anhydride and swirl until proline is dis-
solved. Evap. excess trifluoroacetic anhydride under stream of
dry N. Add 5 mL thionyl chloride, Jet stand 15 min, and evap.
excess under stream of dry N. Dissolve residue in 100 mL
CH 2 C1 2 and refrigerate when not in use. Properly prepd reagent
will give /-amphetamine ratio, 972. 47D, <0.02 with ^-am-
phetamine sulfate. {Caution: Trifluoroacetic anhydride and
thionyl chloride are toxic. Wear rubber gloves and eye pro-
tection and use effective fume removal device for evapn.)
(c) Std soln I. — 0,5 mg USP Ref. Std Dextroamphetamine
Sulfate /mL H 2 0.
(d) Std soln 11. — 0.5 mg ^/-amphetamine sulfate (Sigma
Chemical Co., No. A1263)/mL H 2 0. Recrystallize ^/-am-
phetamine sulfate from alcohol and dry under vac. at 100° be-
fore use.
C. Preparation of Wash Coiumn
Mix 3.0 mL IN NaOH with 5.0 g diat. earth and transfer
to chromatgc tube contg small glass wool plug. Place glass
wool pad on top of diat. earth and tamp firmly.
D. Preparation of Standard Curve
Prep, series of /-amphetamine sulfate stds contg total of ca
10 mg amphetamine sulfate in 20 mL H 2 in 125 mL sepa-
rators as follows: 0% /-amphetamine sulfate from 20 mL std
soln / and mL std soln //; 12.5%, 15 mL / and 5 mL //;
25%, 10 mL / and 10 mL //; 37,5%, 5 mL / and 15 mL //;
and 50%, mL / and 20 mL //. Treat each soln as follows:
Add 5 mL 17V NaOH and ext with two 25 mL portions CHC1 3 .
Filter CHC1 3 exts thru absorbent cotton into dry 1 50 mL beaker.
Add 3 mL proline reagent, (b), and let stand 30 min; then
transfer to wash column. Collect eluate in 150 mL beaker.
Pass addnl 25 mL CHC.I 3 thru column. Evap. on steam bath
with aid of N stream, dissolve residue in 5 mL CHC1 3 , and
transfer to g-s flask. Inject 5 |±L and calc.
/-amphetamine ratio = H { X R } /[(H d x R (I ) + (H l X /?,)]
where H and R — peak ht and retention time, resp., of d- and
/-amphetamine derivatives. Plot /-amphetamine ratio against %
/-amphetamine,
E. Determination
Transfer finely powd sample contg ca 10 mg amphetamine
sulfate to 100 mL beaker, add 2 g diat. earth, and mix. Add
3.0 mL \N NaOH and mix to uniform slurry. Add 3.0 g diat.
AOAC Official Methods of Analysis (1990)
Aminobenzoates
521
earth, mix well, and pack into chromatgc tube contg small
glass wool plug. Place 150 mL beaker under column and elute
with 100 mL CHC1 3 . Proceed as in 972.47D, beginning "Add
3 mL proline reagent, (b), ..." Calc. /-amphetamine ratio
and det. /-amphetamine from std curve.
Ref.: JAOAC 55, 146(1972).
CAS-5 1-63-8 (d-amphetamine sulfate)
CAS-60-13-9 (d/- amphetamine sulfate)
Amphetamine in Presence
of Antihistamines and Barbiturates and Other Drugs
See 974.39.
Mephentermine Sulfate in Drugs
See 978.28.
Calc. absorptivity differential (A a) produced by 1 g/L (1
mg/mL) of std amine base or salt:
Atf std = AA std x 100/wt std
where AA sld = A difference for std soln; 100 = mL std soln
measured; and wt = mg std in aliquot measured.
mg Amine /unit of sample
=(A A samp]e x 25 x 25 x av. wt of unit in mg)/
(Afl sld X 10 X wt sample in mg)
Refs.: JAOAC 40, 824(1957); 41, 509(1958); 48, 170(1965);
49, 166(1966).
CAS-300-62-9 (amphetamine)
CAS-299-42-3 (ephedrine)
CAS- 100-92-5 (mephentermine)
CAS-537-46-2 (methamphetamine)
CAS- 1 483 8- 1 5 -4 (phenylpropanolamine)
CAS-93-88-9 (phenylpropylmethylamine)
957.21 Phenethylamines in Drugs
Spectrophotometric Method
Final Action 1965
(Applicable to amphetamine, methamphetamine, mephenter-
mine, phenylpropylmethylamine (Vonedrine®), ephedrine, and
phenylpropanolamine (Propadrine®))
A. Apparatus
Spectrophotometer . — Suitable for measurement in region
250-270 nm; with 1 cm cells of quartz or fused Si (preferably
matched pair); or recording spectrophtr.
B. Preparation of Standard Solution
Accurately weigh 500-700 mg phenethylamine salt of known
purity, transfer to 100 mL vol. flask, and dissolve in 0.17V
H 2 S0 4 . Dil. to vol. with the H 2 S0 4 and mix well.
C. Determination
Accurately weigh powd sample contg 25-50 mg amine base
and transfer to 40-50 mL g-s centrf. tube contg 3-3.5 g NaCl
and 6-7 glass beads. Dissolve sample by adding 5 mL \N
H 2 S0 4 , and swirl gently to aid escape of any liberated C0 2 .
Test for acidity with litmus paper, adding more acid if nec-
essary. Pipet in 25 mL CHC1 3 and 4 mL 27V NaOH, stopper
securely, and shake 3-5 min. To second 40-50 mL centrf.
tube contg 3-3.5 g NaCl and 6-7 glass beads, add 10 mL std
soln, 957. 21B. Swirl to dissolve salt, pipet in 25 mL CHC1 3
and 1 mL 27V NaOH, stopper securely, and shake 3-5 min.
Centrf. tubes 3-5 min at 1500-1800 rpm. Withdraw 10 mL
clear CHC1 3 layer by closing upper end of 10 mL pipet with
index finger while lowering tip thru aq. layer. Wipe off outer
portion of pipet, and transfer 10 mL CHC1 3 layer to second
40-50 mL centrf. tube contg 25 mL 0.17V H 2 S0 4 and 6-7
glass beads. Stopper securely, shake, and centrf. as above.
Prep, acid blank soln by shaking 25 mL 0. IN H 2 S0 4 with
3-5 mL CHCI 3 and centrfg to obtain clear acid soln. Read A
of portions of clear acid soln obtained from aliquots of std and
of sample solns against acid blank prepd as above in ref. cell,
using 1 cm cells in range 252-255 for first min., 256-258 for
max., and 260-262 for second min.
D. Calculations
Calc. A difference (A A) between A at maximum and A of
2 minima: AA = A max - 0.5(A min , + A min2 ).
AMINOBENZOATES
968.40 Benzocaine in Drugs
Colorimetric Method
First Action 1968
Final Action 1969
A. Principle
Benzocaine is diazotized with NaN0 2 , excess nitrite is re-
moved with NH 4 sulfamate, and product is coupled with 7V-1-
naphthylethylenediamine.2HCl. Colored soln has max. at 540
nm. Method is not applicable in presence of sulfonamides.
Benzocaine must be sepd from inorg. I to avoid interference.
Antipyrine in 10-fold excess does not interfere.
B. Reagents
Benzocaine std solns. — (1) Stock soln. — 0.25 mg/mL. Dis-
solve 25.0 mg benzocaine USP in 25-50 mL H 2 in 100 mL
vol. flask. Add 3 mL HC1, shake gently, and dil. to vol. with
H 2 0. (2) Working soln. — 5 (xg/mL. Pipet 10 mL stock soln
into 100 mL vol. tlask and dil. to vol. with H 2 0. Pipet 20 mL
of this soln into 100 mL vol. flask and dil. to vol. with H 2 0.
C. Preparation of Standard Curve
Pipet 0.0, 2.0, 6.0, and 10.0 mL working std soln into sep.
25 mL vol. flasks. To each flask add 1 mL HC1 (1 + I). Dil.
to 15 mL with H 2 0, add 1 mL 0.1% NaN0 2 soln, prepd fresh
daily, mix, and let stand 5 min, swirling several times during
standing. Add 1 mL 0.5% NH 4 sulfamate soln, and let stand
5 min, swirling several times during standing. Add 1 mL col-
orless 0.1% 7V-l-naphthylethylenediamine.2HCl soln, (prep.
fresh weekly and store in dark glass bottle in refrigerator), let
stand 15 min, swirling several times during standing, and dil.
to 25.0 mL with H 2 0.
Det. A of each soln in matched 1 cm cells in spectrophtr at
540 nm against H 2 as ref. (Avoid collection of N bubbles on
cell walls.) To obtain A A, subtract reading of soln contg no
std from each of other std readings. Plot A. A against benzo-
caine concn.
D. Preparation of Samples
(a) Liquid preparation in water-soluble bases. — Weigh
sample contg 100 mg benzocaine, transfer to 250 mL vol. flask,
and add 75 mL alcohol. Add 3 mL HCl, dil. to vol. with H 2 0,
and mix well. Pipet 10 mL into 100 mL vol. flask, dil. to vol.
522
Drugs: Part I
AOAC Official Methods of Analysis (1990)
with H 2 0, and mix well. Pipet 10 mL of diln into 100 mL
vol. flask, dii. to vol. with H 2 0, and mix well.
(b) Tablets or troches. — Weigh powd sample contg 7.5-
10 mg benzocaine. Transfer to 100 mL beaker, wet with 2-3
mL alcohol, stir with glass rod to slurry, add 5 mL H 2 and
2 mL HO, stir, and let stand at room temp. 5 min. Dil. with
25 mL H 2 0, transfer quant, to 100 mL vol. flask, and dil. to
vol. If soln is cloudy, filter thru dry paper, discarding first 10
mL filtrate. Pipet 10 mL clear filtrate into 100 mL vol. flask,
dil. to vol. with H 2 0, and mix well.
(c) Suppositories in water-soluble bases.— Weigh sample
contg 50-100 mg benzocaine into 100 mL beaker, add 15 mL
H 2 and 3 mL HO, and let stand at room temp. 15 min, stir-
ring occasionally with rod. Transfer quant, to 250 mL vol.
flask, dil. to vol., and mix well. Pipet 10 mL into 100 mL
vol. flask, dil. to vol., and mix well. Pipet 10 mL of diln into
100 mL vol. flask, dil. to vol., and mix well.
E. Determination
Pipet 2 aliquots of final diln specified in 968.40D contg 20-
30 |xg benzocaine into sep. 25 mL vol. flasks. Label one flask
"sample" and the other "sample blank." Proceed as in 968.40C,
beginning "To each flask add 1 mL HO (1 + 1)." except do
not add NaN0 2 to "sample blank."
Det. A at 540 nm for each soln against H 2 as ref. Subtract
A of "sample blank" from "sample" reading. Det. |xg ben-
zocaine in aliquot from std curve.
% Benzocaine = (B x F)/(W x 10)
where B — jxg benzocaine from std curve, F — diln factor,
and W - mg sample.
Ref.: JAOAC51, 612(1968).
CAS-94-09-7 (benzocaine)
968.41* Benzocaine in Drugs
Bromination Method
Final Action
Surplus 1975
See 38.139, 12th ed.
968.42 Benzocaine and Antipyrine
in Drugs
Spectrophotometric Method
First Action 1968
Final Action 1969
(Applicable in presence of glycerol and propylene glycol bases)
A. Principle
Benzocaine and antipyrine are extd by column partition
chromatgy. Antipyrine is retained on FeCl 3 column and ben-
zocaine on HO column. Max. A are detd in CHC1 3 eluates at
272 nm for antipyrine and 283 nm for benzocaine. To identify
compds, 1R spectra of KBr dispersions are compared to stds.
B. Apparatus and Reagents
(a) Chromatographic tubes. — Fuse 6 cm length of 5-6 mm
tubing to piece of 25 mm tubing ca 25 cm long (25 X 200
mm test tube may be used). Constrict stem slightly ca 2 cm
below seal. Com. tubes with dimensions ±10% are satisfac-
tory. Pack wad of Pyrex glass wool in base as support.
(b) Tamping rod. — Flatten end of glass rod to circular head
with clearance of ca 1 mm in tube (a). Or use disk of stainless
steel, Al, etc., of diam. ca 1 mm less than id of column, (a),
attached to 30-45 cm (12-18") rod.
(c) Syringe. — 10 mL syringe with 14 gage 4" laboratory
cannula.
(d) Ferric chloride .—9% . Dissolve 9 g anhyd. FeCl 3 in H 2
and dil. to 100 mL.
(e) Diatomaceous earth. — See 960. 53B.
(f ) Chloroform, washed. — Shake 4 vols CHCl^ with 1 vol.
H 2 0.
(g) Mixed solvent.— H 2 0-satd CHCl 3 -ether-isooctane (10
+ 25 + 65).
(h) Antipyrine std soln. — 1 mg/1.00 mL. Accurately weigh
antipyrine std, previously dried 2 hr at 60°, and dil. with CHC1 3
to give concn of 0.1 mg/mL. Pipet 10 mL into 100 mL vol.
flask contg 10 mL MeOH and 1.0 mL mixed solv., and dil.
to vol. with washed CHC1 3 .
(i) Benzocaine std soln. — 0.4 mg/100 mL. Accurately weigh
USP Benzocaine Ref. Std, previously dried 3 hr over P 2 5 ,
and dil. with CHC1 3 to concn of 0.04 mg/mL. Pipet 10 mL
into 100 mL vol. flask contg 10 mL MeOH and 1.0 mL mixed
solv., and dil. to vol. with washed CHC1 3 .
(j) Photometric blank. — Pipet 10 mL MeOH and 1 mL
mixed solv. into 100 mL vol. flask, dil. to vol. with washed
CHC1 3 , and mix.
(k) Potassium bromide. — Anhyd. spec troph trie grade.
C. Sample Density
(Altho sample is weighed because of viscosity of prepns,
report results on wt/vol. basis.)
Slowly withdraw 10 mL sample with syringe, keeping air
bubbles to min., and transfer to previously weighed 10 mL
vol. flask without touching sides of flask above mark. Let any
air bubbles present rise before filling to final vol. Weigh flask
and contents, and calc. sample density.
D. Preparation of Chromatographic Columns
Loosely pack small amt glass wool uniformly in base of 3
chromatgc tubes to support diat. earth.
Bottom column. — Mix 3 g diat. earth with 2 mL 2/V HC1 to
form uniform fluffy mixt. Transfer mixt. to column and tamp
firmly to uniform mass.
Middle column. — Mix 5 g diat. earth with 3 mL FeCl 3 soln
and transfer to column as above.
Top column. — Accurately weigh sample contg 20 mg an-
tipyrine into 100 mL beaker. Add 2 mL H 2 and then 3 g
diat. earth. Mix thoroly, transfer to column, and tamp firmly
to uniform mass. Dry- wash beaker with 1 g diat. earth and
tamp as above.
Top each column with small loose pad of glass wool.
E. Determination
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
(a) Separation of antipyrine and benzocaine.-— Arrange 3
columns in series. Rinse beaker that contained sample with 50
mL mixed solv. and transfer to top column. Elute columns
with 3 addnl 25 mL portions mixed solv. Discard eluate. (Mid-
dle column contains antipyrine and bottom column contains
benzocaine.) Sep. columns, and elute middle and bottom col-
umns sep. with four 25 mL portions washed CHC.I3, collecting
eluates in 100 mL vol. flasks. Dil. to vol. with washed CHC1 3 .
(b) Determination of antipyrine. — Pipet 5 mL from anti-
pyrine flask into 100 mL vol. flask contg 10 mL MeOH and
dil. to vol. with washed CHC1 3 . Det. A of final diln against
photometric blank, (i), at 272 nm, using 1 cm cells. Similarly,
AOAC Official Methods of Analysis (1990)
Aminobenzoates
523
det. A' of std antipyrine soln and calc. amt of antipyrine in
sample.
(c) Determination of benzocaine. — Pipet aliquot of el u ate
contg 0.4 mg benzocaine into 100 ml vol. flask contg 10.0
mL MeOH and diJ. to vol. with washed CHC1 3 . Det. A of final
diln against photometric blank, (i), at 283 nm. Similarly, det.
A' of std benzocaine soln and calc. amt of benzocaine in sam-
ple.
(d) Identification of antipyrine and benzocaine. — Transfer
remaining CHC1 3 eluates from antipyrine and benzocaine sepn
to sep. 150 mL beakers. Place beakers in 30-40° H 2 bath
and evap. to dryness with gentle air current. {Caution: Ben-
zocaine is volatile.)
Prep. KBr disk of each residue, using 0.8 mg residue and
200 mg KBr. Record IR spectrum of each between 2 and 16
(xm and qual. compare these spectra with IR spectra of anti-
pyrine and benzocaine stds.
Ref.: JAOAC 51, 496, 624(1968).
CAS-60-80-0 (antipyrine)
CAS-94-09-7 (benzocaine)
949.15 Butacaine Sulfate in Drugs
Final Action
A. Reagents
(a) Potassium iodide soln. — 20%. Prep, fresh.
(b) Starch indicator. — Make 1 .5 g sol. starch into paste with
few mL H-,0, and add slowly, with stirring, to 300 mL boiling
H 2 0.
(c) Picrolonic acid soln. — 2.5% in alcohol.
(d) Potassium bromide-br ornate soln. — 0.1N. Prep, as in
947. 13A. Stdze as follows: Transfer 30 mL to I flask, and add
25 mL H 2 0, 5 mL 20% KI soln, and 5 mL HO. Shake thoroly
and titr. with 0.1N Na 2 S 2 3 , using starch indicator (mix ca 2
g finely powd. potato starch with cold H 2 to thin paste; add
ca 200 mL boiling H 2 0, stirring const., and immediately dis-
continue heating. Add ca 1 mL Hg, shake, and let soln stand
over the Hg).
B. Determination
(Caution: See safety notes on distillation, toxic solvents,
and chloroform.)
(a) Ointments containing butacaine sulfate in petrolatum or
other greasy base. — Into 125 mL separator accurately weigh
sample contg ca 50 mg butacaine sulfate. Add 25 mL benzene
and swirl until ointment base dissolves; then add 10 mL HC1
(1 + 7) and shake separator gently ca 1 min. Let layers sep.,
drain aq. phase into second separator, and repeat extn 4 times
with 10 mL portions H 2 0. Wash combined aq. exts with 5 mL
CC1 4 and discard washing. Neutze soln with NH 4 OH, add 2
mL excess, and ext butacaine base by shaking with five 15
mL portions CHC1 3 . Filter each ext thru cotton pledget into
100 mL beaker, and evap. combined exts on steam bath under
air current until no CHC1 3 odor remains.
Rinse down beaker wall with 2 mL alcohol delivered from
pipet, warm until oily base dissolves completely, and add 1
drop HO. Tilt and rotate beaker to wet with acidic soln any
liq. on wall of beaker, and add 1 drop Me red. If soln does
not react strongly acid, add addnl HC1 dropwise. DiJ. with few
mL H 2 0, and wash quant, into 500 mL I flask with more H 2 0.
To soln add, from pipet, 10 mL KBr-KBr0 3 soln, dil. to
200 mL with H 2 0, and add 10 mL HO. Immediately stopper
flask and swirl 5 min or until ppt coagulates. After 5 min, add
5 mL KI soln to flask, stopper, and shake vigorously. Rinse
stopper and neck of flask with little H 2 0, and titr. soln with
0. WNa 2 S 2 3 , 942.27A, until color is discharged. Add 15 mL
starch indicator and 20 mL CHC1 3 , stopper flask, and shake
vigorously. Continue titrn, vigorously shaking stoppered flask
after each addn of Na 2 S 2 3 soln. Add Na 2 S 2 3 soln dropwise
as end point approaches. (During titrn, mixt. passes thru series
of color changes; at end point aq. phase is colorless and emul-
sified CHO3 layer is nearly so.) 1 mL 0AN KBr-KBr0 3 =
0.00889 g (C 18 H 3 oN 2 2 ) 2 .H 2 S0 4 .
To isolate bromination product for identification, transfer titrd
mixt. to separator, make alk. with NH 4 OH, and shake vig-
orously. Drain emulsified CHC1 3 layer, and to break emulsion,
filter with suction thru 0.5 cm layer Hyflo Super-Cel (or sim-
ilar filter-aid) supported on paper in buchner. Shake aq. phase
remaining in separator with 25 mL CHC1 3 , and pass CHCl 3
ext thru filter. Transfer combined filtrates to separator, filter
CHC1 3 layer thru cotton pledget into beaker, and evap. on steam
bath under air current.
To oily residue of dibromobutacaine add 2 mL picrolonic
acid soln and stir. Filter ppt on Hirsch funnel, wash with 2-
3 mL alcohol, dry at 105°, and det. capillary mp, alone and
in admixt. with authentic dibromobutacaine picrolonate (mp
158-160° with decomposition). If ppt does not form on adding
picrolonic acid soln to bromination product, seed with small
crystal of dibromobutacaine picrolonate; if ppt still does not
form, butacaine is absent.
(b) Tablets. — Det. av. wt /tablet. To 125 mL separator add
accurately weighed, finely powd tablet mixt. contg ca 200 mg
butacaine sulfate, add 25 mL H 2 0, and swirl separator until
sample dissolves. Add 2 mL NH 4 OH and ext with six 15 mL
portions CHC1 3 . Shake each ext with 5 mL H 2 in second
separator, and then filter thru cotton pledget into beaker. (If
emulsion forms in aq. phase in first separator, more than 6
extns may be required. Test for complete extn by evapg sev-
enth ext on steam bath; if appreciable residue is obtained, dis-
solve it in CHCI3, combine with previous exts, and continue
extns until complete. If aq. phase in first separator tends to
emulsify, break emulsion by addn of Na 2 S0 4 or by other means.)
Evap. filtrate to small vol. on steam bath and complete detn
by one of following methods:
(7) Quant, transfer coned soln of butacaine base to tared 50
mL beaker with CHC1 3 , remove sol v. on steam bath in air
current, dry 30 min at 105°, cool in desiccator, and weigh. Wt
residue x 1.160 = wt butacaine sulfate, (Ci 8 H 30 N 2 O 2 ) 2 .H 2 SO 4 .
Gravimetric detn may be checked acidimetrically as follows:
Rinse down wall of beaker with 2 mL neut. alcohol delivered
from pipet, warm beaker on steam bath until butacaine base
dissolves completely, add 1 drop Me red, and rinse down beaker
wall with another 2 mL alcohol. Titr. soln with 0.1 N H 2 S0 4 ,
890.01, almost to point of color change; rinse down wall of
beaker with H 2 0, dil. to ca 45 mL, and complete titrn. 1 mL
0.LV H 2 S0 4 - 0.0355 g (C 18 H 30 N 2 O 2 ) 2 .H 2 SO 4 .
(2) Det. gravimetrically as in (1); then proceed as in (a),
beginning "Rinse down beaker wall with 2 mL alcohol ..."
except use 50 mL instead of 10 mL KBr-KBr0 3 soln.
(3) Completely remove solv. on steam bath, and proceed as
in (7), second par. Then wash titrd soln into 500 mL I flask,
pipet in 50 mL KBr-KBr0 3 soln, dil. to 200 mL with H 2 0,
add 10 mL HO, and proceed as in (a), beginning "Immedi-
ately stopper flask ..."
(c) Crystals. — Accurately weigh ca 200 mg sample into 125
mL separator, add 25 mL H 2 0, and swirl separator until sam-
ple dissolves. Continue as in (b), beginning "Add 2 mL NH 4 OH
(d) So Ins. — Transfer to 125 mL separator aliquot contg ca
200 mg butacaine sulfate, and if necessary, dil. to 25 mL with
H 2 0. Proceed as in (b), completing detn by (b)(1), (2), or (3)
524
Drugs: Part I
AOAC Official Methods of Analysis (1990)
if chlorobutanol is absent, and only by (b)(3) if chlorobutanol
is present.
Refs.: JAOAC 32, 548(1949); 33, 206(1950).
CAS- 1 49- 15-5 (butacaine sulfate)
Procaine in Drugs
Qualitative Tests
See 930.40.
975.57 Procaine in Drugs
Quantitative Methods
First Action 1975
Final Action 1976
Method I— with or without Propoxycaine
(Applicable in presence of parabens and phenolic
vasoconstrictors)
A. Apparatus and Reagents
(a) Chromatographic tubes. — Fuse 6 cm length of 5-6 mm
tubing to piece of 25 mm tubing ca 25 cm Jong (25 X 200
mm test tube may be used). Constrict stem slightly ca 2 cm
below seal. Com. tubes with dimensions ± 10% are satisfac-
tory. Pack wad of pyrex glass wool in base as support.
(b) Tamping rod. —Flatten end of glass rod to circular head
with clearance of ca 1 mm in tube (a). Or use disk of stainless
steel, Al, etc., of diam. ca 1 mm less than id of column, (a),
attached to 30-45 cm (12-18") rod.
(c) Bromide -citrate buffer. — Mix equal vols 0.5M Na ci-
trate (147 g 2H 2 0/L) and 0.5A/ citric acid (105 g 1H 2 0/L).
Adjust to pH 4.0 ± 0.2, using pH meter, by addn of appro-
priate citrate soln. Add 10.3 g NaBr/100 mL soln (l.OAf Br)
and mix.
(d) Chloroform-isooctane solvent. — 65% CHCI3 in isooc-
tane. Do not sat. with H 2 before use. Dil. 65 parts CHC1 3
in graduate to 100 parts with isooctane and mix.
(e) Phosphate buffer.— p\\ 7.0 ± 0.2. Mix equal vols 0.5M
KH 2 P0 4 (68.0 g/L) and 0.5M K 2 HP0 4 (87.1 g/L).
(f ) Procaine hydrochloride and propoxycaine hydrochlo-
ride std solns. — Prep. sep. aq. solns contg 10 (xg/mL.
B. Preparation of Sample and Column
Pipet 1 mL sample soln contg 20 mg procaine. HC1 or 4 mg
propoxycaine. HCI into beaker, add 1.0 mL phosphate buffer,
and mix. Add 3 g diat. earth, 960. 53R, and mix.
Place small glass wool plug in base of chromatgc tube. Mix
4 g diat. earth and 3 mL bromide-citrate buffer in small beaker,
transfer to column, and tamp with gentle pressure. Mix 2 g
diat. earth and 1 mL 0.1N NaOH, add to column, and tamp.
Quant, transfer sample mixt. to column and tamp. Scrub beaker
with 1 g diat. earth and 2-3 drops phosphate buffer, add to
column, and tamp. Cover with pad of glass wool.
C. Determination
(Perform elution for propoxycaine even in its absence.)
(a) Propoxycaine. — Place 200 mL vol. flask under column
and elute with 150 mL 65% CHC1 3 in isooctane. Dil. to vol.
with C.HC.I3 and mix. Pipet 10 mL aliquot into 100 mL beaker,
add 4 drops HO Ac, and evap. nearly to dryness on steam bath
under gentle air current, then to dryness with reduced heat.
Pipet 20 mL H 2 into beaker and dissolve residue.
(b) Procaine. — Place 200 mL vol. flask under column and
elute with 125 mL CHC1 ? . Dil. to vol. with CHC1 3 and mix.
Pipet 10 mL aliquot into 100 mL beaker and evap. to dryness
as in (a), but without addn of HO Ac. Dissolve residue in H 2
and transfer quant, to 100 mL vol. flask, dil. to vol. with H 2 0,
and mix.
(c) Spectrophotometry . — Record spectra of sample and std
solns from 350 to 250 nm and det. A of samples and A' of
stds at max., ca 302 and 290 nm, for propoxycaine and pro-
caine, resp.
C - C {A/A')D
where C and C = concns (|xg/mL) of sample and std, resp.,
and D — appropriate diln factor.
Ref.: JAOAC 58, 88, 93(1975).
CAS-59-46-1 (procaine)
CAS-5 1-05-8 (procaine hydrochloride)
CAS-86-43-1 (propoxycaine)
CAS-550-83-4 (propoxycaine hydrochloride)
D. Method II*
—Surplus 1975
(Dets as procaine any p-aminobenzoic acid
formed by decomposition)
See 38.211, 12th ed.
E Method III
(Dets only undecomposed procaine)
See 961. 18B
F. Method IV*
Surplus 1975
(Applicable in presence of chlorobutanol, cocaine, codeine,
heroin, lactose, and morphine)
See 32.096, 10th ed.
976.32 Procainamide Hydrochloride
in Drugs
Spectrophotometric Method
First Action 1976
Final Action 1982
A. Principle
Procainamide is extd from acid soln with CHC1 3 . After evapn
of solv,, residue is dissolved in alk.
ca 272 nm.
soln and max. A detd at
S. Reagent
Procainamide hydrochloride std solns. — Stock soln. — 1 mg/
mL. Assay std as in USP XX. Accurately weigh ca 100 mg
procainamide. HCi (1CN-K&K Laboratories, Inc., A Div. of
ICN Biomedicals, Inc., PO Box 28050, Cleveland, OH 44128-
0250, No. 17158) into 100 mL vol. flask, and dissolve and
dil. to vol. with O.OliV NaOH. Working soln. — 1 mg/100 mL.
Pipet 1 mL stock soln into 100 mL vol. flask and dil. to vol.
with O.OliV NaOH. Prep, fresh daily.
C. Preparation of Sample
(a) Capsules. — Proceed as in 927. 09B. Mix, and transfer
accurately weighed portion contg ca 100 mL procaina-
mide. HCI to 125 mL separator. Add 10 mL HCI (1 + 9) and
shake to disperse. Add 15 mL H 2 0.
(b) Tablets. — Det. av. wt. Reduce tablets to fine powder.
AOAC Official Methods of Analysis (1990)
Synthetics 525
(For tablets with coatings that do not reduce to powder, care-
fully peel off and discard coatings, and reduce tablets to fine
powder.) Mix, and transfer accurately weighed portion contg
ca 100 mg procainamide. HCl to 125 mL separator. Add 10
mL HC1 (1 + 9) and shake to disperse. Add 15 mL H 2 0.
(c) Injections. — Dil. soln, if necessary, with HC1 (1+9)
to give ca 100 mg procainamide. HCl/mL. Pipet 1 mL into
125 mL separator, add 10 mL HCl (1 + 9), and shake. Add
15 mL H 2 0.
D. Determination
(Caution; See safety notes on distillation and chloroform.)
Ext sample soln with three 25 mL portions CHC1 3 and dis-
card CHC1 3 . To aq. soln add 5 mL NH 4 OH and ext with five
25 mL portions CHC1 3 , collecting exts in 250 mL beaker and
rinsing tip of separator into beaker after each extn. Evap. CHC1 3
to dryness on steam bath with air current. Quant, transfer res-
idue to 100 mL vol. flask with 0.0LV NaOH, and dil. to vol.
with same solv. Pipet 1 mL dild soln into another 100 mL vol.
flask and dil. to vol. with 0.0 IN NaOH. Record spectra of std
and sample solns against O.OItV NaOH and det. A at max., ca
272 nm.
mg Procainamide. HCl/capsule or tablet
= (A/A') x C x D x (W/W)
where A and A' refer to sample and std solns, resp.; C = concn
of std soln in mg/mL; D — sample diln factor in mL; W =
av. mg/capsule or tablet; and W = mg sample.
For injections, mg procainamide. HCl/mL
- (A /A') x C x D/V
where V = mL sample aliquot.
Ref.: J AOAC 59, 807(1976).
CAS-61 .4-39-1 (procainamide hydrochloride)
SYNTHETICS
938.14 Phenazopyridine Hydrochloride
in Drugs
Titrimetric Method
Final Action
A. Reagents
(a) Titanium, trichloride std soln. — Prep, as in 948. 28 A and
stdze as in 948.28B.
(b) Light green SF yellowish soln. — Dissolve 1 g (Use C.I.
No. 42095.) in H 2 and dil. to 1 L.
B. Preparation of Solution
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
(a) Solns. — To vol. contg ca 0.1 g phenazopyridine. HCl,
add 10 mL 0.LV HC1 and dil. to 100 mL.
(b) Tablets and jelly. — Accurately weigh sample (powd in
case of tablets) contg ca 0.1 g phenazopyridine. HCl, add 10
mL QAN HCl, and dil. to 100 mL.
(c) Ointments. — Accurately weigh, in 100 mL beaker, sample
contg ca 0. 1 g phenazopyridine. HCl, stir with ether until oint-
ment base dissolves, and wash into separator with ether and
H 2 0. Shake thoroly, and drain aq. layer into second separator
contg 25 mL ether. Shake, and drain aq. layer into third sep-
arator contg 25 mL ether. Shake, and transfer aq. layer to 250
mL beaker. Wash ether layers with alternate 10 mL portions
HCl (1 + 1) and H 2 until no more color is removed, suc-
cessively passing each portion of the HCl or H 2 thru the 3
separators and finally into beaker. Nearly neutze combined acid
exts with NH4OH, cool, wash into separator, make ammonia-
cal, and ext with 25 mL portions CHC1 3 until no more color
is removed, filtering CHC1 3 thru cotton pledget in stem of sep-
arator. Evap. combined CHC1 3 exts just to dryness, take up in
10 mL 0AN HCl, and dil. to" 100 mL.
C. Determination
Heat soln to bp, add 15 g Na acid tartrate, and boil 2 min.
Add 10 mL light green SF yellowish soln and titr. hot with
std TiCl 3 soln in current of C0 2 . End point is change from
green to pale yellow. Perform blank titrn with 10 mL 0.17V
HCl, 90 mL H 2 0, 15 g Na acid tartrate, and 10 mL light green
SF yellowish soln, and subtract from vol. TiCl 3 previously
found. 1 mLO.liV TiCl 3 = 0.00624 g phenazopyridine. HCl,
CnHnNs.HCl.
Ref.: JAOAC 21, 552(1938).
CAS- 136-40-3 (phenazopyridine hydrochloride)
983.28 Amitriptyline in Drug Tablets
and Injectables
Liquid Chromatographic Method
First Action 1983
Final Action 1986
A. Principfe
Amitriptyline content of tablets and injectables is detd by
liq. chromatography, using trifluoperazine as internal std and
UV detection at 239 nm.
B. Apparatus and Reagents
(a) Liquid chromato graph. — Tracor Model 950 solvent pump
(replacement Model 951), Model 970A variable wavelength
detector (replacement Model 971), Model 26325 recorder (re-
placement Model 1200) (Tracor Instruments Inc.,), and 20 [xL
loop injector. Operating conditions: column temp., ambient;
solv. flow rate, 1 .33 mL/min; detector wavelength, 239 nm;
attenuation, 16 AUFS; recorder, 1 mV; chart speed, 1 in./4
min.
(b) Chromatographic column. — Stainless steel, 300 x 3.9
mm id, packed with 10 jxm jmBondapak CN (Waters Associ-
ates, Inc.), or equiv.
(c) Methanol. — AR grade (Fisher Scientific Co.).
(d) Mobile phase. — MeOH-0.005M ammonium acetate (90
+ 10).
(e) Culture tubes. — 95 x 25 mm with screw cap (Kimble
Glass Inc.).
(f ) Internal std soln. —Accurately prepare ca 0.5 mg Tri-
fluoperazine HCl Ref. Std/mL MeOH.
(g) Std soln. — 0.04 mg/mL. Accurately weigh ca 10 mg
USP Amitriptyline HCl Ref. Std and transfer to 250 mL vol.
flask. Dissolve in 1 mL MeOH, add 25.0 mL internal std soln,
dil. with MeOH, and mix.
C. Sample Preparation
(a) Tablets. — Weigh and finely powder >20 tablets. Ac-
curately weigh and transfer amt of powd. equiv. to 10 mg ami-
triptyline HCl into screw-cap culture tube and add 25.0 mL
internal std soln. Tumble on rotator 15 min at ca 30 rpm, and
filter, if necessary. Dil. accurately measured vol. of soln with
MeOH to ca 0.04 mg/mL.
(b) Single tablet. — Place one tablet in 95 X 25 mm screw-
cap culture tube and crush to fine powd. with glass rod. Add
25.0 mL MeOH and mix. Tumble on rotator 15 min at ca 30
526
Drugs: Part I
AOAC Official Methods of Analysis (1990)
rpni, and filter, if necessary. Pipet accurately measured aliquot
(A) of this soln, equiv. to 2 mg amitriptyline HC1, into 50 mL
vol. flask, add 5.0 mL internal std soln, dil. to vol. with MeOH,
and mix.
(c) Injectables. — Accurately pipet vol. of injectable, equiv.
to .10 mg amitriptyline HC1, into 50 mL vol. flask, add 25.0
mL internal std soln, dil. to vol. with MeOH, and mix. Dil.
accurately measured vol. of this soln with MeOH to ca 0.04
mg/mL.
D. Determination
Equilibrate system with mobile phase at 1.33 mL/min, until
baseline is steady. Inject measured vol. of std soln into chro-
matograph by microsyringe or sampling valve. Adjust injec-
tion vol. and operating conditions so amitriptyline HC1 in std
soln injection gives peak ht ca 60% full scale and retention
time ca 7 min. Under these conditions, 5 replicate injections
of std soln should give coefficient of variation of <3% and
resolution factor (R) between the 2 main peaks should be >1.
Make alternate injections of equal vols, of sample and std solns.
Measure peak hts for amitriptyline HO and internal std in sam-
ple and std solns, and det. response ratios.
E. Calculations
Tablets: mg/tablet = RR/RR' x C x T/W
Single tablet: mg/tablet = RR/RR' x C x 5 /A
Injectables: mg/mL = RR/RR' x C/V
where RR and RR' - ratio of amitriptyline HC1 peak ht to
internal std peak ht for sample and std solns, resp.; C = mg
amitriptyline HC1 in 250 mL std soln; T - ay. tablet wt, g;
W = wt sample taken, g; A - aliquot taken, mL; V - vol.
injectable taken, mL.
Ref.: J AOAC 66, 1196(1983).
CAS-549-18-8 (amitriptyline hydrochloride)
Meperidine in Drugs
See 945.69
984.38 Methocarbamol in Drugs
Liquid Chromatographic Method
First Action 1984
Final Action 1988
(Applicable to injectables and tablets)
A. Principle
Methocarbamol is dissolved in aq. MeOH and detd by liq.
chromatgy with acetanilide internal std, H 2 0-MeOH mobile
phase, and detection at 280 nm.
B. Apparatus
(a) Liquid chromato graph. — Equipped with Model U6K
injector, Model 6000A sol v. delivery system, and Model 440
detector (Waters Associates, Inc.) or equiv. Operating con-
ditions: flow rate 1 . 8 mL/min; 280 nm detector at 0.10 AUFS;
temp, ambient; vol. injected 20 jxL.
(b) LC column. — Zorbax ODS, 5-6 fxm particle size, 4.4
mm id by 25 cm long (E.I. DuPont), or equiv.
(c) Integrator.— HP 3380A (replacement Models HP3394A
or HP3396A) (Hewlett-Packard, Avondale Div.), or equiv.
(d) Filters.— Millipore type HAWP, pore size 0.45 fjim
(Mi Hi pore Corp.), or equiv.
C. Reagents
(a) Solvents. — H 2 and MeOH, each of suitable LC grade;
degas before use.
(b) Internal std. — Acetanilide, 6 mg/mL H 2 0-MeOH (75
+ 25).
(c) Methocarbamol std soln. — Transfer ca 50 mg, accu-
rately weighed, USP Methocarbamol Ref. Std to 100 mL vol.
flask, add ca 23 mL MeOH and 10.0 mL internal std, dil. to
vol. with H 2 0, and mix thoroly,
(d) LC mobile phase. ~H 2 0-MeOH (75 + 25); degas.
D. Sample Preparation
(a) Injections. — Transfer accurately measured vol. of
methocarbamol injection equiv. to ca 500 mg to 100 mL vol.
flask, add 25 mL MeOH and dil. to vol. with H 2 0. Transfer
10.0 mL of this diln and 10.0 mL internal std to second 100
mL vol. flask, add 20 mL MeOH, and dil. to vol. with H 2
(sample soln).
(b) Tablets. — Transfer accurately weighed portion of finely
ground tablet composite equiv. to 250 mg methocarbamol to
200 mL vol. flask, add 50 mL MeOH, mix thoroly, and add
H 2 to vol. Transfer 20.0 mL of this initial diln plus 5.0 mL
internal std to 50 mL vol. flask, add 6-7 mL of MeOH, and
dil. to vol. with H 2 (sample soln).
Filter sample soln thru Swinny filter (Millipore Cat. No. XX
30 012 00) contg 0.45 *xm Millipore filter (HAWP 01300) and
attached to Hamilton gas-tight syringe (No. 1010 W) fitted
with Luer-lok tip.
E. Standardization
Set mobile phase flow rate at 1.8 mL/min and inject 20 \xL
methocarbamol std soln. Retention time for methocarbamol (2nd
peak) should be 19 ± 2 min. Resolution, R = 2 (t 2 ~ t ] )/{W 2
+ W|), should be not <6 for methocarbamol and internal stan-
dard peak, where t t = retention of internal std; t 2 = retention
of methocarbamol; W ] = width of base of internal std peak;
W 2 — width of base of methocarbamol peak.
Adjust mobile phase ratios and/or flow rates as necessary
to give required retention times and /or resolution.
F. Determination
Make 20 fxL injections of std and sample solns. Measure
peak areas and calc. response ratios (methocarbamol peak to
internal std peak) for std and sample solns. Calc. methocar-
bamol:
Liqs, mg/mL = (R/R f ) X (C/V) x 1000
Tablets, mg/tab. = (R/R r ) x (C/W) x 500 x T
where R and R' = response ratios for sample and std solns,
resp.; C = concn methocarbamol std soln, mg/mL; V ~ mL
injection taken for analysis; T = av. tablet wt, g; W = sample
wt, g.
Ref.: J AOAC 67, 225(1984).
CAS-532-03-6 (methocarbamol)
926.17* Nitroglycerin in Drugs
Reduction Method
Final Action
Surplus 1980
See 39.104-39.106, 13th ed.
AOAC Official Methods of Analysis (1990)
Synthetics
527
958.13 Nitroglycerin in Drugs
Infrared Spectrophotometric Method
Final Action 1965
A. Reagent
Nitroglycerin std. — Absorbate on lactose contg ca 10% ni-
troglycerin. Stdze as in 958.14 except use k — 89.04 for NaN0 3
and 74.87 for KN0 3 . This product is stable indefinitely in tightly
stoppered bottle.
B. Determination
(Caution: See safety notes on distillation, flammable solvents,
toxic solvents, and carbon disulfide.)
Transfer number of tablets contg ca 5 mg nitroglycerin to
small separator. Dissolve or suspend in 5 mL H 2 0, add 20 mL
CS 2 , shake 1 min, and let sep. Filter CS 2 layer thru pledget
of cotton previously washed with CS 2 and collect in 100 mL
beaker. Repeat extn with three 10 mL portions CS 2 . Evap.
combined exts to ca 3 mL, using gentle current of air, at temp.
<50°. Transfer quant, to 5 mL vol. flask and dil. to vol. with
CS 2 .
Accurately weigh amt std absorbate contg ca 5 mg nitro-
glycerin. Transfer to small separator and ext as above.
Det. baseline A B of sample and std solns relative to CS 2 at
7.89 jmm, drawing baseline between min. at 7.5 and 8.3 fxm.
Calc. nitroglycerin content of sample. Record spectra of sam-
ple and std solns from 2 to 15 u,m and compare for identity
of sample.
Ref.: JAOAC 41, 504(1958).
CAS-55-63-0 (nitroglycerin)
960.54 Nitrate Esters in Drugs
Infrared Spectrophotometric Method
First Action 1960
Final Action 1965
(Applicable to mannitol hexanitrate, erythrityl tetranitrate, or
pentaerythrityl tetranitrate)
A. Apparatus
(a) Recording infrared spectrophotometer. — With two 1.0
mm liq. absorption cells with NaCl windows, preferably
matched or of known A difference, and KBr disk holder.
(b) Chromatographic tube. — 25 X 200 mm with 5 X 40
mm stem.
(c) Die and hydraulic press. — Suitable for prepg KBr disks.
B. Preparation of Standard Solution
Ext ester from com. absorbate (usually 10% on lactose or
other inert diluent) with ether, filter, and evap. to dryness with
aid of air current at temp. <50°. Dry in vac. desiccator 1 hr.
Prep, std soln contg 0.5 mg ester /mL CHC1 3 .
(Caution: Pure crystalline nitrate esters are very explosive, es-
pecially pentaerythrityl tetranitrate. Do not use sample contg
>5 mg pure compd.)
C. Preparation of Sampie
Reduce tablets to fine powder. Weigh sample contg ca 25
mg nitrate ester and transfer to 125 mL separator with ca 5
mL H 2 0. Make distinctly acid with H 2 S0 4 (1 + 9). Proceed
as in (a) in absence of pheno barbital, or (b) in presence of
phenobarbital .
(a) Add 10 mL CHC1 3 to separator, shake vigorously sev-
eral min, and let sep. Transfer CHC1 3 layer to 50 mL vol.
flask. Ext aq. soln with three addnl 10 mL portions CHC1 3 and
transfer each ext to vol. flask. Dil. to vol. with CHC1 3 , mix,
and filter.
(b) Add 15 mL CHC1 3 to separator, shake vigorously sev-
eral min, and let sep. Transfer CHC1 3 layer to chromatgc col-
umn contg 4 mL \M K 3 P0 4 soln adsorbed on 5 g diat. earth,
960. 53B, collecting eluate in 50 mL vol. flask. Ext. aq. soln
with three addnl 10 mL portions CHC1 3 , and pass each ext thru
column, collecting eluate in vol. flask. Dil. to vol. with CHC1 3 ,
mix, and filter.
D. Determination
(Store CHC1 3 to be used in IR measurements in stoppered
flask.)
Transfer 5 mL aliquot CHC1 3 soln to 25 mL g-s erlenmeyer,
evap. to dryness with aid of air current at temp. <50°, and
complete drying in vac. desiccator. Add 5.00 mL CHC1 3 to
residue, stopper flask tightly, and let stand 30 min with oc-
casional shaking to ensure complete soln. Det A of std and
sample solns against CHC1 3 at max. (ca 6.0 (xm) and calc. amt
of ester per tablet.
Evap. another portion CHC1 3 soln to dryness as above. Prep.
KBr disk by grinding together in agate mortar 1 mg residue
with 200 mg IR spectral grade KBr and pressing in die and
hydraulic press. Record spectrum from 2 to 15 |mm and com-
pare with spectrum of std nitrate ester to det. identity of sam-
ple.
Ref.: JAOAC 43, 259(1960).
CAS-7297-25-8 (erythrityl tetranitrate)
CAS- 15825-70-4 (mannitol hexanitrate)
CAS-78-11-5 (pentaerythrityl tetranitrate)
958.14 Mannitol Hexanitrate;
Mannitol Hexanitrate and Phenobarbital in Drugs
Spectrophotometric Methods
Final Action
(Ascorbic acid interferes. See also 960.54.)
A. Reagents
(a) Phenoldisulfonic acid. — Heat 5 g colorless phenol, 30
mL H2SO4, and 15 mL fuming H 2 S0 4 (ca 20% free S0 3 ) on
steam bath 2 hr. (Caution: See safety notes on sulfuric acid
and fuming acids.)
(b) Nitrate std soln. — Dissolve 100 mg KN0 3 or NaN0 3 in
ca 1 mL H 2 and dil. to 100 mL with HO Ac.
(c) Phenobarbital std soln. — In 100 mL vol. flask dissolve
100 mg phenobarbital and dil. to vol. with HOAc. Pipet 5 mL
of this soln and 15 mL HOAc into 100 mL vol. flask, dil. to
vol. with H 2 0, and filter, discarding first 5 mL filtrate.
B. Preparation of Sample
Transfer accurately weighed sample contg ca 30 mg man-
nitol hexanitrate to 50 mL vol. flask, and dil. to vol. with
HOAc. Shake well and filter, discarding first 5 mL filtrate.
C. Determination of Mannitol Hexanitrate
Transfer 1.0 mL aliquots of sample, std, and HOAc (blank)
to individual 100 mL vol. flasks, add 2.0 mL phenoldisulfonic
acid to each, and let stand 15 min. Dil. with H 2 to ca 60
mL, add NH 4 OH (ca 10 mL) until max. yellow color appears,
cool to room temp., dil. to vol. with H 2 0, and mix. Det. A
of sample and std, A', relative to blank at 408 nm.
528
Drugs: Part I
AOAC Official Methods of Analysis (1 990)
% Mannitol hexanitrate = (A x R 2 x k x 50)/(A' x /?,)
where Ri is mg sample, R 2 is mg std/mL, and fc is 88.66 for
NaN0 3 and 74.56 for KN0 3 std.
D, Determination of Phenobarbital
Pipet 10 mL aliquot sample soln into 50 mL vol. flask, dil.
to vol. with H 2 0, shake, and filter, discarding first 5 mL fil-
trate. Prep, blank by dilg 10.0 mL HOAc to 50 mL with H 2
and filtering. DiL sep. 20 mL aliquots of std, sample, and
blank solns to 100 mL with NH 4 OH (1 + 9), adjusting to room
temp, before dilg. to vol. (Final pH of soln, 9.0-9.6.) Det. A
of sample and std relative to blank at 240 nm, and calc. phe-
nobarbital content.
Ref.: JAO AC 41, 493(1958).
CAS- 15825-70-4 (mannitol hexanitrate)
CAS-50-06-6 (phenobarbital)
964.25 Pentaerythrityl Tetranitrate
in Drugs
Spectrophotometric Method
Final Action 1965
(Applicable in presence of meprobamate)
Accurately weigh powd sample contg ca 40 mg pentaery-
thrityl tetranitrate into 50 mL g-s vol. flask. Add ca 30 mL
HOAc, shake ca 1 min, and dil. to vol. with HOAc. Filter,
discarding first 5 mL filtrate. Proceed as in 958. 14C.
% Pentaerythrityl tetranitrate = (A x R' x k x 50) /(A' x R)
where A and A' refer to sample and std, resp.; R and R ! are
mg sample and mg std/mL, resp.; and k is 92.99 for NaN0 3
or 78.18 for KN0 3 std.
Ref.: JAOAC 47, 469(1964).
CAS-78-11-5 (pentaerythrityl tetranitrate)
970.79 Pentaerythrityl Tetranitrate
and Meprobamate in Drugs
Infrared Spectroscopic Method
First Action 1970
Final Action 1971
(Caution: PETN may explode when heated strongly, even when
dissolved.)
A. Apparatus
See 960.54A
B. Reagents
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
(a) Dilute phosphoric acid.— 3 + 1. Dil. 3 vols 85% H3PO4
with 1 vol. H 2 0.
(b) Water-washed benzene. — Shake equal vols benzene and
H 2 1 min in separator. Discard lower phase. Use within 2
days of prepn.
(c) Water-washed chloroform. — Shake equal vols. CHC1 3
and H 2 1 min in separator. Discard upper layer. Use within
2 days of prepn.
(d) Anhydrous chloroform. — Filter H 2 0- washed CHC1 3 thru
anhyd. Na 2 S0 4 ,
(e) Pentaerythrityl tetranitrate (PETN) std soln. — 10 mg/
50 mL. Ext PETN from com. PETN (usually 10% on lactose
or other inert diluent) with CHC1 3 to give ca 20 mg pure PETN.
Filter and evap. to dryness under air current with little or no
heat. Dry in vac. desiccator 2 hr. Accurately weigh ca 10 mg,
using microbalance, dissolve in anhyd. CHC1 3 , and dil. to 50
mL with this solv. Destroy excess PETN by dissolving in ace-
tone and burning in large vessel behind safety barrier, using
effective fume removal device.
(f ) Meprobamate std soln. — 80 mg/100 mL. Dissolve 80
mg USP Meprobamate Ref. Std in anhyd. CHC1 3 and dil. to
100 mL with this solv.
(g) Diatomaceous earth. — See 960. 53B.
C. Determination
(Use H 2 0- washed solvs unless designated anhyd. Caution: See
safety notes on distillation, flammable solvents, benzene, and
chloroform.)
Loosely pack small amt of fine glass wool in base of chro-
matgc tube to support diat. earth. Weigh 3 g diat. earth into
100 mL beaker, add 2.0 mL IN NaOH, mix with metal spatula
until fluffy, and pack uniformly in tube. Weigh 5 g diat. earth
into 250 mL beaker, add 7.0 mL dil. H 3 P0 4 , mix until fluffy,
and pack uniformly on column. (Do not pack too tightly as
column will elute too slowly.) Accurately weigh portion powd
sample contg ca 10 mg PETN into 150 mL beaker. Add 4 mL
benzene and heat gently with swirling ca 1 min. Cool, add 4
g diat. earth, mix until fluffy, transfer quant, to column, and
pack uniformly. Dry- wash beaker with 1 g diat. earth and 0.5
mL benzene, transfer to column, and pack uniformly. Wipe
sample beaker and all app. used in column prepn with glass
wool, and pack on column.
Pass 75 mL benzene thru column and collect eluate in 150
mL beaker until elution ceases. Rinse column tip with small
portions benzene into beaker and set aside. This fraction con-
tains PETN.
Place 250 mL beaker under column. Add 4.0 mL H 2 to
column and let it be absorbed. Pass 150 mL CHC1 3 thru col-
umn and collect eluate in 250 mL beaker. Rinse column tip
with CHC1 3 into beaker. This fraction contains meprobamate.
Evap. each fraction on steam bath under gentle air current
to ca 10 mL and take to dryness with little or no heat from
steam bath. Place beakers in vac. oven 30 min at 30° and <380
mm (15") Hg. Remove from oven. Add ca 10 mL anhyd. CHC1 3
to PETN beaker and heat gently to dissolve residue. Quant,
transfer with anhyd. CHC1 3 to 50 mL vol. flask and dil. to
vol. with anhyd. CHC1 3 .
Dissolve meprobamate residue with ca 20-25 mL anhyd.
CHCI3. If theoretical wt of meprobamate in sample wt taken
is 100 mg, transfer quant, to 100 mL vol. flask with anhyd.
CHCI3 and dil. to vol. with this solv. If theoretical wt of me-
probamate is ca 200 mg, use 250 mL vol. flask and proceed
as above.
Scan sample and std solns in 1.0 mm cells from 5.0 to 6.5
fjim (2000-1540 cm" 1 ) on IR spectrophtr against anhyd. CHC1 3
as ref.
Calc. PETN by subtracting A at 5.5 fxm (1818 cm"') from
A at ca 6.02 fxm (1660 cm" 1 ) and compare with std A. (Note:
PETN sample solns may contain very small peak at ca 5.8 (xm
(1722 cm" 1 ). This is contaminant of meprobamate and does
not interfere with PETN detn. Also, a peak may appear at ca
6.25 |xm (1600 cm" 1 ). This is H 2 peak. Disregard this peak
in calcg PETN net A.)
Calc. meprobamate by subtracting A at 5.5 fxm (1818 cm" 1 )
from A at ca 5.82 fxm (1718 cm" 1 ) and compare with std A.
AOAC Official Methods of Analysis (1 990)
Synthetics
529
D. Identification
(a) PETN. — Prep, both std and sample KBr disks from re-
spective assay solns. Evap. 4-5 mL of each soln in small mor-
tar, add 200 mg KBr, mix thoroly, and press. Scan spectrum
from 2 to 15 |xm (5000-667 cm" 1 ). Compare sample and std
curves. (Note: Sample IR curve may deviate from std curve.
This deviation is caused by meprobamate contaminant. How-
ever, all major peaks in std and sample should be evident.)
(b) Meprobamate. — Prepare KBr disks as above from 1 mL
sample and std assay solns. Scan and compare as in (a).
Ref.: J AOAC 53, 594(1970).
CAS-57-53-4 (meprobamate)
CAS-78-11-5 (pentaerythrityl tetranitrate)
980.34 Nitroglycerin
in Sublingual Drug Tablets
Semiautomated Method
First Action 1980
Final Action 1982
A Principle
Nitroglycerin is hydrolyzed in aq. alkali to nitrite which di-
azotizes procaine. HC1. Product is coupled with 7V-l-naphthyl-
ethylenediamine.2HCl, and A is measured at 550 nm.
B. Apparatus
(a) Automatic analyzer. — With following modules (Techni-
con Instruments Corp., or equiv.): Sampler with 30/hr (2:1)
cam; proportioning pump; const temp, bath (50°) with one 40'
(12 m) x 1.6 mm id coil; colorimeter with 15 x 2.0 mm id
flowcell and matched 550 nm filters, or spectrophtr with 10
mm flowcell; recorder; manifold (see Fig. 980.34). Use glass
interconnecting tubing thruout, unless otherwise specified.
(b) Shaker. — Wrist action.
C. Reagents
(Use deionized or distilled H 2 thruout.)
(a) Strontium hydroxide soln. — 1%. Add 20.0 g
Sr(OH) 2 .8H 2 to 1.8 L recently boiled H 2 0, and heat to dis-
solve. (Turbidity due to SrC0 3 may remain.) Cool, dil. to 2
L with H 2 0, and mix. Let stand overnight, filter thru paper,
and protect from C0 2 .
(b) Procaine soln. — 0.3%. Dissolve 3.0 g procaine. HC1 in
H 2 0, dil. to 1 L, and mix.
(c) Hydrochloric acid. — (1 + 4).
(d) Coupling reagent. — 0.1% aq. Af-naphthylethylenedi-
amine. 2HCI. Prep, fresh weekly and store in dark glass bottle
in refrigerator.
(e) Nitroglycerin std soln. — 15 |xg/mL. Accurately weigh
75 mg 10% nitroglycerin lactose absorbate, stdzd as in 958.14,
except use k = 89.04 for NaN0 3 and 74.87 for KN0 3 , into
500 mL vol. flask. Add 300 mL H 2 0, shake vigorously, and
dil. to vol. with H 2 0.
D. Preparation of Sample
Place individual tablet or weighed composite into suitable
g-s container, and add accurately measured vol. H 2 to give
nitroglycerin concn ca 15 |mg/mL. Shake vigorously mech. until
drug is in soln (5-15 min).
E. Analytical System
Sample is withdrawn, segmented with air, and hydrolyzed
with Sr(OH) 2 in 50° heating bath. Soln is resampled, seg-
mented with air, and mixed with procaine. HO, HC1, and cou-
pling reagent in succession. After time delay, A is read at 550
nm.
F. Start-Up and Shut-Down Operations
Pump H 2 thru all lines for 5 min; then place all lines in
resp. solns and pump until steady baseline is obtained. For
shut-down, pump H 2 thru all lines for 5 min; then remove
lines and pump dry.
G. Determination
Fill 2 mL sample cups in following order: 3 cups std soln,
5 cups sample soln, 1 cup std soln, 5 cups sample soln, etc.,
ending with 2 cups std soln. (First 2 cups of std soln are used
to equilibrate system, but are not included in calcns.) Start
Sampler II or IV. After last cup has been sampled, let system
operate until steady baseline is obtained. Draw tangent to ini-
tial and final baselines. Subtract baseline to det. net A and A f
for each sample and std peak, resp. Using av. of 2 stds which
bracket sample peak, calc. nitroglycerin as follows:
mg Nitroglycerin in portion taken = (A /A') X C X D
where C = concn of std in mg/mL and D = diln factor.
Ref.: JAOAC 63, 696(1980).
CAS-55-63-0 (nitroglycerin)
972.48 Dichlorophene in Drugs
Spectrophotometric Method
First Action 1972
Final Action 1974
A. Principle
Dichlorophene is extd from acid soln with CHC1 3 and detd
by measuring A in ale. NaOH soln at 305 nm.
B. Reagent
Dichlorophene std solns. — (1) Stock soln. — 600 u>g/mL.
Dissolve 60 mg dichlorophene (Sigma Chemical Co., Inc.) in
alcohol and dil. to 100 mL. (2) Working soln. — 15 (xg/mL.
Dil. 5.0 mL stock soln to 200 mL with 0.17V NaOH. Prep,
fresh daily.
C. Preparation of Sample
(a) Soft gelatin capsules. — Select representative number (5-
20) of capsules. Treat each capsule, one at a time, as follows:
Using sharp scalpel, cut capsule lengthwise and transfer to funnel
placed in neck of 500 mL vol. flask. Thoroly rinse capsule
contents, scalpel, and funnel with alcohol. Remove funnel and
carefully transfer opened capsule to vol. flask. Dil. to vol.
with alcohol and mix. Prep, soln contg ca 0.3 mg dichloro-
phene /mL by stepwise diln with alcohol.
(b) Suspension. — Mix thoroly. Using 10 mL "to contain"
pipet with wide orifice (Duopette pipet, Scientific Products,
Inc., No. P4615-1X, or equiv.), withdraw suspension to TC
mark. Drain pipet into 250 mL vol. flask, using pressure ap-
plied from rubber bulb. Thoroly rinse pipet into flask with al-
cohol, dil. to vol. , and mix. Prep, soln contg ca 0.3 mg dichlo-
rophene/mL by stepwise diln with alcohol.
D. Determination
Transfer 10 mL aliquot prepd sample soln to 125 mL sep-
arator contg 10 mL H 2 0. Add 3 mL IN NaOH, mix well, and
ext with 10 mL w-hexane. Let sep. and transfer lower aq. layer
to second separator. Ext with another 10 mL portion n-hexane.
Transfer aq. layer to third separator. Combine «-hexane exts
in first separator and ext with 10 mL 50% alcohol. Discard
hexane. Add ale. wash to aq. layer in third separator. Add 7
530
Drugs: Part I
AOAC Official Methods of Analysis (1990)
■*- To Weir Box
0.80T
0.58RA
JACKETED MIXER
c
JSSSL
2.00T
HEATING BATH
0.32T
jmmsL
A10
jftpft
A10
JUL
LOOT
Q.32T
0.42T
0.42T
042T
WASTE
RECORDER
WASTE -<j-
1.00T
Sr(OH) 2
Air (air bar)
-r WASTE
Resample
Air (air bar)
Procaine HCI
HCI
Coupling Reagent
FIG. 980.34— Flow diagram for semiautomated analysis of nitroglycerin: B, injection fitting, No. 116-0489-01; C, 10-turn coil, No.
157-0226; D, heating bath equipped with 40 ft x 1.6 mm id coil; E, jacketed coil (tap H 2 0), 4 turns x 1 mm id; F, injection fitting,
No. 116-0492-01; G, 5-turn coil, No. 170-0103-01; H, 20-turn coil with center tap, No. 157-B089; J, phasing coil, 28-turn x 2.4 mm
id, No. 116-152-4; RA, red acidflex pump tube; T, tygon pump tube. Pump tube sizes in mL/min; Technicon part numbers— use
these or equiv.
mL \N HCI and mix well. (Soln should be distinctly acid to
litmus paper.) Ext acid soln with four 25 mL portions CHCl 3 .
Filter each ext thru CHCl 3 -washed cotton into 250 mL beaker.
Rinse funnel and cotton with ca 5 mL CHC.l 3 and carefully
evap. just to dryness on steam bath, using gentle air jet. Let
beaker and contents cool. Dissolve residue of dichlorophene
in 5.0 mL alcohol and transfer to 200 mL vol. flask, using
0. IN NaOH. Dil. to vol. with 0AN NaOH and mix.
Record spectra of sample and std solns from 370 to 225 nm,
using 1 cm cells and 5 mL alcohol dild to 200 mL with 0.1W
NaOH as ref .
Det. A of std and sample solns at peak wavelength ca 305
nm and calc. dichlorophene content of sample.
E. Identification
Dil. aliquots of sample assay soln, working std soln, and
ref. soln with equal vols 0AN NaOH. Obtain A between 370
and 225 nm.
Spectra should be similar and exhibit maxima at 245 and
305 nm. Ratio (A 24 5/A 305 ) of sample does not differ appreci-
ably from that of std.
Ref.: JAOAC55, 163(1972).
CAS-97-23-4 (dichlorophene)
920.210 Methenamine in Tablets
Titrimetric Method
Final Action
A. Reagent
(a) Modified Nessler reagent. — (/) Dissolve 10 g HgCl 2 ,
30 g KI, and 5 g acacia in 200 mL H 2 0, and filter thru cotton;
(2) dissolve 15 g NaOH in 100 mL H 2 0. Mix 20 mL soln (/)
with 10 mL soln (2).
(b) Starch indicator. — Mix ca 2 g finely powd. potato starch
with cold H 2 to thin paste; add ca 200 mL boiling H 2 0, stir-
ring const., and immediately discontinue heating. Add ca 1
mL Hg, shake, and let soln stand over the Hg.
B. Determination
Weigh 0.5 g powder, prepd as in 927.09A, into r-b flask,
and add 100 mL H 2 and 25 mL HCI (1 + 2.5). Connect with
reflux condenser (preferably of worm type) and boil gently 15
min. Cool, wash condenser tube with little H 2 0, transfer con-
tents of flask to 250 mL vol. flask, and dil. to vol.
Chill 30 mL Nessler reagent and add 10 mL aliquot of hy-
dro! yzed sample soln. Wash neck of container with jet of H 2
and let stand >1 min. Add 10 mL HO Ac (1 + 1.5) so that
inside of neck is completely washed by reagent, mix quickly
AOAC Official Methods of Analysis (1 990)
Synthetics
531
and thoroly by rotating and tilting flask, and immediately add
20 mL 0.1A r I from buret or pipet. Titr. excess I with OAN
Na 2 S 2 3 , adding 5-10 drops starch indicator toward end of
titrn, until blue disappears. Final color of soln is pale straw-
green. If preferred, end point may be detd by reappearance of
faint blue when drop of the I soln is added. 1 mL 0.17V I =
0.00117 g methenamine.
Ref.: JAOAC 3, 374(1920).
CAS- 100-97-0 (methenamine)
973.71 Methenamine
and Methenamine Mandelate in Drugs
Automated Method
First Action 1973
Final Action 1974
A. Principle
Methenamine is hydrolyzed to HCHO and NH 4 + in acid soln.
Free HCHO condenses with chro mo tropic acid in strong acid
soln to form colorless hydroxydiphenylmethane derivative which
is further oxidized to colored /?-quinoidal compd with max. A
at 570 nm. Method is applicable to methenamine, methena-
mine mandelate, and methenamine with NaH 2 P0 4 .
B. Reagents and Apparatus
(Use deionized H 2 or equiv. thruout.)
(a) Dilute ammonia. — Dil. 5 mL NH 4 OH to 1 L with H 2 0.
(b) Dilute sulfuric acid. — 72%. Slowly add H 2 S0 4 to 600
mL H 2 to total vol. of 1.5 L (when cool).
(c) Chromotropic acid (CTA) color reagent. — Suspend 500
mg CTA, di-Na salt (Sigma Chem Co., Inc., No. D5144,
Eastman Kodak Co. , or equiv.), in 20 mL H 2 and slowly add
30 mL H 2 S0 4 in small portions (overheating produces deep
violet color and inactivates reagent). Cool, and mix into 1.5
L 72% H 2 S0 4 .
(d) Methenamine std soln. — Dissolve enough methena-
mine, previously dried over P 2 5 and stdzd (USP), in dil.
NH 4 OH to give concn of std appropriate for dosage level ana-
lyzed {see table).
(e) Automatic analyzer. — Auto Analyzer with following
modules (Technicon Instruments Corp.): Sampler II with 20/
hr (2:1) cam; proportioning pump I; heating bath set at 90°
with two 40' coils, 1 .6 mm id; colorimeter with 15 mm tubular
flowcell and matched 570 nm filters; recorder with semilog
paper; manifold (Fig. 973.71). Wire down all tube connections
carrying H 2 S0 4 .
C. Preparation of Sample
Disintegrate uncoated tablets contg equiv. of 250-500 mg
methenamine by intermittent shaking in 100 mL dil. NH 4 OH.
Crush coated tablets and hard uncoated tablets before addn of
solv. Ultrasonic bath may be used to hasten soln. Dil. 1.0 mL
sample soln to 50 mL with dil. NH4OH.
Samples of all dosage levels can be prepd in 1 25 mL (4 oz)
glass, snap-cap vials provided with Parafilm seal if appropriate
sampling pump tube is used (see table sizes). A 1 + 50 diln
may be accomplished with 1.00 mL Thomas-Seligson vac. diln
pipet mounted beneath automatic 50.0 mL delivery pipet. Est.
increase in vol. resulting from dissolving 1 tablet in 100 mL
solv., using vol. flask and graduated pipet.
D. Analytical System
See Fig. 973.71 and the table. Sample solns are withdrawn
from sample cups, segmented with air, and dild in manifold
with H 2 0. CTA soln is added and stream is passed thru beaded
coil into 90° heating bath for color development. Stream is
cooled in H 2 0-jacketed coil and equilibrated at room temp, in
mixing coil. Stream is debubbled and passed into colorimeter
equipped with 570 nm filters and 15 mm tubular flowcell for
A measurement. Inlet and outlet tubing of flowcell should be
ca same id.
Pump
Dosage,
Tube
Std,
mg/
Size,
mg/100
Product
tablet
mL/min
mL
Methenamine mandelate
250
1.00
2.50
Methenamine mandelate
500
0.60
5.00
Methenamine
300, 325
0.60
6.00, 6.50
Methenamine
500
0.23
5.00
Methenamine mandelate
1000
0.23
10.00
E. Start-Up and Shut-Down Operations
Turn on heating bath (3 hr), cooling H 2 in jacketed coil
(30 min), and colorimeter (30 min) in advance. Prewash sys-
tem 5 min with H 2 and then pump all reagents thru their resp.
lines. Let equilibrate 20—30 min and adjust colorimeter and
recorder to produce steady baseline. To shut down system,
flush 10-15 min with H 2 and pump all lines dry.
F. Determination
Fill 8.5 mL sample cups with prepd solns and aspirate thru
0.034" stainless steel probe at 20 /hr with sample-to-wash ratio
of 2:1. Include 1 std soln between each 5 sample solns, and
insert 3 std solns at beginning and end of each 10-30 samples.
Draw line between baseline at beginning and end of run. Sub-
tract av. baseline A from max. A to obtain net A (A A) for each
peak. Calc. av. A A' for std solns, disregarding first and last
2 std peaks.
mg Methenamine /unit dose = (LA/ A A 1 ) X C x D
where C = mg methenamine /mL std soln and£> - diln factor.
Ref.: JAOAC 56, 647, 1295(1973).
CAS-100-97-0 (methenamine)
CAS-5 87-23-5 (methenamine mandelate)
923.11 Methylene Blue in Drugs
Titrimetric Method
Final Action
A. Preparation of Solution
(a) Foreign material absent. — Into 50 mL beaker weigh 0.1-
0.14 g powd sample, 927. 09 A, and transfer to 200 mL vol.
flask with 100-140 mL H 2 0. Dissolve completely by heating
30 min on steam bath with frequent shaking.
(b) Oils or water-insoluble material present. — (Caution: See
safety notes on toxic solvents, and carbon tetrachloride.) To
50 mL beaker transfer weighed amt of prepd sample, 927.09A,
contg 0.1-0.14 g methylene blue. Add 15 mL CC1 4 , warm on
steam bath few min, and stir with glass rod to dissolve oils.
Transfer to 100 mL separator, using ca 50 mL hot H 2 and
little CCI4, if necessary. Cool, shake, and let sep. Transfer
CCI4 with undissolved material to second separator for further
treatment. (Clear aq. soln of dye should now remain in first
separator. If not clear, ext with another 15 mL portion CC1 4 ,
transferring any remaining insol. material in similar manner to
second separator.) Add ca 10 mL CC1 4 to second separator and
remove methylene blue by shaking vigorously with 20-40 mL
portions H 2 until practically no more dye is extd. (Few drops
532 Drugs: Part I
AOAC Official Methods of Analysis (1990)
To
Waste
C3
Mixing Coil
jmmsL
H1
28 Turn
2.6 mm
I
. Upper
' Level
I Tube SizeX
Lower I ml/min
Level 023 1 00 T ,
— -h3h— s
©
©
^-v 3.9T Diluent (H 2 0)
<2>-— *
ja.
- 2.0TAfr
0.6T
Heating
Bath
H 2
Cooled Jacketed
Mixing Coil
14 Turn
4 mm
Mixing
Coil
14 Turn, 2.6 mm
Double
Coi
To Waste
Mixing Coil
__MftQ»
H1
14 Turn
4 mm. Beaded
•&■
<9>
2.03A CTA Soln
^ LOT Air
To Waste
Colorimeter
8 mm Tubular f/c
570 nm Filters
Recorder
To Sampler
II Wash -*-
Receptacle
©
Kj>
2.03A
2.70TH ?
Proportioning
Pump
3 See Table
SAMPLER II
RATE (per hour) 20/hr
Sample Cam 2:1
FIG. 973.71— Flow diagram for automated analysis of methenamine and methenamine mandelate
of HO Ac hasten this extn.) To aq. exts in 400 mL beaker, add
main soln from first separator, cover with inverted watch glass
on glass rods, and evap. to ca 50 mL. Proceed as in (c). CC1 4
soln may be reserved for qual. tests for oils.
(c) Water-soluble material present. — Either use aq. soln from
(b), or weigh portion of sample contg 0.1-0.14 g methylene
blue into 1 50 mL beaker, add ca 50 mL H 2 0, and heat 30 min
on steam bath with occasional shaking. Transfer to 100 mL
separator, keeping vol. as small as possible. Ext with a-dichlo-
rohydrin, using 10, 5, 3, and 2 mL portions. Combine dichlo-
rohydrin exts in 200-300 mL separator, add 3 or 4 times their
vol. CC1 4 , and ext dye with H 2 by repeated vigorous shaking
with 30-50 mL portions. (Few drops of HO Ac hasten re-
moval.) From combined aq. exts, remove any traces of dichlo-
rohydrin by shaking once with ca 15 mL CC1 4 and draining
after settling 5-10 min. Evap. aq. exts to ca 50 mL over flame,
covering beaker as in (b) with inverted watch glass. Transfer
to 200 mL vol. flask. Dissolve completely by heating 30 min
on steam bath with frequent shaking.
B. Determination
Conduct blank as in detn, including filtration. Cool soln,
923.11A(a) or (c), add 50 mL HOAc, shake thoroly, and let
stand >25 min. Add 30 mL 0.2N I, 939.13A, from buret,
adding first 10 mL by fast drops with const rotating of flask
and remaining 20 mL at full speed, and continue shaking.
Stopper flask and let stand 50 min, shaking thoroly 5 or 6
times during interval. Dil. to vol. with H 2 0, shake, and let
stand 10 min longer. Filter rapidly thru dry, folded, 12 cm
paper. Titr. 100 mL aliquot with 0.LV Na 2 S 2 3 , with or with-
out starch indicator as desired. Correct for blank titrn. 1 mL
0.2N 1 - 0.01496 g methylene blue, C I6 H I8 N 3 C1S.3H 2 0; or
0.01279 g anhyd. methylene blue, C, 6 H ]g N 3 ClS.
Ref.: J AOAC 7, 20(1923).
CAS-61-73-4 (methylene blue)
969.50 Fluorescein Sodium in Drugs
Fluorometric Method
First Action 1969
Final Action 1971
(Applicable to solns. Caution: See safety notes on photofluo-
rometers and hazardous radiations.)
A. Apparatus
(a) Fluorometric apparatus. — Spectrophotofluorometer or
fluorocolorimeter. With cell path >1 cm, excitation wave-
length 460 nm, emission wavelength 515 nm, and sensitivity
to yield >85% T for most coned std soln. Warm lamp >20
min before making measurements.
(b) Thin layer sheets. — Silica gel (100 |mm) with fluores-
cent indicator (Eastman Kodak Co. Chromagram sheets for
TLC, No. 13188, or equiv.).
B. Reagents
(a) Acriflavine hydrochloride soln. — Dissolve 5 mg salt (J.
T. Baker Inc., No. A381, or equiv.) in 0.5 mL H 2 and dil.
to 5 mL with alcohol.
(b) Fluorescein diacetate. — Mp 206-208° (Eastman Kodak
Co. No. 1688, or equiv.). If material is impure, indicated by
low mp or other evidence, recrystallize from alcohol.
(c) Fluorescein sodium std solns.— (1) Stock soln. — 903.6
|xg fluorescein Na/mL. Accurately weigh 100 mg fluorescein
diacetate (equiv. to 90.36 mg fluorescein Na), dried 1 hr at
100°, and transfer to 100 mL vol. flask with ca 10 mL alcohol.
Add 2 mL 10% NaOH and heat on steam bath at ca boiling
temp. 20 min. Swirl frequently. After hydrolysis, cool flask,
dil. to vol. with H 2 0, and mix. (2) Intermediate soln. — 0.9036
jxg fluorescein Na/mL. Dil. 1 mL clear stock soln to 1 L with
H 2 and mix. (3) Working solns.— Q.Q0O, 0.009, 0.018, 0.027,
0.036, and 0.045 u,g fluorescein Na/mL. Transfer 0.0, 1.0,
2.0, 3.0, 4.0, and 5.0 mL intermediate soln to sep. 100 mL
AOAC Official Methods of Analysis (1990)
MlCROCHEMlCAL TESTS
533
vol. flasks, add 20 m'L pH 9 buffer to each, and dil. to vol.
with H 2 0.
(d) Boric acid buffer.— pH 9. Prep, ca 200 mL soln 0.05M
in boric acid and 0.05M in KC1. Adjust to pH 9 with 0.2M
NaOH.
C. Preparation of Sample
Quant, dil. sample with H 2 to obtain ca 1 |xg fluorescein
Na/mL and transfer 3.0 mL aliquot to 100 mL vol. flask contg
20 mL pH 9 buffer. Dil. to vol. with H 2 and mix.
D. Determination
Measure fluorescent intensity (I) of all working std solns and
plot std curve (jxg fluorescein Na against /). Det. / of sample
soln and calc. concn of sample.
E. Purity and Identification
Dil. coned sample and hydrolyzed stock solns with alcohol
to contain ca 1 mg fluorescein Na/mL.
Spot 10 jjlL each of above solns and acriflavine.HCl soln on
fluorescent silica gel sheets and develop with n-BuOH-alco-
hol-H 2 (2 + 1 + 1). Dry sheet and view under longwave
UV light. Sample and std should exhibit only one spot, which
has similar but different R f from spot obtained with acrifla-
vine.HCl.
Ref.: J AOAC 52, 110(1969).
CAS-5 18-47-8 (sodium fluorescein)
961.17 Piperazine in Drugs
Gravimetric Method
First Action 1961
Final Action 1971
(Applicable to aq. solns)
A. Apparatus
Chromatographic tube. — 40 X 300 mm, with stopcock and
fritted glass disk or plug of glass wool as support.
B. Determination
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Prep, layered column with tamped layer of 5 g diat earth,
960.53B, on bottom; add layer of 5 g diat. earth thoroly mixed
with 5 mL H 2 0, and tamp. Thoroly mix 25 g diat. earth and
5 g NaHC0 3 in 600 mL beaker, add 25 mL aliquot of piper-
azine soln contg ca 100 mg piperazine, and again mix thoroly.
Add 2 mL Ac 2 and mix 5 min, transferring to another beaker
to ensure thoro mixing. Add mixt. to column, using large fun-
nel to prevent loss, and tamp. Dry- wash the 2 beakers with 5
g diat. earth, add to column, and tamp. Place pad of glass
wool on top.
Pass 200 mL CHC1 3 thru column, adjusting flow to ca 7
mL/min, and collect eluate in 250 mL beaker, previously dried
at 80°, cooled in desiccator, and weighed. Evap. CHC1 3 on
steam bath with air current, and dry to const wt in convection
oven at 80° (ca 3 hr). Piperazine = diacetylpiperazine x 0,5061.
Check for complete extn by passing another 100 mL portion
CHC1 3 thru column, evapg to dryness, and noting if residue is
present.
Det. mp of diacetylpiperazine, which should be ca 140°.
Ref.: J AOAC 44, 312(1961).
CAS-1 10-85-0 (piperazine)
966.25 Piperazine in Drugs
Near-Infrared Method
First Action 1966
Final Action 1971
A. Apparatus and Reagents
(a) Near-infrared spectrophotometer. — With 5 cm Si cells.
(b) Drying tube. — Approx. 3.5 cm diam. x 9 cm long. Pack
with glass wool and ca 6 cm granular anhyd. Na 2 S0 4 pre-
wetted with ca 30 mL reagent grade CHC1 3 .
(c) Piperazine dihydrochloride . — Anal. std. (Available from
Pfaltz & Bauer Inc., 375 Fairfield Ave, Stamford CT 06902.)
Store above Si gel.
B. Preparation of Standard
Accurately weigh std piperazine equiv. to ca 3.5-3.8 g an-
hyd. base, transfer to 100 mL vol. flask with H 2 0, dil. to vol.,
and mix. Transfer 10.0 mL of this soln and exactly 5 mL H 2
to separator. Add 25 mL NaOH soln (1 + 1) and swirl. Final
concn of NaOH must be >30%. Cool separator under tap, add
30 mL CHCI3, and shake carefully ca 2 min. Drain CHC1 3
layer thru drying tube, (b), into 100 mL vol. flask. Ext with
three 20 mL portions CHC1 3 , draining thru drying tube into
vol. flask. Rinse tube with CHC1 3 and dil. to vol. Prep, blank
as above, using 15 mL H 2 0.
C. Preparation of Sample
(a) Powders. — Transfer sample contg ca 250-300 mg pi-
perazine base thru small funnel to separator contg exactly 5
mL H 2 0. Rinse funnel with exactly 10 mL H 2 from pipet
Mix, and proceed as in 966.25B, beginning, "Add 25 mL NaOH
soln ..." Dil. CHCI3 exts to 100 mL.
(b) Sirups. — Transfer sample contg ca 500 mg piperazine
base to separator. Add H 2 to total vol. of exactly 15 mL.
Proceed as in 966. 25B, beginning "Add 25 mL NaOH soln
..." except use 200 mL vol. flask to collect CHC1 3 exts, and
ext with three 50 mL portions CHC1 3 , finally rinsing inside of
separator with several 10-15 mL portions CHC1 3 before dilg
to 200 mL.
D. Determination
Using 5 cm cells, scan from 1600 to 1450 nm against blank.
(Max. is ca 1520 nm.) Draw baseline between min. at ca 1460
and 1565 nm and det. net A.
(A /A') X (mg base in std/mL CHC1 3 )
= (mg base in sample) /(mL final CHC1 3 soln)
where A and A' refer to sample and std, resp. Convert sample
from base to known salt formula, if desired.
Ref.: JAOAC 48, 590(1965).
CAS-1 10-85-0 (piperazine)
MlCROCHEMlCAL TESTS
930.40 Alkaloids and Related Amines
in Drugs
Microchemical Tests
Final Action
A. Reagents
(a) Ammoniacal silver nitrate soln. — Mix 2.5 mL 4% AgN0 3
soln with 2.5 mL NH 4 OH (1 + 5). Prep, fresh.
(b) Ammonium hydroxide soln. — 10% NH 3 (2 + 3).
(c) Ammonium thiocyanate soln. — Dissolve 5 g NH 4 SCN
in 100 mL H 2 0.
(d) Bismuth iodide soln. — (7) Prep, stock coned Bi(N0 3 ) 3
534
Drugs; Part I
AOAC Official Methods of Analysis (1990)
Table 930.40 Characteristics of Microchemical Tests for Alkaloids and Related Amines
Alkaloid
Reagent
Description of Crystals
Aconitine ( 1 )
Sodium carbonate
In 1 :3000 soln heated to 50° in test tube. Small, transparent, hexagonal plates; also rods
in contact.
Amylocaine (2)
1 drop HCI and 1
gold chloride
drop
1 :50. Dendritic crystals.
Apomorphine (3)
Potassium iodide
Gold chloride
Hydrochloric acid
1 :50. Small crystals that have sharp, clear-cut angles like those of diamond.
Red-brown, fine needles, in dense masses in all solns to 1:10,000.
1 :50. Small rods singly and in clusters.
Arecoline ( / )
Bismuth iodide
Red, rhombic crystals.
Atropine (4)
Iodine potassium
odide
Small, dark rods and triangular plates form in great numbers, singly and in groups.
Benzylmorphine (5)
(Peronine)
Potassium iodide
Ammonium thiocyanate
Hydrochloric acid
1:200. Dense rosettes of needles. Crystals are formed readily in dil. solns (1:1000) in
form of sheaves of needles.
1 :200. Rosettes and sheaves of needles in acid or neut. soln.
1:100. Rods, usually notched at ends and often in rosettes, are formed on stirring.
Berberine (6)
Hydrochloric acid
Satd soln; fine yellow needles. (Avoid excess reagent.)
Brucine (7)
Potassium iodide
Mercuric chloride
Long masses of transparent, rectangular plates; also rosettes of thin plates.
Small, dense rosettes.
Choline (8)
Reinecke salt
Add 1 drop acetone to 1 drop H 2 soln of base. Stir, add 1 drop reagent, and stir again.
1:100. Thin, hexgonal plates and star-shaped forms.
1:1000-1:10,000. Six-sided, more coffin-shaped plates; sometimes rosette aggregates of
plates on edge, resembling needles.
Platinic chloride and
sodium iodide
1:100 in H 2 0. Add 1 drop H 2 PtCI 6 soln, stir, and add small drop Nal soln without stirring.
Small black rectangular prisms and slender black rods.
Cinchonidine {9)
Sodium benzoate
Platinic chloride
Sodium carbonate
Rosettes and sheaves of needles spreading to large size.
Rosettes of transparent plates.
Spherical crystals, but not needles as in cinchonine.
Cinchonine (9)
Sodium carbonate
Disodium phosphate
Dark rosettes, composed of radiating needles, form immediately.
Similar to crystals formed by Na 2 C0 3 , but more burr-shaped.
Cocaine {10)
Platinic chloride
Delicate, feathery crystals, later becoming heavier in structure.
Codeine (TO)
Potassium cadmium iodide
Iodine potassium iodide
Silvery, circular masses, crystg into dark rosettes of irregular outline.
Heavy, red-brown ppt; crystallizes very slowly in yellow blades extending in branches
(never red).
Cotarnine (6)
Platinic chloride
Mercuric chloride
Potassium ferrocyanide
1:200. Hair-like crystals, yellow and curving.
Colorless, long, branching needles.
Acidify with 1 drop 5% HCI; globules that develop into dense, burr-shaped crystals; also
amber-brown plates.
Diacetylmorphine {11)
(Heroin)
Platinic chloride
Spherical clusters of golden yellow needles form slowly around nucleus; cluster
disintegrates on standing.
Ephedrine {12)
Bismuth iodide in dild sulfuric acid 1:200. Long, brownish orange, radiating and interlacing needles and branching rods.
Ethylhydrocupreine {13)
Ammonium thiocyanate
1:100 in 0.1 N HCI. Long, straight needles.
Ethy (morphine (5)
Iodine potassium iodide
Mercuric chloride
1:200. Groups of yellow needles, branching later.
Transparent plates, often with notched ends; singly and in groups. Stir to start crystn.
Homatropine {14)
Gold chloride
1:200. Green-gold blades, often with pointed ends and united in pairs; surfaces appear
etched on long standing.
Hydrastine (3)
1 drop 5% HCI and 1 drop
potassium ferrocyanide
1:100. Spheres of radiating crystals. Shake slide to start crystn. Avoid excess reagent.
Hydrastinine (5)
Potassium permanganate
Mercuric chloride
1 drop 5% HCI and 1 drop
potassium ferrocyanide
1:500. Immediate red plates, often with serrated edges. In coned soln, great number of
large red or brown plates with deeply cut edges.
1 :500. Transparent needles forming branches rapidly in neut. and acidified solns.
1 :200. Yellow rhombic plates and tree-like crystals.
Hydromorphone {15)
Sodium nitroprusside
To minute amt (<1 mg) in 2 drops H 2 add minute fragment of reagent. Elongated 6-
sided prisms; also in aggregates.
Thin, transparent, nearly colorless irregular plates, often curved. Crystals form slowly in
1:100 to 1:200 soln. Shaking slide aids crystn.
Hyoscyamine (74)
Gold chloride
Morphine {10)
Potassium cadmium iodide
Iodine potassium iodide
Silvery, gelatinous ppt, crystg in dense masses of fine needles.
Small drop of reagent produces heavy, red-brown ppt, slowly crystg in shining, red,
overlapping plates extending in branches.
Narceine {6)
Iodine potassium iodide
or zinc potassium iodide
Platinic chloride
1 :400. Blue, radiating needles, sometimes with yellow dichroism.
Beautiful feathery rosettes develop in all solns.
Nicotine {16)
Mercuric chloride
Mercuric chloride-sodium chloride
Radiating, transparent blades form in presence of slight excess of H 2 S0 4 ; feather-like
blades form in presence of HCI.
Radiating, transparent blades.
AOAC Official Methods of Analysis (1990)
MlCROCHEMICAL TESTS 535
Table 930.40 Characteristics of Microchemical Tests for Alkaloids and Related Amines — Continued
Alkaloid
Reagent
Description of Crystals
Noscapine (6)
(/-Narcotine)
Potassium hydroxide or
ammonium hydroxide
1 :200. White, amorphous ppt that crystallizes slowly; dense rosettes of needles.
Papaverine (17)
Zinc chloride
Thin, rectangular plates in excess HCI.
Physostigmine (18)
Lead iodide
Gold bromide in HCI
1:100. Radiating, serrated plates.
1 mg in 1 drop H 2 0. Brown, dendritic aggregates.
Pilocarpine (4)
Platinic chloride
Crystals form slowly; layers of thin, yellow, triangular plates of delicate structure.
Procaine (17)
Platinic chloride
Gold chloride and HCI
Spherical crystals of radiating branches.
Irregular, radiating branches.
Quinidine (9)
Potassium iodide
Small, triangular crystals in great numbers; best in 1:1000 diln; sol. in excess reagent.
Quinine (9)
Disodium phosphate
Silvery, sheaf-like crystals.
Racephedrine (19)
(d/-Ephedrine)
Bismuth iodide in dild
sulfuric acid
1 :200. Large orange plates and red prisms and grains.
Scopolamine (14)
(Hyoscine)
Gold chloride
Clusters of pale yellow, transparent blades, with coarse, saw-toothed edges form
immediately on shaking slide. Crystals grow to large size in 1 :200 soln.
Sparteine (16)
Gold chloride
Large numbers of blade-like crystals varying in size according to concn.
Strychnine (20)
Platinic chloride
Potassium cadmium iodide
Crystals form immediately in clusters and singly in small, wedge-shaped needles that
move about field.
Silvery masses, slowly forming rosettes.
Yohimbine (1)
Sodium carbonate
In 1:1000 soln heated to 50°. Fine needles in sheaf-like bundles and rosettes.
soln by dissolving 50 g Bi subnitrate in 70 mL HN0 3 (1 + 1)
and dilg to 100 mL with H 2 0. (2) Dissolve 1.25 g KI in 4.5
mL H 2 and add 0.5 mL stock coned Bi(N0 3 ) 3 soln. Prep.
fresh when soln darkens appreciably.
(e) Bismuth iodide in diluted sulfuric acid soln. — Dissolve
1.25 g KI in 2.0 mL H 2 0, and add 2.5 mL H 2 S0 4 (1 + 3)
and 0.5 mL stock coned Bi(N0 3 ) 3 soln, (d)(7). Prep, fresh
daily.
(f) Disodium phosphate soln. — Dissolve 5 g Na 2 HP0 4 .
12H 2 in 100 mL H 2 0.
(g) Gold bromide in hydrochloric acid soln. — Dissolve 1 g
HAuCl 4 .3H 2 and 1.5 mL 40% HBr in 18 mL HCI. (Satd aq.
NaBr soln may be substituted for the HBr.)
(h) Gold chloride soln.— Dissolve 1 g HAuCl 4 .3H 2 in 20
mL H 2 0.
(i) Hydrochloric acid. — 5% (1 + 6).
(j) Iodine -potassium iodide soln. — Dissolve 1.27 g 1 and 2
g KI in 5 mL H 2 0, and dil. to 100 mL.
(k) Lead iodide soln. — To aq. KOAc soln (1 + 3) add 1
drop Me red and HO Ac until yellow changes to orange; then,
while gently warming, sat. with Pbl 2 , cool, and filter.
(1) Mercuric chloride soln. — Dissolve 5 g HgCl 2 in 100 mL
H 2 0.
(m) Mercuric chloride -sodium: chloride soln. — Dissolve 5
g HgCl 2 and 0.75 g NaCl in 100 mL H 2 0.
(n) Platinic chloride soln. — Dissolve 5 g H 2 Pt0 6 .6H 2 in
100 mL H 2 0.
(o) Potassium cadmium iodide soln. — Dissolve 3 g Cdl 2 in
18 mL H 2 contg 6 g KI.
(p) Potassium ferrocyanide soln. — Dissolve 5 g
K 4 Fe(CN) 6 .3H 2 in 100 mL H 2 0.
(q) Potassium hydroxide soln. — Dissolve 5 g KOH in 100
mL H 2 0.
(r) Potassium iodide sc/az.— Dissolve 5 g KI in 100 mL H 2 0.
(s) Potassium permanganate soln. — Dissolve 1 g KMn0 4
in 100 mL H 2 0.
(t) Reinecke salt soln. — Dissolve 0. 1 g NH 4
[Cr(NH 3 ) 2 (SCN) 4 ].H 2 and 0.03 g H 2 NOH.HCl in 10 mL al-
cohol. Filter, and store in refrigerator. (Reagent is stable >6
months.)
(u) Sodium benzoate soln. — Dissolve 5 g Na benzoate in
100 mL H 2 0.
(v) Sodium carbonate soln. — Dissolve 5 g Na 2 C0 3 .H 2 in
100 mL H 2 0.
(w) Sodium iodide soln. — Dissolve 5 g Nal in 100 mL H 2 0.
(x) Sodium nitroprusside . — Na 2 Fe(CN) 5 N0.2H 2 crystals.
(y) Zinc chloride soln. — Dissolve 5 g ZnCl 2 in 100 mL H 2 0.
(z) Zinc potassium iodide soln. — Dissolve 5 g
Zn(OAc) 2 .3H 2 and 20 g KI in 100 mL H 2 0.
B. Preparation of Samples
(a) Usual controls. — Dissolve 0.4 or 0.2 mg pure alkaloid
salt in 0.04 mL H 2 to make ca 1:100 or 1:200 soln.
(b) Alkaloids in compounds. — Sep. alkaloid in pure form
by extg it from ammoniacal soln with suitable immiscible solv.,
and evap. solv. Dissolve little of residue in min. of 0.17V HCI
and dil. with H 2 0, if necessary, to ca alkaloid concn specified
in (a) or in test.
(c) Hypodermic tablets. — Dissolve portion of tablet in drop
of H 2 to ca same alkaloid concn specified in (a) or in test.
C. Identification
Place drop (ca 0.04 mL) of alkaloid soln on glass slide, add
drop of reagent, and without stirring or covering, examine un-
der microscope, using magnification of ca 100-500X. Note
kind of crystals formed. Compare their characteristics with de-
scriptions given, Table 930.40, and with a control. Use po-
larizing microscope if available, and note characteristics such
as birefringence and dichroism.
Refs.: (/) JAOAC 15, 413(1932).
(2) JAOAC 23, 746(1940).
(3) JAOAC 20, 551(1937); 21, 91(1938).
(4) JAOAC 11, 353(1928); 14, 316(1931);
521(1935).
(5) JAOAC 21, 525(1938).
(6) JAOAC 22, 706(1939).
(7) JAOAC 13, 315(1930).
(8) JAOAC 26, 96(1943).
(9) JAOAC 12, 282(1929).
(70) JAOAC 10, 370(1927); 11, 353(1928).
(77) JAOAC 5, 154(1921); 10, 370(1927).
(72) JAOAC 14, 316(1931).
(75) JAOAC 20, 553(1937).
18,
536
Drugs: Part I
AOAC Official Methods of Analysis (1990)
(14) JAOAC 18, 521(1935).
(15) JAOAC 24, 830(1941).
(16) JAOAC 16, 345(1933).
(17) JAOAC 17, 433(1934).
(18) JAOAC 23, 746(1940); 24, 830(1941).
(19) JAOAC 43, 262(1960); 61, 1435(1978).
(20) JAOAC 11, 353(1928).
962.21 Barbiturates in Drugs
Microchemical Tests
First Action 1962
Final Action 1972
A. Reagent
Iodine-potassium, iodide soln. — Dissolve 5 g I and 80 g KI
in enough H 2 (ca 78 mL) to make 100 mL. Dil. with 2 parts
by vol. of H3PO4. Prep, dild reagent every 2-3 weeks.
0, Identification
Dissolve little barbiturate in drop H 2 on slide. If present
as Na salt, it dissolves readily; if present as acid, add little
droplet 1% NaOH on stirring rod and mix. Add 1 full drop
reagent and let stand until crystn occurs (immediate with some
compds, 0.5-1 hr with secobarbital). Free acid may ppt or
crystallize. However, I reaction crystals are easily distin-
guished by their color, often coupled with strong dichroism.
Det. birefringence with polarizing microscope. Cover glass is
usually not needed but may be used for observation at high
magnification and when slide stands >1 hr; on standing, KI
may crystallize as sq, colorless, isotropic crystals.
Note crystals formed and compare characteristics with de-
scriptions, Tables 962. 21 A and B.
Ref.: JAOAC 45, 600(1962).
985.44 Phenothiazine Drugs
Microcrystalline Identification
First Action 1985
Final Action 1988
(Applicable to perphenazine, promethazine, thiethylperazine,
and triflupromazine pure drug substances)
A. Reagents
(a) Gold bromide soln. — Dissolve 1 g HAuCl 4 .3H 2 and 1
g NaBr in 30 mL H 2 S0 4 (2 + 3). Before use, mix 3 vols of
this soln with 1 vol. of glacial HO Ac. Store in dark bottle.
(b) Iodine-potassium iodide acidic soln. — Dissolve 5 g I and
30 g KI in 100 mL H 2 0. Mix 1 vol. of this soln with 1 .5 vols
of HC1 and L5 vols of H 3 P0 4 . Store in dark bottle.
B. Standard Solutions
Prep, individual solns of perphenazine, promethazine, thi-
ethylperazine, and triflupromazine by dissolving in HO Ac (2
+ I) to final concn of ca 2 |xg/|xL. If necessary, dil. further
with HO Ac (2+1) for optimum results. Exact concn depends
on compd being tested.
C. Sample Solutions
Dissolve sample in HO Ac (2 + 1) to final concn of ca 2
|mg/|xL. If necessary, dil. further with HOAc (2 + 1) to obtain
solns with strength equiv. to std solns.
D. Procedure
Place small drop (ca 10 |xL) of sample soln on each of 2
clean microscope slides. Place small drop (ca 10 |xL) of re-
agent soln (a) or (b) on 2 cover slips, resp.; invert cover slips
and place over sample solns on slides. Let stand until crystn
occurs (immediately with some compds; 1-20 min with oth-
ers). Examine slide under polarizing microscope at ca 100-
300x. If microcrystn product is formed, note shape and rate
of formation of crystals. See Table 985.44. Repeat tests with
corresponding std soln of comparable strength, and compare
product with that obtained with sample soln.
Ref.: JAOAC 68, 527(1985).
CAS-58-39-9 (perphenazine)
CAS-60-87-7 (promethazine)
CAS- 1420-55-9 (thiethylperazine)
CAS- 146-54-3 (triflupromazine)
960.55 Sympathomimetic Drugs
Microchemical Tests
Final Action 1970
A. Reagents
(a) Bismuth iodide in diluted sulfuric acid soln. — See
930.40A(e).
(b) Gold chloride in diluted phosphoric acid soln. — Dis-
solve 1 g HAuCI 4 .3H 2 in 20 mL H 3 P0 4 (1+2).
(c) Platinic chloride in diluted phosphoric acid soln. — Dis-
solve 1 g H 2 PtCl 6 .6H 2 in 20 mL H3PO4 (1 + 3).
(d) Sodium tetraphenylboron soln. — Aq. soln (1 + 20).
B. Identification
(a) Direct test. — Add drop of reagent to little of powd solid
or crushed tablet and spread out on slide with little stirring.
Do not stir to homogeneity as local concns and dilns will assist
crystn. Let stand to evap. to higher acid concn if necessary
for crystal formation.
(b) Volatility test. — Place small amt of substance or crushed
tablet in depression of cavity slide, add drop 5% NaOH soln,
and stir briefly. Place very small drop of reagent on thin slide,
invert over cavity slide, and let stand. As crystals appear, ex-
amine with inverted slide in place. After observing crystals or
after >1 hr exposure, if only few or no crystals form, reinvert
thin slide with hanging drop, and let stand for gradual evapn
of H 2 from reagent drop. Examine for crystals. Compare with
descriptions, Table 960.55.
Refs.: JAOAC 43, 262(1960); 61, 1435(1978).
Table 985.44 Characteristics of Microchemical Tests for Synthetics
Synthetic
Solvent
Concn of
Synthetic
Reagent
Description of
Test and Crystals
Perphenazine
HOAc (2 + 1 )
1:500
Iodine-potassium iodide acidic
X-shape serrated blades. Pos.
elongation
Thiethylperazine
HOAc (2+1)
1:500
Iodine-potassium iodide acidic
Branching clusters. Neg. elongation
Promethazine
HOAc (2+1)
1:500
Gold bromide in H 2 S0 4 /HOAc
Small rods; clusters of rods
Triflupromazine
HOAc (2 + 1)
1:500
Gold bromide in H 2 S0 4 /HOAc
Large X-shape segmented plates;
small swirling tufts
AOAC Official Methods of Analysis (1990)
MlCROCHEMICAL TESTS 537
Table 962.21 A Characteristics of Microchemical Tests for Barbiturates
Barbiturate
Crystal Form
Dichroism or Pleochroism
Remarks
Allobarbital (Diallylbarbituric acid,
5,5-Diallylbarbituric acid)
5-Allyl-5-(2-cyclopenten-1 -yl)
barbituric acid (cyclopal)
Crystallizes quickly in branching twigs,
splinters, and blades.
Extreme black to "white"
dichroism by polarized light.
Golden-beetle iridescence by
reflected light.
Gradual crystn in dichroic straight-edged
blades, brown-yellow to brown-orange.
Very bright birefringence; free
acid as colorless rods,
splinters, needles.
Amobarbital (5-Ethyl-5-
isoamylbarbituric acid)
Dil. soln: fairly large brown blades.
Coned soln: multitudes of little pale-
colored flakes.
Examine at 200 x; sensitive test.
Aprobarbital (5-Allyl-5-
isopropylbarbituric acid)
Light orange-brown rod-blades,
birefringent.
Yellow to brown-orange
dichroism.
—
Barbital (5,5-Diethylbarbituric
acid)
Form very soon; fairly large, rectangular
or splinter blades.
Extreme pleochroism by
transmitted polarized light.
Beetle-green iridescence by
reflected light.
* Bemegride (4-Ethyl-4-methyl-
2,6-piperidinedione)
Small light-colored dichroic rods or
blades and flakes or plates, orangish
brown to colorless or yellowish.
Birefringence is bright and plates
that are sq or nearly so
extinguish diagonally.
Butabarbital Sodium (Sodium 5-
sec-butyl-5-ethylbarbiturate)
Dil. soln: red-brown irregular plates.
Coned soln: brown blades in clusters.
Slightly dichroic; dichroism yellow
to red-brown.
Free acid: colorless blades.
Butalbital (Allylbarbital, 5-Allyl-5-
isobutylbarbituric acid,
"Itobarbital")
Immediate crystn in rods, splinters, and
leaflike crystals with pointed ends.
Red to black dichroism.
Free acid may crystallize out.
Butethal (5-Butyl-5-ethylbarbituric
acid)
Small plate crystals basically rhomboids.
"Propeller-type" of elongate pointed
blades.
Strong dichroism, light yellow to
black.
Cyclobarbital (5-(1 -Cyclohexenyl)-
5-ethylbarbituric acid)
Dil. soln: rosettes of little pointed
crystals; larger are red-brown plates.
Red-brown plates of variable
dichroism frequently four-
bladed.
Sensitive test.
Heptabarbital (5-(1 -Cyclohepten-
1-yl)-5-ethylbarbituric acid)
Little red-brown plates in great numbers,
often 4-parted; good birefringence.
__
Sensitive.
Hexobarbital Sodium (Sodium 5-
(1-cyclohexen-1-yl)-1,5-
dimethylbarbiturate
Dichroic blades and broad splinters in
groups, varying to curving threads and
needles in rosettes.
Very strong dichroism; black to
light brownish yellow.
Sensitive for i reaction crystals
as well as for free acid.
Metharbital (5,5-Diethyl-i-
methylbarbituric acid)
Dark needles, small to large, and
splintery narrow blades.
Dichroism black to brown.
Good birefringence with crossed
nicols.
Pentobarbital Sodium (Sodium 5-
ethyl-5-(1-methylbutyl)
barbiturate)
Crystallizes quickly in great numbers of
small red-brown plates.
Minute light-colored flakes exhibit
dichroism; dark brown or black
to yellow.
Phenobarbital (5-Ethyl-5-
phenylbarbituric acid)
Soon crystallizes in little dark grains;
also a few larger red blades and dark
splinter-rods in clusters.
Free acid may also crystallize
out.
Phenylmethylbarbituric acid (5-
Methyl-5-phenyibarbituric acid)
Red-brown irregular platy forms appear
after free acid is pptd.
Gradually strongly dichroic rods
or blades
Test fairly sensitive for dil. soln.
Probarbital (5-Ethyl~5-
isopropylbarbituric acid)
Scattered iodine-reaction crystals form in various jagged shapes, color dark
brown to black dichrosim, or red-black with but little dichroism.
Free acid thrown out, forming
long rods with pointed ends.
Secobarbital (5-Allyl-5-(1-
methylbutyl)barbituric acid)
Crystallizes in plates or elongate and
rectangular but mostly distorted into
any shape after 1 hr.
Light yellow to orange or red
dichroism by polarized light.
Distinctly birefringent.
Talbutal (5-Allyl-5-sec-
butytbarbituric acid)
Amorphous ppt crystallizes in large
needles and dichroic blades, lighter to
deeper brown, in dendrites; then gray-
black curled sheaves of threads.
Excellent test. Both types of
crystals have good
birefringence
Vinbarbital (5-Ethyl-5-(1 -methyl-1 -
butenyl) barbituric acid)
Multitudes of small dark crystals, tiny grains and rods with dichroism brown to
black. In quite dil. soln possible to get good small crystals, little dark rods
with dichroism red to black, and small plates tending to be sq, generally
appearing red but with same red to black dichroism, and with sq extinction
(not diagonal).
Very sensitive.
* This drug has barbiturate -type formula (although there is only one N) but is central nervous stimulant instead of depressant.
538
Drugs: Part I
AOAC Official Methods of Analysis (1990)
Table 962.21 B Characteristics of Microchemical Tests for Synthetics
Synthetic
Solvent
Concentration
of Synthetic
Reagent
Description of Tests
and Crystals
Acetanilid (1)
HCI (1 + 3)
HCI (1 + 3)
1:100
1:100
Phosphotungstic
acid
Bromide-bromate
soln
Rosettes of prisms.
Small prisms.
Allobarbital
(Diallybarbituric acid)
(2)
Benzoic acid (6)
Diphenhydramine
hydrochloride (8)
Dry powder Lead Stir small amt of synthetic into 1 drop reagent. Rods
triethanolamine singly and in clusters.
Dry powder Barium hydroxide Stir small amt of synthetic into 1 drop reagent. Rods
singly and in groups.
Aminopyrine (3)
H 2
1:100
Mercuric chloride
Potassium cad-
mium iodide
Long, slender, radiating crystals, often curved.
Groups of spiny branches.
Amobarbital (4)
NH 4 OH (1 + 9)
NH 4 OH (1 + 9)
1:50
1:25
Acetic acid
Acetic acid
Long, branching needles; some hexagonal plates in
groups.
Groups of rectangular plates.
Antipyrine (5)
H 2
1:100
Potassium
ferrocyanide
Add 1 drop HCI (1 + 39). Acicular and prismatic
crystals form.
Aspirin (Acetylsalicylic
acid) (6)
2% triethanolamine
1:50
Silver nitrate
Fine, curling, hair-like crystals form first near edge of
drop.
Barbital (4)
NH4OH (1 + 9)
Approx. 1 mg
powder
1:50
Ammoniacal silver
nitrate
Acetic acid
Stir to aid soln and crystn. Very small, twined crystals
and larger tufts.
Dark burrs (stirring hastens crystn).
Benzocaine (Ethyl
aminobenzoate) (7)
0.1 A/ HCI
1:100
Potassium
ferrocyanide
Colorless, irregular plates and rods.
Dry powder Lead Stir small amt of synthetic into 1 drop reagent. Stir
triethanolamine thoroly to induce crystn. 4-sided plates, singly and in
groups.
Dry powder Zinc pyridine Stir small amt of synthetic into 1 drop reagent. Stir
thoroly to induce crystn. Hexagonal crystals.
2% triethanolamine
1:100 to 1:200
Silver nitrate
Rods or curving blades with irregular ends.
Cinchophen (7)
0.1/VNaOH. Add H 2
and make slightly
acid with HCI
1:1000
Gold chloride
Dark clusters of needles. Few short, rhombic crystals.
Dinitrophenol (3)
Small amt of 0.1 N
NaOH
1:100
HCI
Plates with 4 branches. In more dil. soln, single
rectangular plates.
Glycerol-alcohol (1 +
1) or H 2
Approx. 0.2 mg Platinic chloride Aggregates of platy crystals form readily in glycerol-
powder or tablet alcohol, gradually in H 2 0. Plates with jagged edges,
material or tendency to twin, forming X-shaped aggregates, hour-
1:100 glass forms, and dendritic structures. First order gray
polarization colors; symmetrical or parallel extinction.
Plates show pos. elongation.
Hydrochlorothiazide (9)
5% NaOH
Iodine-potassium Burrs, with iodine-colored centers and highly
iodide, 962.21 A birefringent peripheral blades or dichroic rods, iodine-
colored to colorless 3
8-Hydroxyquinoline
sulfate (7)
Phenacetin
(Acetophenetidin (?)
Dissolve salt in H 2 0.
Dissolve free base
in HCI (1 + 3),
avoiding excess
1 :500 Magnesia mixt. Small, elliptical grains. Few burr-shaped crystals on
standing.
Mandelic acid (2)
H 2
H 2
1:100
1:100
Lead acetate
Mercurous nitrate
Rosettes of thin, curving plates.
Burr-shaped groups of needles.
Methenamine (5)
H 2
1:500
Silicotungstic
acid
Thin, transparent, rectangular crystals.
Neocinchophen (1)
HCI (1 + 3)
HCI (1 + 3)
Satd soln
Satd soln
Ammonium
thiocyanate
Platinic chloride
Rosettes of needles. (Gentle agitation by tipping slide
back and forth hastens crystn,)
Needles in clusters.
Pentylenetetrazol (10)
H 2
H 2
1:100
Mercuric chloride
(1:10)
Silicotungstic
acid
Rods, many almost needle-like; frequency in groups;
also in radiating aggregates.
Amorphous, changes to elongated prisms; also long
needles.
HCI (1 + 3)
Approx. 1 mg
powd material
Satd soln
HNO3
Iodine-potassium
iodide
Add 1 drop HN0 3 , let stand few sec, then add 1 drop
H 2 0. Bright yellow, curving, branched crystals.
Large, irregular plates.
Phenazopyridine.HCI (7)
Dissolve salt in H 2 0.
Dissolve free base
in HCI (1 + 3),
avoiding excess
1:1000
Ammonium
thiocyanate
Small, red-brown, dense sheaves.
AOAC Official Methods of Analysis (1990)
MlCROCHEMICAL TESTS 539
Table 962.21 B Characteristics of Microchemicai Tests for Synthetics— Continued
Synthetic
Solvent
Concentration
of Synthetic
Reagent
Description of Tests
and Crystals
Phenobarbital (4)
—
Approx. 1 mg
powder
Ammoniacal
nickel acetate
Stir to aid soln and crystn. Single rectangular crystals.
Pyrilamine maleate (8)
Glycerol-alcohol (1 +
1)or H 2
1:1000 or ca 0.1
mg powder
Platinic chloride
Needles in rosette aggregates, sheaves, and singly.
Needles show second order blue and green, and first
order red and yellow polarization colors; parallel
extinction and neg. elongation.
Salicylic acid (6)
HCI (1 + 3) Dry powder Bromide-bromate Stir few crystals into 1 drop of the HCI. Add 1 drop
soln reagent. Fine needles appear to grow from the
crystals of salicylic acid.
— Dry powder Lead Stir few crystals into 1 drop reagent. Rods or needles
triethanolamine grow from the crystals of salicylic acid.
2% triethanolamine 1:100 to 1:200 Silver nitrate Small, irregular plates; few short rods.
Sulfadiazine (11)
H 2
—
Gold bromide in
HCI
Red, circular masses composed of fine needles.
Sulfanilamide (2)
0.1WHCI
Dry powder
Satd soln
Benzaldehyde
Sodium nitrite
Thoroly stir small amt into 1 drop reagent. 4-sided
plates.
Yellow needles.
Sulfapyridine (12)
Acetone + H a O
—
Gold chloride
Yellow rods or blades; also X-shaped aggregates.
Sulfapyridine sodium
monohydrate (12)
H 2
1:100
Gold chloride
Yellow rods in X-shaped aggregates.
Sulfathiazole (10)
50% alcohol
50% alcohol (or no
solv.)
Picric acid Long, fine, yellow needles, many curved, occur in
dense rosettes; also short, stout rods in groups or
singly.
Picrolonic acid Distinct rosettes of very fine needles; also single
needles.
Trolamine
(Triethanolamine) (5)
H 2
1:100
Bismuth iodide
Oily globules changing to large, red, hexagonal plates
and prismatic crystals.
Tripelennamine
hydrochloride (8)
Glycerol-alcohol (1 +
1) or H 2
1:1000 or ca 0.1 Platinic chloride Small needles and bladed crystals in dense rosette
mg powder or aggregates and singly. Needles show first order white
tablet material and yellow polarization colors, parallel extinction, and
pos. elongation.
a Official First Action.
Refs.:
(7) J AOAC 19,
(2) J AOAC 22,
(3) JAOAC 18,
(4) JAOAC 20,
JAOAC 17,
JAOAC 21,
JAOAC 16,
JAOAC 35,
JAOAC 61,
JAOAC 25,
JAOAC 26,
(-5)
(6)
(7)
(8)
(9)
(10)
(11)
(12) JAOAC 24,
514(1936).
709(1939).
523(1935).
553(1937).
435(1934).
528(1938).
391(1933).
576(1952).
1435(1978).
830(1942).
96(1943).
830(1941).
540
Drugs: Part I
AOAC Official Methods of Analysis (1990)
Table 960.55 Characteristics of Microchemical Tests for Sympathomimetics
Sympathomimetic
Reagent
Test
Description of Crystals
Volatile Substances
d/-Amphetamine
Gold chloride in dild
phosphoric acid
Platinic chloride in dild
phosphoric acid
direct or volatility Very irregular plates, with irregular blade-arms especially after
evapn; sq if perfect,
volatility Irregular blades and needles, very low birefringence; after
evapn, characteristic plates with narrow irregular arms of
blades.
Dextroamphetamine
(d-Amphetamine)
Gold chloride in dild
phosphoric acid
Platinic chloride in dild
phosphoric acid
direct or volatility Long yellow rods and blades; with evapn, some crystals as
with di may form.
volatility Long needles, often bent, very little birefringence; after some
evapn, long rectangular blades. (/-Ephedrine in direct test
gives similar crystals which are more sol.; it is less volatile
and does not normally form crystals in hanging drop.)
(o7-Methamphetamine)
o7-Desoxyephedrine
Bismuth iodide in dild volatility
sulfuric acid
Freshly prepd gold chloride volatility
in dild phosphoric acid
Aged (>4 months) gold volatility
chloride in dild
phosphoric acid
Drops, crystg in orange-red prisms with conspicuously slanting
ends; inclined extinction ca 20°; also "mossy" formation of
grains and some large deep red grains.
Right-angled crossed blades with serrated and/or lobed edges.
X blade formation with highly birefringent ribs visible in thin
crystals or thickened X blades. Right-angled crossed blades
are not present.
Ephedrine ((-)-Ephedrine)
Gold chloride in dild
phosphoric acid
Bismuth iodide in dild
sulfuric acid
direct or volatility
volatility
Long needles or splinters and long jointed forms; strong bire-
fringence.
Long brownish orange needles, often branching or in sheaves;
also, especially with evapn, orange irregular blades.
Epinephrine
Sodium tetraphenylboron
volatility
MeNH 2 liberated; birefringent X's or 4-arm crystals; also thick
blades with central rib, pointed ends, pos. elongation.
Isoproterenol
Sodium tetraphenylboron
volatility
isopropylamine liberated; plates tending to nonregular
hexagons; no birefringence where plates lie flat but there are
rods which are birefringent.
Methamphetamine and
cf/-Methamphetamine
{d- and oV-Desoxyephedrine)
Gold chloride in dild
phosphoric acid
direct or volatility
Platinic chloride in dild
phosphoric acid
volatility
Methamphetamine
(d-Desoxyephedrine)
Bismuth iodide in dild
sulfuric acid
volatility
Freshly prepd gold chloride
in dild phosphoric acid
volatility
Aged (>4 months) gold
chloride in dild
phosphoric acid
volatility
Long blades and jointed crystals, fairly high birefringence.
Grains with sharp edges which aggregate in chains and short
prisms. Birefringent.
Drops, long orange splinters, blades, needles; also deep red
angular grains (red prisms only after evapn).
Numerous multiple "V"-shaped blades, few single "V"-shaped
blades.
Numerous single "V"-shaped blades.
Pseudoephedrine
Gold chloride in dild
phosphoric acid
direct or volatility (2 hr)
Thin branching sticks, many like combs; some broaden to
blades or spear-head plates; very high birefringence.
Slightly Volatile Substances
Racephedrine (ctf-Ephedrine)
Gold chloride in dild
phosphoric acid
Bismuth iodide in dild
sulfuric acid
direct or volatility Irregular plates based on the sq, growing along diagonals in 4
arms; some birefringent, some not.
volatility Orange rods or sticks, short and stubby, some plates; more
irregular plates on evapn.
Phenylpropanolamine
Phenmetrazine
Gold chloride in dild direct Plates and blades of extremely high birefringence, elongate
phosphoric acid hexagonal or diamonds, very bright colors. Branch into 4 or
6 irregular arms,
volatility (2 hr) After definite drying, pyramidal grains to blades and plates with
irregular arms, very birefringent.
Gold chloride in dild
phosphoric acid
Bismuth iodide in dild
sulfuric acid
direct or volatility Rectangular plates joined in jagged arms of strongly
birefringent crystals, often in X forms, very characteristic.
volatility Orange-red blades, usually pointed ends, often in rosettes; also
with needles in branching aggregates; also red prisms.
935.68 Synthetic Drugs
Microchemical Tests
Final Action
(See Table 962.21B.)
(a) Acetic acid. — Dil. 6 mL HOAc to 100 mL with H 2 0.
(b) Ammoniacal nickel acetate so In. — Mix 1 vol. 5%
Ni(OAc) 2 .4H 2 soln with 1 vol. NH 4 OH (2+3). Use clear
supernate.
(c) Ammoniacal silver nitrate soln. — See 930.40A(a).
(d) Ammonium thiocyanate soln. — See 930.40A(c).
(e) Barium hydroxide soln. — Satd aq. soln.
(f ) Benzaldehyde. — USP quality.
(g) Bismuth iodide soln. —See 930.40A(d).
AOAC Official Methods of Analysis (1990)
Microscopy
541
(h) Br omide-br ornate soln. — Dissolve 0.3 g KBr0 3 and 1.2
g KBr in H 2 0, and dil. to 100 mL.
(i) Glycerol-alcohol mixture. — (1 + 1).
(j) Gold bromide in hydrochloric acid soln. — See
930.40A(g).
(k) Gold chloride soln. —See 930.40A(h).
(I) Iodine-potassium iodide soln. — See 930.40A(j).
(m) Lead acetate soln. — Dissolve 5 g Pb(OAc)2.3H 2 in
H 2 and dil. to 100 mL.
(n) Lead triethanolamine soln. — Add 1 mL triethanolamine
(tech. 90% is satisfactory) to soln of 1 g Pb(OAc) 2 .3H 2 in
20 mL H 2 0. Slight turbidity does not interfere.
(o) Magnesia mixture. — Dissolve 5.5 g MgCl 2 .6H 2 and
14.0 g NH 4 C1 in H 2 0. Add 13.05 mL NH 4 OH and dil. to 100
mL with H 2 0.
(p) Mercuric chloride soln. — See 930.40 A(I).
(q) Mercurous nitrate soln. — Dissolve 15 g HgN0 3 .H 2 in
mixt. of 90 mL H 2 and 10 mL HNO3 (1 + 9). Store in dark,
amber bottle contg small globule of Hg.
(r) Nitric acid. — (1 + 1).
(s) Phosphotungstic acid soln. — Dissolve 5 g P 2 5 .24W0 3 .
xH 2 in 100 mL H 2 0.
(t) Picric acid. — Crystals.
(u) Picrolonic acid soln. — Dissolve 250 mg l-(p-nitro-
phenyl)-3-methyl-4-nitropyrazolone in 25 mL alcohol.
(v) Platinic chloride soln. — See 930.40A(n).
(w) Potassium cadmium iodide soln. — See 930.40A(o).
(x) Potassium f err ocyanide soln. — See 930.40A(p).
(y) Silicotungstic acid soln. — Dissolve 5 g 4H 2 0.
Si0 2 .12W0 3 .22H 2 in 100 mL ca 6N H 2 S(V
(z) Silver nitrate soln. — Dissolve 1 g AgN0 3 in 20 mL H 2 0.
(aa) Sodium nitrite soln. — Dissolve 10 g NaN0 2 in H 2
and dil. to 100 mL.
(bb) Zinc pyridine soln. — Add 1 mL pyridine to soln of 1
g Zn(OAc) 2 .2H 2 in 20 mL H 2 0.
960.56 Xanthine Group Alkaloid Drugs
Microchemical Tests
First Action
A. Reagents
(a) Bismuth iodide soln.— See 930.40A(d)(2).
(b) Gold bromide in dilute hydrochloric acid. — Dissolve 1
g HAuCl 4 .3H 2 in 1.5 mL 40% HBr and add HC1 (1 + 3) to
make 45 mL.
(c) Iodine-potassium iodide soln (5-14). — Dissolve 5
and 14 g Kl in H 2 and dil. to 100 mL with H 2 0.
B. General Test
gl
(Murexide reaction)
To small amt of substance in small porcelain crucible add
very small crystal KC10 3 and 1 drop HC1 (1 + 1). Set on hot
plate at ca 100°, or hot enough to boil off H 2 in short time.
Soon after drying, residue becomes orange to red. Add 1 drop
NH4OH. Purple color is produced in presence of caffeine,
theobromine, theophylline, and related xanthine derivatives.
C. Identification
(a) Bismuth iodide soln. — Add 1 drop reagent to little dry
material on slide and cover.
(b) Gold bromide in dilute hydrochloric acid. — Place 1 drop
reagent beside very small amt of dry substance on slide and
apply cover glass so that reagent flows over substance.
(c) Iodine -potassium iodide soln (5-14). — In depression of
cavity slide dissolve little of substance in small drop 1 % NaOH
soln and stir in excess NaHC0 3 (some undissolved). Add large
drop reagent and stir slightly. Add several crystals KC1. Ex-
amine center and edge as soln evaps.
See Table 960.56.
Refs.: JAOAC 43, 262(1960); 61, 1435(1978).
MICROSCOPY
960.57 Crystalline Substances
Optical-Crystal lographic Examination
Final Action 1970
(General knowledge of microscopy and crystallography is nec-
essary for application of this technic. Some of std works on
this subject are listed in References. Optical-cry stallographic
properties of antihistamines, alkaloids, antibiotics, barbitu-
rates, hallucinogens, steroids, sulfonamides, sympatho-
mimetic amines and tranquilizers are given in Tables 955.57
and 955.58.)
A. Apparatus
(a) Polarizing microscope. — Fitted with polarizing prisms
below and above rotating, graduated circular stage and with
accessories (Bertrand lens or pinhole eyepiece, first order red
Table 960.56 Characteristics of Microchemical Tests for Xanthine Alkaloids
Alkaloid
Reagent
Description of Crystals
Caffeine Gold bromide in dil. HCI
Bismuth iodide
Iodine-potassium iodide (5-14)
Outer part: brownish needles with bright white birefringence. Inner part: small rods to sticks, little
grains and plates with weak yellow birefringence.
Small brownish orange rods or blades growing from sample or nearby in rosettes; also some orange
grains.
Grains, dark red to black, sometimes yellow or orange-brown; generally sq or cubical; birefringent
with fairly strong light; some irregular dichroic blades.
Dyphylline
Gold bromide in dil. HCI
Bismuth iodide
Iodine-potassium iodide (5-14)
Needles, scattered and in rosettes; fairly bright birefringence.
Very small grains, flakes, blades in multitudes, birefringent.
Fuzzy brown dense rosettes thruout drop, birefringent around rims; excess reagent must be used; 5
min required to form crystals.
Theobromine
Gold bromide in dil. HCI
Bismuth iodide
Iodine-potassium iodide (5-14)
Grains or plates in dense groups; bright birefringence at edge of cluster,
Brown needles in rosettes.
Orange-brown chips; also rectangular plates with opposite sides incised; smaller crystals: grains,
often lens shaped or diamonds; birefringent, somewhat dichroic.
Theophylline
Gold bromide in dil. HCI
Bismuth iodide
Iodine-potassium iodide (5-14)
Long needles in sheaves; fairly bright birefringence.
Grains and short prisms, often rectangular; brightly birefringent.
Black needles in rosettes around edge; birefringent; when larger, blades or rods, dichroic black
vertically to yellow horizontally.
542
Drugs: Part I
AOAC Official Methods of Analysis (1990)
or quartz wedge compensators) for observation of interference
figures, optic sign, and sign of elongation.
(b) Refractometer . — For measuring refractive indices of liqs
at 20° or 25° from 1.300 to 1.840 with precision of ±0.0005.
B. Reagents
Immersion media. — Ideally immersion media for refractive
index detn should have same color and intensity of color as
substance being examined and be chemically stable. Refractive
indices should not vary perceptibly with ordinary changes of
temp, with exception of special liqs used in index- variation
methods. Permanent set of liqs covering range 1.430-1.790
in 0.005 intervals made with following mixts is useful for both
inorg. and org. substances:
Mixture
n D (20r)
Kerosene and mineral oil
Mineral oil and a-monochloronaphthalene
a-Monochloronaphthalene and methylene
iodide
Methylene iodide and sulfur
1.435-1.480
1.485-1.640
1.645-1.740
1 .740-1 .790
Substances sol. in these liqs require prepg special set of liqs.
C. Determinations
Refractive indices. — Det. refractive indices by mounting
cry st. material in suitable immersion liqs and observing Becke
line. Successively suspend crystals or crystal fragments of sub-
stance in immersion liqs of known refractive indices. Greater
the difference between refractive indices of crystal and liq.,
the more prominently one stands out in bold relief from other.
By repeatedly mounting such crystals in oils of successively
lower or higher index, ultimately zone of contact of crystal
and liq. becomes practically invisible, demonstrating that re-
fractive indices of liq. and solid have been matched.
In case of substances crystg in isometric (cubic) system, there
is only 1 refractive index, designated by n. Such substances
are not doubly refractive when examined with crossed nicols.
Substances crystg in other systems, hexagonal, tetragonal,
monoclinic, triclinic, and orthorhombic, in ideal cases, have
>1 measurable refractive index. With uniaxial substances such
as those crystg in hexagonal and tetragonal systems, 2 signif-
icant indices can be detd, designated as n e and n a . Substances
crystg in monoclinic, triclinic, and ortho-rhombic systems, in
ideal cases, have 3 refractive indices, designated as n aJ « p , and
n y .
Extinction and extinction angle of anisotropic substances. —
Anisotropic crystals, when rotated through 360° on stage, be-
come dark 4 times. Positions of darkness are known as ex-
tinction positions and correspond to positions in which vibra-
tions of birefringent rays produced by crystal are mutually
parallel to vibration directions of polarizer and analyzer indi-
cated by cross hairs in eyepiece. If crystal extinguishes when
crystal edge or face is parallel to one of cross hairs, extinction
is parallel. If bisector of silhouette angle is parallel to one of
cross hairs, extinction is symmetrical. Crystals showing ex-
tinction differing from these 2 have inclined extinction. Mea-
sure extinction angles on those crystals showing inclined ex-
tinction by rotating crystal so that crystal edge or face is parallel
to 1 of cross hairs. Rotate stage until crystal extinguishes. Read
on stage vernier extinction angle between face or edge at ex-
tinction and nearest cross hair. Express extinction angles with
relationship to principal vibration directions of light and crys-
tallographic axes.
Elongation. — Many crystals are frequently elongated in 1
direction. Relationship between direction of elongation and vi-
bration directions of slow and fast rays of anisotropic crystal
is sometimes of determinative value. If substance is length slow,
i.e., slow ray or higher refractive index is parallel to direction
of elongation, sign of elongation is pos.; if substance is length
fast, sign is neg.
Sign of elongation ( + or — ) is detd with gypsum plate and
crossed nicols. A long and narrow crystal, showing very little
color with crossed nicols, is so oriented that its long dimension
is parallel to direction "z" of plate (slow ray) which is inserted
in slit of microscope tube. (Direction "z" is indicated by arrow
on plate.) If crystal appears blue or other color of higher order
than red-violet due to plate, elongation is + ; if crystal appears
yellow, white, or gray, i.e., of lower order color than red-
violet field, elongation is — .
Optic character and optic sign. — Det. optic character (uni-
axial or biaxial) and optic sign (+ or -), using first order red
or quartz wedge compensators in conjunction with interference
figures. Obtain interference figures from conoscopic images
of crystals suitably oriented. In absence of interference fig-
ures, det. these properties from relationship of principal re-
fractive indices. When (n p — n a ) is <(n y — n p ), optic sign is
+ . When («p — n a ) is >(n y — n p ), optic sign is -.
Optic axial angle (2V). — Calc. axial angle (2V) from values
of 3 refractive indices (here designated a, p, and 7) according
to formulas:
Cos 2 V a -
Cos 2 V.
7 2 (P 2 ~ <* 2 )
P 2 (7 2 " a 2 )
P V " a 2 )
(for — optic sign), or
(for + optic sign)
where 2V a is axial angle about a, and 2V y is axial angle about
7. Alternatively, est. approx. value of 2V from curvature of
isogyre referring to diagrams of substances with known an-
gles. Angle ranges from small (0-25°, sharply curved) to me-
dium (26-60°, moderately curved) to large (61-90°, nearly
straight isogyre).
Refs.: Stewart and Stolman, editors, "Toxicology, Mecha-
nisms and Analytical Methods," Vol. I, pp. 660-
713(1960). Hartshorne and Stuart, "Crystals and the
Polarizing Microscope," 3rd ed., 1960. Chamot and
Mason, "Handbook of Chemical Microscopy," Vol. 1,
1983. Bloss, "Introduction to the Methods of Optical
Crystallography," 1961. Wahlstrom, "Optical Crystal-
lography," 1979. McCrone and Delly, "The Particle
Altas," Vols IV and V, 1973. Sunshine, editor, "Hand-
book of Analytical Toxicology," pp. 289-330, 1969.
MISCELLANEOUS
985.45 Trimethobenzamide Hydrochloride
in Drugs
Bon-Pair Column Chromatographic Method
First Action 1985
Final Action 1988
(Applicable to capsules and injections)
A. Principle
Sample is dild in H 2 0, acidified, mixed with Celite, and
packed in chromatgc column. Breakdown products and pre-
AOAC Official Methods of Analysis (1990)
Antifungal
543
servatives are removed with ether. Trimethobenzamide HC1 is
eluted with CH 2 C1 2 , and detd by UV spectrophotometry at 261
nm.
B. Apparatus
(a) Chromatographic column and tamping rod, — See
968.42B.
(b) Filter paper. —Schleicher & Schuell, Inc., no. 589 White
ribbon, or equiv.
(c) Ultraviolet spectrophotometer. — With matched 1 cm
cells.
C. Reagents
(a) C elite 545. —See 960.53B
(b) Hydrochloric acid soln. — \M. Dil. 8.3 mL HC1 to 100
mL with H 2 0.
(c) Water-saturated ether .—Shake, equal vols ether and H 2
in separator 1 min. Discard lower phase.
(d) Methylene chloride. — Distilled-in-glass grade.
(e) Water-saturated methylene chloride. — Shake equal vols
CH 2 C1 2 and FLO in separator. Discard H 2 phase.
(f ) Trimethobenzamide HCl (TMBH) std soln.— -0.02 mg/
mL. Accurately weigh 10 mg USP TMBH, previously dried
4 h at 105°, and transfer to 100 mL vol. flask. Add 70 mL
CH 2 C1 2 and sonicate until dissolved. Dil. to vol. with CH 2 C1 2 .
Transfer 10.0 mL of this soln to 50 mL vol. flask and dil. to
vol. with CH 2 C1 2 .
D. Preparation of Sample
(a) Capsules. — Det. av. wt of contents per capsule and re-
duce to powder passing No. 60 sieve. Accurately weigh por-
tion of powder contg ca 200 mg TMBH into 50 mL vol. flask.
Add 30 mL H 2 0, mech. shake 10 min, and dil. to vol. with
FLO. Mix and filter, discarding first 10 mL filtrate.
(b) Injections. — Transfer accurately measured vol. soln,
contg ca 200 mg TMBH, into 50 mL vol. flask. Dil. to vol.
with H 2 0.
E. Preparation of Column
Pack pledget of fine glass wool in base of chromatgc tube.
Mix 1 g Celite 545 with 0.5 mL 1M HCl in 50 mL beaker,
transfer to column, and tamp. Transfer 2.0 mL prepd sample
soln to 100 mL beaker, add 4 drops coned HCl, and swirl
gently. Add 3 g Celite 545, mix well, and transfer to column.
Scrub beaker with 1 g Celite 545, transfer Celite to column,
wipe beaker and spatula with pledget of glass wool, and tamp
glass wool on column.
F. Determination
Elute column with 50 mL FLO-satd ether. Rinse column tip
with ether, and discard eluate and rinses. Place 200 mL vol.
flask under column, and elute column with four 50 mL por-
tions of FLO-satd CH 2 C1 2 . Rinse column tip with CH 2 C1 2 , add
rinse to flask, and dil. to vol. with CH 2 C1 2 . Transfer 25.0 mL
of this soln to 50 mL vol. flask and dil. to vol. with CH 2 C1 2 .
Det. A of sample and std solns at 261 nm, using CH 2 C1 2 as
blank. If recording spectrophtr is used, scan from 340 to 235
nm.
mg TMBH/capsule = (A/A') x (C) x (10 000) x (K/W)
mg TMBH/mL injection = {A/ A') x (C) x (10 000/V)
where A and A' refer to sample and std solns, resp.; C = concn
std soln, mg/mL; K = av. capsule contents wt, g; W = sample
wt, g; and V = vol. injection, mL.
Ref.: JAOAC 68, 1055(1985).
CAS- 1 38-56-7 (trimethobenzamide)
ANTIFUNGAL
988.21 Flucytosine in Drug Capsules
Liquid Chromatographic Method
First Action 1988
A. Principle
Flucytosine content of capsules is detd by liq. chromatgy on
Ci 8 reverse phase column, using H 2 0-MeOH-HOAc mobile
phase contg 1 -octanesulfonic acid Na salt, p-aminobenzoic acid
as internal std, and UV detection at 285 nm.
B. Apparatus
(a) Liquid chromatograph.— Model 8800 sol v. pump with
variable wavelength detector capable of monitoring elution at
285 nm (Du Pont Instrument Products Div.), injection valve
with 20 fxL sample loop (Valco Instruments Co. , Inc. , PO Box
55603, Houston, TX 77055), and suitable strip chart recorder.
Equiv. LC system may be used. Operating conditions: flow
rate 1.5 mL/min; detector 285 nm, 0.32 AUFS; chart speed
0,5 cm/min; temp, ambient; injection vol. 20 |xL. To detect
fluorouracil, main degradation/precursor product of flucyto-
sine, monitor sepn at 266 nm to maximize sensitivity.
(b) Chromatographic column. — Stainless steel, 300 mm X
3.9 mm id, packed with 10 jjtm pJBondapak C 18 (Waters As-
sociates, Inc.), or equiv. column that meets LC system suit-
ability requirements.
C. Reagents
(a) 1 -Octanesulfonic acid N A salt. — (Eastman Kodak Co.).
(b) p-Aminobenzoic acid. — Certified ACS Grade (Fisher
Scientific Co.), or equiv.
(c) Mobile phase.— H 2 0-MeOH-HOAc (785 + 200 + 15
v/v/v) contg 2 g/L of 1 -octanesulfonic acid Na salt.
(d) Internal std soln. — Accurately weigh ca 160 mgp-ami-
nobenzoic acid and transfer to 200 mL vol. flask. Add 100
mL mobile phase, sonicate 5 min, shake mech. 25 min, dil.
to vol. with mobile phase, and mix well.
(e) Flucytosine std soln. — Accurately weigh ca 30 mg USP
Flucytosine Ref. Std and transfer to 50 mL vol. flask. Add 25
mL mobile phase, sonicate 5 min with gentle swirling, shake
mech. 25 min, dil. to vol. with mobile phase, and mix. Trans-
fer 10.0 mL of this soln to 100 mL vol. flask, add 5.0 mL
internal std soln, dil. to vol. with mobile phase, and mix. Prep.
this soln fresh daily.
D. Sample Preparation
Accurately weigh contents of >20 flucytosine capsules and
det. av. wt/capsule. Accurately weigh portion of powder equiv.
to ca 100 mg flucytosine and transfer to 100 mL vol. flask.
Add 50 mL mobile phase, sonicate 5 min with gentle swirling,
shake mech. 25 min, dil. to vol. with mobile phase, and mix
well. Filter portion of soln thru suitable paper or 0.45 fxm
membrane filter, discarding first 10 mL filtrate. Transfer 6.0
mL filtrate to 100 mL vol. flask, add 5.0 mL internal std soln,
dil. to vol. with mobile phase, and mix well. From this point,
complete detn on same day.
E Suitability Test and Determination
Inject 20 |uUL each of sample and std solns into LC system
by using sampling valve or high pressure microsyringe. Op-
erate system as described in Apparatus. Adjust detector sen-
sitivity so peak response for flucytosine is between 40 and 75%
full scale. In suitable chromatogram, CV of peak ht (or area)
ratios for 6 replicate injections of std soln should be <2.0%,
and resolution factor, R, for flucytosine peak and internal std
peak should be >2.
544
Drugs; Part
AOAC Official Methods of Analysis (1990)
Calc. resolution factor as follows:
R = \2(h - h)\/{W 2 + WO
where t 2 and t { = retention times of 2 components, and W 2
and W] = corresponding widths of peaks, measured by ex-
trapolating sides of peaks to baseline.
F. Calculation
Calc. amt flucytosine in dosage form, using response ratios
based on either peak hts or peak areas, according to following
equation:
Flucytosine, mg/capsule = 1.667C x (R/R') X (T/W)
where C = concn, |xg/mL, of flucytosine in std soln; R and
R' - response ratios for flucytosine peak to internal std peak
for sample and std, resp.; T = av. capsule wt, mg; W = wt
sample taken for assay, mg.
Ref.: JAOAC 71, 33(1988).
CAS-2022-85-7 (flucytosine)
ANTIPARKINSONIAN
988.22 Levodopa and Levodopa-
Carbidopa in Solid Dosage Forms
Liquid Chromatographic Method
First Action 1988
A. Principle
Levodopa in tablets or capsules and levodopa-carbidopa in
tablets are detd by reverse phase liq. chromatgy on C )8 column
with 3% HO Ac as mobile phase, and UV detection at 280 nm.
Methyldopa is internal std for levodopa tablets or capsules;
acetaminophen is internal std for levodopa-carbidopa tablets.
B. Apparatus
(a) Liquid chromatography — Isocratic system equipped with
detector capable of monitoring A at 280 nm, suitable strip chart
recorder, and injection valve with 20 |xL sample loop.
(b) Chromatographic column. — 300 X 3.9 mm id, |xBond-
apak Cis, 10 |xm particle size (Waters Associates, Inc), or equiv.
column that meets suitability requirements,
(c) Membrane filters. — 0.45 fxm porosity (Millipore, or
equiv.).
C. Reagents
(a) Mobile phase. — 3% aq. HO Ac.
(b) Methyldopa internal std soln. — 2 mg/mL. Accurately
weigh ca 200 mg USP Methyldopa Ref. Std into 100 mL vol.
flask, add 50 mL 0. IN HO, and sonicate to dissolve std. Dil.
to vol. with mobile phase, and mix.
(c) Acetaminophen internal std soln. — 0.5 mg/mL. Accu-
rately weigh ca 125 mg USP Acetaminophen Ref. Std into 250
mL vol. flask, add 75 mL MeOH, and sonicate to dissolve
std. Dil. to vol. with mobile phase, and mix.
(d) Levodopa std soln. — Just prior to use, dry USP Levo-
dopa Ref. Std 4 h at 105°. Store in tightly covered, light-re-
sistant container. Accurately weigh ca 100 mg dried std into
50 mL vol. flask. Add 30 mL 0.1N HC1, and sonicate to dis-
solve. Dil. to vol. with 0.1N HO, and mix. Filter soln thru
0.45 |xm membrane filter, discarding first 5 mL filtrate. Pipet
5 mL filtrate and 10 mL methyldopa internal std soln into 100
mL vol. flask, dil. to vol. with mobile phase, and mix.
(e) Levodopa-carbidopa std soln. — Dry USP Carbidopa Ref.
Std to const wt at 100° under reduced pressure not exceeding
5 mm Hg. Store in tightly covered, light-resistant container.
Accurately weigh ca 100 mg dried USP Levodopa Ref. Std
(d) into 50 mL vol. flask. Add accurately weighed amt of dried
carbidopa std so that carbidopa-to-levodopa ratio corresponds
to that found in com. levodopa-carbidopa tablet. Add 30 mL
0.1N HO, sonicate to dissolve, dil. to vol. with 0.1N HO,
and mix. Filter soln thru 0.45 u,m membrane filter, discarding
first 5 mL filtrate. Pipet 10 mL filtrate into 100 mL vol. flask,
and add vol. of acetaminophen internal std soln so that acet-
aminophen concn is 1.25 times carbidopa concn. Dil. to vol,
with mobile phase, and mix.
D. Sample Preparation
(a) Levodopa tablets or capsules. — Weigh and finely pow-
der >20 tablets or composite contents of 20 capsules. Weigh
portion of powder equiv. to ca 100 mg levodopa into 50 mL
vol. flask, and proceed as directed under levodopa std soln (d),
beginning "Add 30 mL (UN HC1 . . ."
(b) Levodopa-carbidopa tablets. — Weigh and finely pow-
der >20 tablets. Weigh portion of powder equiv. to ca 100
mg levodopa into 50 mL vol. flask, and proceed as directed
under levodopa-carbidopa std soln (e), beginning "Add 30 mL
0.1N HC1 ..."
(c) Levodopa-carbidopa tablets for content uniformity de-
termination. — Dissolve 1 tablet in sufficient 0.1N HO to prep,
soln contg 2 mg levodopa/mL. Filter soln thru 0.45 |mm mem-
brane filter, discarding first 5 mL filtrate. Pipet 10.0 mL fil-
trate into 100 mL vol. flask, add acetaminophen internal std
soln (15 mL for levodopa-carbidopa 100/25 tablets, 5 mL for
all other dosage levels), dil. to vol. with 0.1N HC1, and mix.
E. System Suitability Test and Assay
Equilibrate LC system with mobile phase at 1.5 mL/min.
Inject 20 jxL std soln. Approx. retention times are levodopa,
3 min; methyldopa, 4.5 min; carbidopa, 5 min; and acetami-
nophen, 9 min. Calc. resolution factor, /?, as follows:
R - [2(t 2 - t x )\/(W 2 + WJ
where t 2 and t { = retention times of the 2 components, and
W 2 and W\ = corresponding widths of bases of peaks obtained
by extrapolating relatively straight sides of peaks to baseline.
R between levodopa and carbidopa and between carbidopa and
acetaminophen should be >3.5. R between levodopa and
methyldopa should be >2.
Change flow rate to improve resolution. For levodopa-car-
bidopa tablets, change detector sensitivity between levodopa
peak (approx. 0.64 AUFS) and carbidopa peak (approx. 0.08
or 0.04 AUFS). Set detector sensitivity to 35-95% AUFS. If
necessary, adjust vol. of internal std soln added to sample soln
and std soln to obtain satisfactory detector response for std
soln. Inject std soln 5 times and compare peak fits. Calc. CV
as follows:
CV, % = ■
100
X
S^-*) 2
N - 1
1/2
where X = mean of set of N measurements, and X x = an in-
dividual detn of ratio of peak ht of analyte to peak ht of in-
ternal std. In suitable system, CV = <2.0%.
Proceed with sample analysis by injecting 20 |xL each of
sample soln and corresponding std soln.
F. Calculation
Using peak ht ratios R and R f relative to internal std, calc.
mg drug/tablet or capsule as follows:
mg/tablet or capsule = (R/R') x C X (D/W) X T
AOAC Official Methods of Analysis (1990)
Antihypertensive
545
where R and R f = peak ht ratios for sample and std solns,
resp.; C — concn of std soln, mg/mL; W = wt of sample
taken, mg; D - sample diln; and T = av. tablet or capsule
wt, mg.
Ref.: J AOAC 70, 987(1987).
CAS-59-92-7 (levodopa)
CAS-38821-49-7 (carbidopa)
CAS-28860-95-9 (carbidopa anhydrous)
ANTIHYPERTENSIVE
989.07 Hydralazine Hydrochloride
in Drug Tablets
Spectrophotometric Method
First Action 1989
Method Performance:
Tablets, 10 mg:
s r = 0.53;s R = 1.01; RSD r = 0.55%; RSD R = 1.06%
Tablets, 100 mg:
s r = 0.26; s R - 0.77; RSD r - 0.26%; RSD R = 0.77%
Synthetic mixt., 10 mg:
s r = 0.78; s R - 0.96; RSD r - 0.77%; RSD R - 0.95%
A. Principle
Hydralazine HC1 is converted to tetrazolo[5,l-a]phthala-
zine, which is detd by UV detection at 274 nm.
B. Apparatus and Reagents
(a) Spectrophotometer. — UV-vis., recording, with matched
1 cm quartz cells, to measure A at 274 nm.
(b) NaN0 2 soln.—\% w/v NaN0 2 (ACS grade or equiv.).
Prep, fresh daily.
(c) Hydralazine HCl std soln. — Accurately weigh ca 25 mg
USP Hydralazine Hydrochloride Ref. Std previously dried un-
der vac. over P 2 5 for 8 h, and transfer to 250 mL vol. flask.
Dil. to vol. with 0.1N HCl and mix well. Pipet 20.0 mL dild
soln into 100 mL vol. flask, add 1 .0 mL 1% NaN0 2 soln, mix,
and heat mixt. ca 1 h on steam bath. Cool soln to room temp.,
dil. to vol. with H 2 0, and mix.
C. Preparation of Sample
Weigh and finely powder >20 tablets. Accurately weigh
portion of powder (or crushed tablet) equiv. to ca 25 mg hy-
dralazine HCl, and transfer to 250 mL vol. flask. Add ca 125
mL 0. IN HCl and mech. shake 20 min. Dil. to vol. with 0. IN
HCl, mix, and filter, discarding first 20 mL filtrate. Pipet 20.0
mL filtrate into 100 mL vol. flask, add 1.0 mL 1% NaN0 2
soln, mix, and heat ca 1 h on steam bath. Cool soln to room
temp., dil. to vol. with H 2 0, and mix.
D. Determination
Use suitable spectrophtr (a) to concomitantly det. A of sam-
ple and std solns contg ca 20 |xg hydralazine HCl/mL.
Calc. mg hydralazine HCl /tablet as follows:
Hydralazine HCl, mg/tablet - (A u /A s ) x (W s /W u ) x T
where A u and A s — A of sample and std solns, resp.; W s and
W u = mg ref. std and sample taken for std and sample solns,
resp.; and T = av. tablet wt, mg.
Ref.: JAOAC71, 1121(1988).
CAS-86-54-4 (hydralazine)
CAS-304-20-1 (hydralazine monohydrochloride)
Common and Chemical Names of Drugs in this Chapter
Common Name
Chemical Name
Acetanilide
Aconitine
Allobarbital
Aminopyrine
Amitriptyline hydrochloride
Amobarbital
Amphetamine sulfate
Amylocaine hydrochloride
Antipyrine
Apomorphine
Aprobarbital
Arecoline
Atropine
Aspirin
Barbital sodium
Benzocaine
Bemegride
Berberine
Brucine
Butabarbital sodium
Butacaine sulfate
Butalbital
Butethal
Caffeine
Calomel
Carbidopa
Chlorobutanol
Chlorpheniramine maleate
Choline
Cinchonidine
Cinchonine
Cinchophen
Cocaine
Codeine phosphate
Cotarnine
A/-Phenylacetamide
1 6-Ethyl-1 ,16,1 9-trimethoxy-4-(methoxymethyl)aconitane-3,8, 1 0, 1 1 , 1 8-pentol 8-acetate 1 0-benzoate
5,5-Di-2-propenyl-2,4,6(1H,3H,5H)-pyrimidinetrione
4-(Dimethylami no)- 1,2-di hydro- I.S-dimethyi^-phenyl-SH-pyrazol-S-one
3- (1 0,1 1 -Dihydro-5H-dibenzo(a,cr)-cyclohepten-5-ylidene)-A/,A/-dimethyl-1-propanamine hydrochloride
5-Ethyl-5-(3-methylbutyl)-2,4,6-(1H,3H f 5H)-pyrimidinetrione
(±)-a-Methylphenethylamine sulfate
1-(Dimethylamino)-2-methyl-2-butanol benzoate hydrochloride
1 ,2-Dihydro-1 ,5-dimethyl-2-phenyl-3H-pyrazole-3-one
5,6,6a,7-Tetrahydro-6-methyl~4H-dibenzo(o'e,g)quinoline- 10,11 -diol
5-{1-Methylethyl)-5-(2-propenyl)-2,4 ) 6(1H,3H,5H)-pyrimidinetrione
1,2,5,6-Tetrahydro-1-methyi-3-pyridinecarboxylic acid methyl ester
a-(Hydroxymethyl) 8-methyl-8-azabicyclo[3.2.1]oct-3-yl ester benzeneacetic acid
2-(Acetyloxy)benzoic acid
5,5-Diethyl-2,4,6(1H,3H,5H)-pyrimidinetrione sodium salt
p-Aminobenzoic acid ethyl ester
3-Ethyl-3-methylglutarimide
5,6-Dihydro-9, 1 0-dimethoxybenzo(a>1 ,3-benzodioxolo(5,6-a)quinolizinium
2,3-Dimethoxystrychnidin-1 0-one
5-Ethyl-5-(1 -methylpropyl)-2,4,6(1 H,3/-/,5H)-pyrimidinetrione monosodium salt
4-Aminobenzoate 3-dibutylamino-1-propanol sulfate
5-(2-Methylpropyl)-5-(2-propenyl)-2,4,6(lH,3H,5H}-pyrimidinetrione
5-Butyl-5-ethyl-2,4,6(1H,3H,5H}-pyrimidinetrione
3,7-Dihydro-1 ,3,7-trimethyH H-purine-2,6-dione
Mercurous chloride
(-)-L-a-Hydrazino-3,4-dihydroxy-a-methylhydrocinnamic acid monohydrate
1,1,1 -Trichloro-2-methyl-2-propanol
7-(4-Chlorophenyl)-/V,A/-dimethylpyridine propanamine-2-butenedioate
2-Hydroxy-A/,A/,W-trimethylethanaminium hydroxide
(8a,9fl)-Cinchonan-9-ol
(9S)-Cinchonan-9-ol
2-Phenylcinchoninic acid
3-(Benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester
(5a, 6«)-7,8-Didehydro-4,5-epoxy-3-methoxy*17-methylmorphinan-6-ol phosphate (salt) hemihydrate
5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-dioxolo(4,5-gf)isoquinolin-5-ol
546
Drugs: Part I
AOAC Official Methods of Analysis (1990)
Common and Chemical Names of Drugs in this Chapter {Continued)
Common Name
Chemical Name
Cyclobarbital
Dextroamphetamine
Dextroamphetamine sulfate
Dextromethorphan hydrobromide
Dichlorophen
Diphenhydramine hydrochloride
Dyphylline
Ephedrine
Ephedrine sulfate
Epinephrine bitartrate
Erythrityl tetranitrate
Ethylhydrocupreine
Ethylmorphine
Flucytosine
Fluorescein sodium
Heptabarbital
Heroin
Hexobarbital sodium
Homatropine
Hydralazine hydrochloride
Hydrastine
Hydrastinine
Hydrochlorothiazide
Hydromorphone
8-Hydroxyquinoline sulfate
Hyoscyamine
Iodoform
Isoproterenol
Mandelic acid
Meperidine hydrochloride
Mephentermine sulfate
Meprobamate
Merbromin
Methamphetamine hydrochloride
Methapyrilene hydrochloride
Metharbital
Methenamine
Meth en amine mandelate
Methocarbamol
Methyldopa
Methylene blue
Morphine
Narceine
Neocinchophen
Nicotine
Nitroglycerin
Nitromersol
Norepinephrine
Noscapine
Papaverine
Pentaerythritol tetranitrate
Pentobarbital sodium
Pentylenetetrazol
Perphenazine
Phenacetin
Phenazopyridine hydrochloride
Pheniramine maleate
Phenmetrazine
Phenobarbital
Phenylephrine hydrochloride
Phenylmethybarbituric acid
Phenylpropanolamine hydrochloride
Phenylpropylmethylamine
Physostigmine
Pilocarpine
Potassium guaiacolsulfonate
Probarbital
Procainamide hydrochloride
Procaine hydrochloride
Promethazine hydrochloride
Propoxycaine hydrochloride
Pyrilamine maleate
Quinidine
Ringer's Injection
Ringers Injection, lactated
Salicylic acid
Scopolamine
Secobarbital
Sodium cacodylate
Sparteine
5-(1-Cyclohexen-1-yl)-5-ethyl-2,4,6(1H,3H,5H)-pyrimidinethone
cf-a-Methylphenethylamine
d-a-Methylphenethylamine sulfate
3-Methoxy-1 7-methyl-9a, 1 3a, 1 4a-morphinan hydrobromate monohydrate
2,2'-Methylenebis(4-chlorophenol)
2-Diphenylmethoxy-N,/V-dimethylethanamine hydrochloride
7-(2,3-Dihydroxypropyl)-3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione
a-[1-(Methylamino)ethyl]benzenemethanol
a[1 -(Methylamino)ethyl]benzenemethanol sulfate
(~)-3,4-Dihydroxy-a-[(methylamino)methyl] benzyl alcohol (+)-tartrate (1:1) salt
1 ,2,3,4-Butanetetrol, tetranitrate
8a, 9fl-6'-Ethoxy-10,11-dihydro-cinchonan-9-ol
(5a, 6a)-7,8-Didehydro-4,5-epoxy-3-ethoxy-17-methylmorphinan-6-ol
5-Fluorocytosine
3\6'-Dihydroxyspiro[isobenzofuran-1 (3H),9'-[9/-/]xanthen]-3-one disodium salt
5-(1-Cyclohepten-1-yl)-5-ethyl-2,4,6(1H,3H,5H)-pyrimidinetrione
(5a, eaJ^^-Didehydro^.S-epoxy-^-methylmorphinan-S.G-diol diacetate (ester)
5-(1-Cyclohexen-1-y)-1 ,5-dimethyl-2,4,6(1H,3H,5H)-pyrimtdetrione monosodium salt
a-Hydroxy-8-methyl-8-azabicyclo[3.2.1]oct-3-yl ester benzeneacetic acid
1 -Hydrazinophthalazine monohydrochloride
6,7-Dimethoxy-3-[5,6,7,8-tetrahydro-6-methyl-1,3-dioxolo(4,5-p)tsoquinolin-5-yl]-1(3H)-isobenzofuranone
5,6,7,8-Tetrahydro-6-methyl-1,3-dioxolo(4,5-sf)isoquinolin-5-ol
6-Chloro-3,4-dihydro-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 , 1 -dioxide
4,5a-Epoxy-3-hydroxy-17-methylmorphinan-6-one
8-Quinolinol sulfate
a-(Hydroxymethyl)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl ester benzeneacetic acid
Triiodomethane
4-[1-Hydroxy-2-[(1-methylethyl)-amino]ethyl]-1,2-benzenediol
a-Hydroxybenzeneacetic acid
1-Methyl-4-phenyl-4-piperidinecarboxylic acid, ethyl ester, hydrochloride
A/,a,a-Trimethylphenethylamine sulfate
2-Methyl-2-propyl-1 ,3-propanediol dicarbamate
2,7-Dibromo-4-hydroxymercurifluoresceine dibromide salt
(+)-A/,a-Dimethylphenethylamine hydrochloride
2-[[2-(Dimethylamino)ethyl]-2-thenylamino]pyridine monohydrochloride
5,5-Diethyl-1 -methyl-2,4,6(1 H,3H,5H)-pyrimidinetrione
1 ,3,5,7-Tetraazatricyclo[3.3. 1 . 1 a7 ]-decane
a-Hydroxybenzeneacetic acid, compd. with 1,3,5,7-tetraazatricyclo-[3.3.1.1 3 ' 7 ] decane (1:1)
3-(o-Methoxyphenoxy)-1 ,2-propanediol 1 -carbamate
L-3-(3,4-Dihydroxyphenyl)-2-methylalanine sesquihydrate
3,7-Bis(dimethylamino)phenothiazin-5-ium chloride
(5a, 6a)-7,8-Didehydro-4,5-epoxy-1 7-methylmorphinan-3,6-diol
6-[[6[2-(Dimethylamino)ethyl]-4-methoxy-1,3-benzodioxol-5-yl]acetyl]-2,3-dimethoxybenzoic acid
6-Methyl-2-phenylquinoline-4-carboxylic acid ethyl ester
3-(1-Methyl-2-pyrrolindinyl)pyridtne
1 ,2,3-Propanetriol trinitrate
5-Methyl-2-nitro-7-oxa-8-mercurabicyclo[4.2.0]octa-1,3,5-triene
4-(2-Amino-1 -hydroxyethy!)-1 ,2-benzenediol
6,7-Dimethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo(4,5-g)isoquinolin-5-yl)-1(3H)-tsobenzofuranone
1-[(3,4-Dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline
2,2-Bis[(nitrooxy)-methyl]-1,3-propanediol dinitrate (ester)
5-Ethyl-5-(1 -methyibutyl)-2,4,6(1 H,3H,5H)-pyrimidinetrione monosodium salt
6,7,8,9-Tetrahydro-5H-tetrazolo(1 ,5-a)azepine
4-[3-(2-Chlorophenothiazin-10-yl)propyl]-1-piperazineethanol
A/-(4-Ethoxyphenyl)acetamide
3-(Phenylazo)-2,6-pyridinediamine monohydrochloride
2[[a-(2-Dimethylamino)ethyl]benzyl]pyridine bimaleate
3-Methyl-2-phenylmorpoline
5-Ethyl-5-phenyi-2,4,6(1H,3H,5tf)-pyrimidinetrione
3-Hydroxy-a-[(methyamino)methyl]benzene-methanol hydrochloride
5-Methyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione
a-(1 -Aminoethyl)benzenemethanol hydrochloride
N, p-Dimethylphenethylamine
1 ,2,3,3a,8,8a-Hexahydro-1 ,3a,8-trimethylpyrrolo(2,3-b)indol-5-ol methylcarbamate (ester)
3-Ethyldihydro-4-[(1-methyl-1H-imidazol-5-yl)methyl]-2(3H)-furanone
Potassium hydroxymethoxybenzenesulfonate hemihydrate
5-Ethyl-5-isopropylbarbituric acid
4-Amino-[2-(diethylamino)ethyl]-benzamide monohydrochloride
2-(Diethylamino)ethyl ester-4-Aminobenzoic acid, monohydrochloride
1 0-[2-(Dimethylamino)propyol]phenothiazine monohydrochloride
4-Amino-2-propoxybenzoic acid, 2-{diethylamino)ethyl ester monohydrochloride
2-[(2-(DimethyIamino)ethyl](p-methoxybenzyl)amino pyridine maleate
6'-Methoxycinchonan-9-ol
Sodium chloride (compound solution)
Sodium lactate (compound solution)
2-Hydroxybenzoic acid
a-(Hydroxymethyl)9-methyl-3-oxa-9-azatricyclo[3.3,1 .0 2,4 ]-non-7-yl benzeneacetic acid
5-(1-Methylbutyl)-5-(2-propenyl)-2,4,6(1H,3H,5H)-pyrimidinetrione
Sodium dimethylarsonate
Dodecahydro-7,14-methano-2H,6H-dipyrido(1 ,2-a:1 ', 2'-e)(1 .5)diazocine
AOAC Official Methods of Analysis (1 990)
Chemical Names
547
Common and Chemical Names of Drugs in this Chapter
Common Name
Chemical Name
Strychnine
Sulfadiazine
Sulfanilamide
Sulfapyridine monohydrate
Sulfathiazole
Talbutal
Theobromine
Theophylline
Thiethylperazine
Thonzylamine hydrochloride
Triflupromazine
Trimethobenzamide hydrochloride
Tripelennamine citrate
Tripelennamine hydrochloride
Trolamine
Vinbarbital
Yohimbine
Strychnidin-10-one
4-Amino-/V-2-pyrimidinylbenzenesulfonamide
4-Aminobenzenesulfonilamide
4-Amino-/V-2-pyridinylbenzen6sulfonamide monohydrate
4-Amino-/V-2-thiozolybenzenesulfonamide
5-(1-Methylpropyl)-5-(2-propenyl)-2,4,6(1H,3H,5H)-pyrimidinetrione
3,7-Dihydro-3,7-dimethyl-1/-/-purine-2,6-dione
3,7-Dihydro-1 ,3-dimethyl-1 H-piirine-2,6-dione
2-(Ethylthio)-10-[3-(4-methyl-1^piperazinyl)propyl]phenothiazine
2-[(2-Dimethylaminoethyl)(p-methoxybenzyl)amino pyrimidine hydrochloride
10-[3-(Dimethylamino)propyl]-2-(trifluoromethyl)phenothiazine monohydrochloride
A/-[p-[2-(Dimethylamino)ethoxy]benzyl]-3,4,5-trimethoxybenzamide monohydrochloride
N, A/-Dimethyl-/V'-(phenylnethy!)-A/'-2-pyridinyl-1 ,2-ethanediamine-2-hydroxy-1 ,2,3-propanetricarboxylate citrate {1 :1 )
A/,A/-Dimethyl-A/'-(phenylmethyl)-A/ f -2-pyridinyl-1,2-ethanediamine monohydrochloride
2,2',2"-Nitrilotriethanol
5-Ethyl-5-(1-methyl-1-butenyl)barbituric acid
17a-Hydroxyyohimban-16a-carboxylic acid methyl ester
Source: USAN and the USP Dictionary of Drug Names, (1983; 1989) U.S. Pharmacopeia! Convention, Rockville, MD.
19. Drugs: Part " II
SaSvafore M. IVSarchese, Associate Chapter Editor
Food and Drug Administration
ACIDS
945.98* Benzoic and Salicylic Acids
in Drugs
Titrimetric Method
Final Action
Surplus 1972
(Applicable to ointments. Caution: See safety notes on distil-
lation, flammable solvents, toxic solvents, diethyl ether, and
chloroform.)
See 37.001, 12th ed.
967.31 Benzoic and Salicylic Acids
in Drugs
Chromatographic Method
First Action 1967
Final Action 1968
A. Apparatus
(a) Chromatographic tubes. — Fuse 6 cm length of 5-6 mm
tubing to piece of 25 mm tubing ca 25 cm long (25 x 200
mm test tube may be used). Constrict stem slightly ca 2 cm
below seal. Com. tubes with dimensions ±10% are satisfac-
tory. Pack wad of Pyrex glass wool in base as support.
(b) Tamping rod. — Flatten end of glass rod to circular head
with clearance of ca 1 mm in tube (a). Or use disk of stainless
steel, Al, etc., of diam. ca 1 mm less than id of column, (a),
attached to 30-45 cm (12—18") rod.
B. Reagents
(a) Ferric chloride-urea soln. — Dissolve, without heating,
18 g reagent grade urea in 2.5 mL 60% FeCl 3 .6H 2 (EM Sci-
ence, No. FX0210) and 12.5 mL 0.057V HC1. Prep, fresh daily.
(b) Phosphoric acid.— 30%. Dil. 30 mL 85% H 3 P0 4 to 85
mL with H 2 0.
(c) Sodium bicarbonate. — \N. Dissolve 2.5 g NaHC0 3 in
30 mL H 2 0. Use only freshly prepd soln.
(d) Diatomaceous earth. — See 960. 53B.
(e) Benzoic acid std soln. — Accurately weigh 40-100 mg
benzoic acid (depending on concn benzoic acid in sample),
dissolve in CHC1 3 , and dil. to 100 mL with CHC1 3 . Shortly
before use, transfer 5 mL to 50 mL vol. flask; add 4 drops
HC1, 1 mL HOAc, 5 mL ether, and 7 mL MeOH; and dil. to
vol. with CHC1 3 .
(f ) Salicylic acid std soln. — Dissolve 50 mg salicylic acid
in CHCh and dil. to 100 mL. Transfer 5 mL to 100 mL vol.
flask; add 2 drops HC1, 2 mL HOAc, 10 mL MeOH, and 20
mL ether; and dil. to vol. with CHC1 3 .
C. Preparation of Sample
(a) Ointments. — Dissolve ca 1 g sample, accurately weighed,
in CHCI3 and dil. to 100 mL. If necessary, dil. aliquot to prep,
final soln contg 0.15-0.25 mg salicylic acid/mL CHCI 3 .
(b) Liquids. — Dil. aliquot of liq. contg 150-250 mg sali-
cylic acid to 100 mL with MeOH. Dil. 10 mL methanolic soln
to 100 mL with CHC] 3 .
D. Preparation of Columns
Column I. — Lower stage: Mix 1 g diat. earth with 0.5 mL
30% H 3 P0 4 to uniform fluffy mixt. Transfer to tube and tamp
to uniform mass with gentle pressure. Upper stage: Similarly
mix 5 g diat. earth with 3 mL FeCi 3 urea reagent. (Mix tho-
roly, as nonuniform column may cause difficulty in elution of
salicylic acid.) Transfer to tube directly above H3PO4 layer.
Cover with glass wool.
Column II. — Mix 2 g diat. earth with I mL freshly prepd
IN NaHC0 3 soln.
E. Determination
(Use H 2 0-satd solvs.)
Mount Column I directly above Column II. Pipet 10 mL dild
sample into small beaker. Pour onto upper column, washing
beaker with 10 mL CHC1 3 in small portions. Let sample sink
into column and wash column with 75 mL CHC! 3 . If purple
salicylic acid band reaches H 3 P0 4 layer, repeat with smaller
sample. Sep. columns and wash Column II with 50 mL ether.
Discard wash. Elute Column I into 100 mL vol. flask (contg
10 mL MeOH and 2 drops HC1) with 2 mL HOAc in 20 mL
ether followed by enough 1% HOAc in CHC1 3 to bring to vol.
Measure A of eluate and salicylic acid std at max., ca 306 nm,
and calc. concn salicylic acid.
Elute Column II into 50 mL vol. flask (contg 7 mL MeOH
and 4 drops HC1) with 0.5 mL HOAc in 5 mL CHC1 3 followed
by enough 1% HOAc in CHC1 3 to bring to vol. Measure A of
eluate and benzoic acid std at max., ca 275 nm, and calc.
concn benzoic acid.
Ref.: JAOAC 50, 666(1967).
CAS-65-85-0 (benzoic acid)
CAS-69-72-7 (salicylic acid)
930.41* Salicylic Acid in Drugs
in Presence of Other Phenols
Final Action
Surplus 1965
See 32.184-32.185, 10th ed.
939.16 Mandelic Acid in Drugs
Final Action
A. Qualitative Tests
(Applicable to free acid)
See Microchemical Tests, Table 962. 21B.
548
AOAC Official Methods of Analysis (1990)
Phenolics
549
B. Determination
(Caution: See safety notes on distillation, flammable solvents,
toxic solvents, diethyl ether, and chloroform.)
(a) Tablets. — Weigh amt of powd sample contg 0.4-0.5 g
mandelic acid and transfer to separator contg 10 mL H 2 0.
Acidify with HC1 (1 +3) and add 2 mL excess. Ext with six
20 mL portions CHCl 3 -ether (2 + 1); wash each portion in
second separator with 2 mL H 2 0, and pass soln thru cotton
plug, previously satd with solv., into 250 mL beaker. Wash
outer surface of separator stem with few mL solv. and add to
main portion. Test for complete extn with 15 mL addnl solv.
and evap. in sep. beaker. Wash any residue thus obtained into
beaker contg main ext with few mL solv. Evap. to dryness at
<40 D with aid of air current. Dissolve residue in 25 mL C0 2 -
free H 2 0, 936.16B(a), and titr. with 0. IN NaOH, using phthln.
1 mL 0AN NaOH - 0.01522 g mandelic acid
(C 6 H 5 CHOHCOOH); 0.01692 g NH 4 mandelate, 0.01741 g
Na mandelate, 0.01712 g Ca mandelate, and 0.01633 g Mg
mandelate.
After titrn, mandelic acid may be re-extd and ext used for
mp detns or qual. tests.
(b) Liquid preparations. — Measure 1 mL sample, or ali-
quot of diln contg 0.4-0.5 g mandelic acid, into separator and
acidify with HC1 (1 + 3). Proceed as in (a).
Ref.: JAOAC 22, 757(1939).
CAS-90-64-2 (mandelic acid)
PHENOLIC DRUGS
958.15 p-Aminosalicylic Acid
and Isoniazid in Drugs
Spectrophotometric Method
Final Action
A. Reagents
(a) Benzaldehyde. — USP or reagent grade.
(b) Concentrated phosphate buffer. — pH 7. Dissolve 34 g
anhyd. KH 2 P0 4 in 136 mL IN NaOH and dil. to 1 L with
H 2 0.
B. Extraction of Tablets
(a) Accurately weigh sample of powd tablets contg 35-40
mg INH and transfer to 150 mL beaker. Stir with 20 mL H 2 0,
add 1.5 g NaHC0 3 , and continue stirring until effervescence
stops. Filter with vac. thru medium porosity fritted glass filter
(3.5 cm diam. is convenient) precoated with ca 3 mm layer
of diat. earth, 960.53B. Rinse beaker thoroly with 5 mL H 2 0,
break vac, transfer rinsings to funnel, washing down inside
wall, and reapply vac. Repeat washing of beaker and funnel
with 3 addnl 5 mL portions H 2 0. Quant, transfer filtrate to 50
mL vol. flask with aid of small portions H 2 0, dil. to vol., and
mix. Proceed immediately with detn of PAS. Det. INH as soon
as practicable, preferably ^4 hr after prepn of NaHC0 3 soln.
(b) (Applicable when filtration with vacuum is not feasi-
ble.) — Weigh sample as in (a), and transfer quant, to 40-50
mL r-b centrf. tube. Cautiously add, in small portions, freshly
prepd soln of 1 .5 g NaHC0 3 in 20 mL H 2 0. Agitate well after
each addn, avoiding loss from foaming by occasionally adding
few drops of ether. After all NaHC0 3 soln is added, continue
agitation until effervescence stops. Centrf. 5-10 min at ca 2000
rptn and decant supernate into 50 mL vol. flask. Add 10 mL
H 2 to tube, using rubber policeman to wash down wall, to
disintegrate residual cake, and to secure uniform suspension.
Centrf. as before, and combine supernate wash with original
ext. Repeat washing with three 5 mL portions H 2 0, dil. com-
bined aq. phases to vol., mix, and filter thru fluted paper. Pro-
ceed as in (a).
C. p-Aminosalicylic Acid (PAS)
From aq. NaHC0 3 ext, transfer aliquot contg ca 150 mg
PAS to 500 mL vol. flask and dil. to vol. with H 2 0. Transfer
10 mL aliquot to 250 mL vol. flask, add 12.5 mL coned pH
7 buffer, and dil. to vol. with H 2 0. Measure A of this diln in
1 cm cell at 299 (max.), 244 (min.), and 325 nm against 1 +
19 diln of the buffer. (With instruments suitable for A readings
in range 1 .0-1.5, use 2 cm cell thruout method or modify diln
so that concn of substance is twice that specified.) Calc. base-
line A B :
A B = A 299 - (0.3210 A 244 + 0.6790 A 32S )
Accurately weigh ca 50 mg finely powd pure PAS, dissolve
in 2 mL alcohol, add 5 mL 0.1W NaOH, and dil. with H 2
to exactly 500 mL. Transfer 25 mL aliquot to 200 mL vol.
flask, add 10 mL coned pH 7 buffer, and dil. to vol. with H 2 0.
Det. A at 244, 299, and 325 nm as above. Det. A B , and from
this value and that obtained from sample soln, calc. amt PAS
in sample.
D. Isoniazid (INH)
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Transfer 20 mL aliquot of the NaHC0 3 ext to 125 mL sep-
arator, add 0.5 mL benzaldehyde, shake 15 min, and let stand
10 min. Ext with six 20 mL portions CHC1 3 , filter exts thru
compact pledget of absorbent cotton into 150 mL beaker, and
evap. filtrate on steam bath in air current until residue has only
faint odor of benzaldehyde. Rinse down wall of beaker with
little CHC1 3 to cone, residue at bottom, and evap. to dryness.
Add 1-2 mL CHC1 3 , evap. again to dryness on steam bath in
air current, and heat residue few min. Repeat CHC1 3 and heat-
ing treatment until hot residue of benzylidine isoniazid (BINH)
is odorless, or has at most very faint odor of benzoic acid
(there must be no sweet odor or odor of benzaldehyde; take
care to avoid loss from spattering).
Dissolve residue in CHC1 3 and transfer quant, to separator
with addnl solv. Add CHC1 3 to vol. of 20-30 mL, shake with
10 mL freshly prepd 5% NaHC0 3 , and filter CHC1 3 layer thru
compact pledget of absorbent cotton. Wash aq. soln with three
10 mL portions CHC1 3 , passing each wash thru filter, and evap.
combined CHC1 3 exts to dryness on steam bath in air current.
Dissolve residue of BINH in alcohol without heat, and dil.
to exactly 100 mL with alcohol. Dil. 5 mL aliquot of this soln
to exactly 200 mL with alcohol, and det. A of diln (1 cm cell;
alcohol blank) at 302 (max.) and 375 nm. Subtract reading at
375 (background A from impurities) from that at 302 nm. Dif-
ference represents A from BINH at 302 nm.
Dissolve ca 20 mg, accurately weighed, of pure BINH in
alcohol and dil. to exactly 100 mL. Dil. 10 mL aliquot of this
soln to exactly 250 mL with alcohol and det. A at 302 nm.
Using this value and that due to BINH obtained from sample,
calc. equiv. amt of BINH in sample. BINH x 0.6088 = isoni-
azid.
Ref.: JAOAC 41, 496(1958).
CAS-65-49-6 (^-aminosalicylic acid)
CAS-54-85-3 (isoniazid)
550
Drugs: Part
AOAC Official Methods of Analysis (1990)
935.69* Dinitrophenoi
(or Its Sodium Compound) in Drugs
Bromination Method
Final Action
Surplus 1965
See 32.331-32.332, 10th ed.
974.40
Guaifenesin in Drugs
Polarographic Method
Final Action 1974
(Not applicable in presence of salicylate)
A. Apparatus
(a) Polarograph. — Any voltammetric or polarographic in-
strument with necessary accessories (cells, electrodes, Hg,
capillaries) capable of scanning up to 3.0 V in either pos.
or neg. direction.
(b) Micro or std cell, H-shaped. — Satd calomel electrode,
with 3% KCl-agar plug.
(c) Water bath. — Maintain at 65 ± 1° in freely circulating
H 2 bath.
B. Reagents
(a) Potassium nitrate soln. — IM. Weigh 50.5 g KN0 3 into
500 mL vol. flask, dil. to vol. with H 2 0, and mix.
(b) Dilute sulfuric acid soln.—\0% (v/v). Dil. 20 mL H 2 S0 4
(1 + 1) to 100 mL with H 2 and mix.
(c) Gelatin maximum suppressor. — 1 mg/mL. Accurately
weigh 100 mg gelatin (Difco Laboratories No. 0143; or Kind
& Knox Pharmaceutical Grade Gelatin, Kind & Knox, Div.
of Knox Gelatin, Inc., Park 80 West, Plaza Two, Saddle Brook,
NJ 07662) into 100 mL vol. flask, and dissolve in small amt
H 2 on steam bath. Cool, dil. to vol. with H 2 0, and mix.
Prep, fresh daily, as needed.
(d) Supporting electrolyte. — pH 10.4. Weigh 53.5 g NH 4 C1
into 1 L vol. flask, add 400 mL NH4OH, mix to dissolve, and
dil. to vol. with H 2 0.
(e) Guaifenesin std soln. — 1 mg/mL. Accurately weigh 25
mg guaifenesin std into 25 mL vol. flask. Dil. to vol. with
H 2 and mix.
C. Preparation of Sample
(a) Sirups. — Quant, transfer accurately measured portion
of sample contg ca 100 mg guaifenesin to 125 mL separator,
add 10 mL dil. H 2 S0 4 , and ext with four 20 mL portions CHC1 3 ,
and then with 15 mL CHC1 3 . Collect CHC1 3 exts in second
separator and wash with 10 mL H 2 0. Filter CHC1 3 layer thru
pledget of CHCl 3 -washed cotton into 100 mL vol. flask. Rinse
separator with 2-3 mL CHC1 3 and add wash to vol. flask. Dil.
to vol. with CHCI3 and mix.
(b) Tablets. — Det. av. wt/tablet. Grind without loss to pass
No. 60 sieve. Accurately weigh powder contg ca 50 mg guai-
fenesin and transfer to 125 mL separator. Add 10 mL H 2 and
shake 2 min. Proceed as in (a), beginning "... add 10 mL
dil. H 2 S0 4 , . . ."
D. Derivative Formation
Pipet duplicate 10 mL aliquots for sirups or 20 mL aliquots
for tablets of prepd soln into sep. 100 mL vol. flasks and care-
fully evap. to dryness with aid of air only. Add 10 mL H 2
to each and shake to dissolve guaifenesin. Label flasks as sam-
ple and blank. Pipet 10 mL guaifenesin std soln into third 100
mL vol. flask and label as std.
Pipet 3 mL H 2 S0 4 (1 ■+- 1) into each flask. Pipet 3 mL \M
KN0 3 into std and sample flasks and 3.0 mL H 2 into blank
flask. Place flasks in 65° const temp. bath. When solns reach
65°, heat addnl 60 min. Remove from bath and cool to room
temp. Into each flask pipet 25 mL electrolyte soln and 5 mL
gelatin soln, cool to room temp., dil. to vol. with H 2 0, and
mix thoroly,
£ Poiarography
Transfer soln to polarographic cell and bubble N thru for 5
min with micro H cell or 10 min with std H cell at moderate
rate. Polarograph from ~0.2 to ~0.9 v against satd calomel
ref . electrode. Measure ht of diffusion current (/ d ) at half-wave
potential as follows: Draw line tangent to top of residual cur-
rent extending to half- wave potential point. Draw line along
top of limiting current extending to half-wave potential point.
Measure vertical drop at half-wave potential between the 2 lines
in convenient units.
Det. guaifenesin concn by comparing wave ht of sample soln
with those of std and blank solns.
Subtract diffusion current (/ d ) of blank, if any, from sample
only. Perform all detns at same current sensitivity and within
same time span.
F. Caicuiations
(a) Sirup. — mg Guaifenesin /mL
- 100 x (/ d - /*) x C/(/ d x V)
(b) Tablets. — mg Guaifenesin /tablet
= 50 x (/ d -/S)XCXW, /(/ d x W s )
where / d , 7 d , and I d = diffusion current of sample, blank, and
std solns, resp.; C = mg guaifenesin /mL std soln; W t and W s
= av. tablet wt and wt sample taken, resp.; and V = mL liq.
prepn taken.
Ref.: JAOAC 57, 756(1974).
CAS-93-14-1 (guaifenesin)
938.15
Guaiacol in Drugs
Titrimetric Method
Final Action
A. Reagent
Hydr iodic acid. — Sp gr 1.7. Boil HI 30 min under reflux
with excess of hypophosphorous acid. When cool, transfer to
dark, g-s bottle. Do not leave bottle unstoppered more than
few min.
B. Apparatus
Methoxyl apparatus .See Fig. 956. 07B.
C. Determination
■ Place aliquot of alk. guaiacol soln (guaiacol dissolved in 1%
NaOH) contg 0.03-0.06 g guaiacol in boiling flask and evap.
soln just to dryness on steam bath in air current. For solid
guaiacol compds, weigh 0.06^0.10 g and transfer directly to
flask. Complete detn by method for methoxyl group, 956.07C,
beginning "Add 2.5 mL melted phenol from wide-tip pipet
..." Boil 30 min and titr. with 0.1/V Na 2 S 2 3 . I mL 0.17V
I = 0.00207 g guaiacol; 0.00229 g guaiacol carbonate; 0.00404
g K guaiacol sulfonate.
Refs.: JAOAC 21, 543(1938); 22, 721(1939).
CAS-90-05-I (guaiacol)
AOAC Official Methods of Analysis (1990)
Phenolics 551
933.10 Hexylresorcinol in Drugs
Titrimetric Method
Final Action
A. Reagents
(a) Sodium thiosulfate std soln. — 0.17V. Prep, as in 942.27 A.
(b) Purified methanol. — Purify if necessary as follows: Add
enough Br to com. MeOH to give bright yellow soln, heat to
bp on H 2 bath, and boil 5 min. Cool, and carefully decolorize
by adding 10% NaHS0 3 soln dropwise until just colorless.
(c) Potassium bromide ~br ornate soln. — 0.1N. Prep, as in
947.13A. Stdze as follows: Transfer 30 mL to I flask, and add
25 mL H 2 0, 5 mL 20% KI soln, and 5 mL HC1. Shake thoroly
and titr. with 0.1N Na 2 S 2 3 , using starch indicator (mix 2 g
finely powd. potato starch with cold H 2 to thin paste; add ca
200 mL boiling H 2 0, stirring const., and immediately discon-
tinue heating; add ca 1 mL Hg, shake, and let soln stand over
the Hg).
B. Standardization of Thiosulfate
Add 30 mL 0. IN KBr-KBr0 3 , and 10 mL purified MeOH
to 150 mL g-s flask. Wet stopper. Add 5 mL HO, stopper
flask, immediately place under running tap H 2 0, and swirl un-
til flask cools to room temp.; continue to shake 5 min after
adding HC1. Cautiously loosen stopper and add 5 mL 20% KI
soln. Swirl gently to liberate I, wash stopper, and titr. with
Na 2 S 2 3 soln. Add starch paste when soln is pale yellow.
C. Determination
Transfer 0.07-0.09 g sample to 150 mL g-s flask. Add 10
mL MeOH, (b), and swirl gently to dissolve sample. Add 30
mL 0.1/V KBr-KBr0 3 . Moisten stopper, add 5 mL HC1, stop-
per flask, and immediately hold under running H 2 while
swirling vigorously. When cooled to room temp, (ca 1 min),
remove from tap and shake vigorously 5 min after adding HO.
Cautiously loosen stopper and add 5 mL 20% KI soln. Swirl
gently, wash stopper with little H 2 0, add 1 mL CHC1 3 , and
titr. with Na 2 S 2 3 soln while swirling flask gently. Near end
point, stopper flask and shake vigorously to remove halogen
from CHC1 3 . When soln becomes pale yellow, add starch paste
and continue titrn. End point is reached when starch -I color
does not return during 30 sec of vigorous shaking. 1 mL 0AN
KBr-KBr0 3 soln = 0.00486 g hexylresorcinol.
Refs.: JAOAC 16, 384(1933); 20, 564(1937).
CAS- 136-77-6 (hexylresorcinol)
945.97 Oxyquinoline Sulfate in Drugs
Titrimetric Method
Final Action 1965
Method I
(For amts of oxyquinoline sulfate between 25 and 250 mg. Use
this method whenever nature of sample permits.)
A. Extraction
(a) Interfering substances absent. — Dissolve sample in ca
75 mL H 2 and add 5 mL HO.
(b) Nonoily preparations. — Ext preferably from soln alk.
with NaHC0 3 or borax. If extn from such medium is imprac-
ticable, or if compds of NH 3 or heavy or alk. earth metals are
present, add Me red, 936.150(a), and adjust with NaOH and/
or HC1 to slight acidity. Add NaOAc.3H 2 in proportion of
1 g/100 mL soln. If heavy or alk. earth metals are present,
also add 2 mL HOAc/100 mL soln.
Ext adjusted soln with enough 20 mL portions CHC1 3 . For
alk. or slightly acid soln, usually 6 extns suffice; when extra
HO Ac has been added, 10—12 extns are needed. Test for com-
plete extn by adding little HO (1 + 9) to last portion, evapg
CHC1 3 on steam bath, adjusting to 70°, and adding drop of
0.0 IN KBr-KBr0 3 and then drop of Me red; Me red should
be bleached immediately.
Ext combined CHC1 3 exts with five 10 mL portions HO
(1+9). If salicylic acid, volatile oils, etc., are present, wash
each acid portion with same 10 mL ether. If sample contains
phenol or other volatile interfering substances not completely
removed by preceding process, boil acid soln to remove them,
keeping vol. ca const by adding more H 2 0.
(c) Ointments, etc. — Transfer sample to separator with 50
mL ether, and ext with five 10 mL portions HO (1 + 9). If
salicylic acid, etc., is present, wash each acid portion with
same 10 mL ether. Add Me red; make just alk. with 10% NaOH
soln, then just acid with di'LHCl, and proceed as in (b), be-
ginning "Add NaOAc.3H 2 . . ."
B. Determination
Adjust acid soln (a), (b), or (c), to 50° and keep at this
temp, during titrn by reheating occasionally. Add drop (or more)
Me red 936.15D(a), from buret and titr. with 0.1JV KBr-KBr
3 , 933.10A(g). (Color of liq. gradually changes from brown-
orange to yellow; add more indicator whenever soln becomes
yellow. At slightly beyond halfway point, dibromohy-
droxyquinoline may crystallize and adsorb dye. Disregard color
of ppt and judge by that of soln. By dilg to a concn <0.1 g
oxyquinoline/ 100 mL, formation of ppt can be avoided.) End
point is reached when, after waiting 10 sec for absorption of
last drop of KBr-KBr0 3 soln and adding drop of indicator, it
is bleached almost immediately. Timing for addn of drop of
indicator at end point is important, as proper conditions prevail
only brief period.
Read vols of 2 solns consumed. Measure 10 mL Me red
into erlenmeyer, add 2 mL HO, and titr. with 0. \N KBr-KBr0 3 .
* Correct main titrn for vol. of Br consumed by measured vol.
indicator used in titrn. 1 mL 0.1/V KBr-KBr0 3 - 0.00508 g
(C 9 H 7 NO) 2 .H 2 S0 4 .H 2 0.
Method II
(For amts between 2 and 10 mg)
C. Determination
Ext as in 945.71A. Start titrn as in 945.71B, using 0.0LV
KBr-KBr0 3 , and dild Me red (1 vol. Me red, 936.15D(a), 4
vols H 2 0, and enough NaOH to dissolve dye) instead of stronger
reagents. Use as little indicator as possible. When near end
point, shown by more rapid consumption of indicator, heat to
70°, and complete titrn at this temp.
Refs.: JAOAC 28, 699(1945); 29, 280(1946).
CAS- 134-3 1-6 (oxyquinoline sulfate)
960.58 Methyl Salicylate in Drugs
Spectrophotometric Method
First Action 1960
Final Action 1961
A. Reagents
(a) Salicylic acid std soln. — 20 |xg/mL. Dissolve 0.2500 g
reagent grade salicylic acid in 95 mL CHC1 3 in 250 mL vol.
552
Drugs-. Part II
AOAC Official Methods of Analysis (1990)
flask and dil. to vol. with alcohol. Dil. 2.00 mL to 100 mL
with alcohol.
(b) Sodium bicarbonate soln. — Dissolve 5 g NaHC0 3 in 100
mL of H 2 to which 1 drop HC1 has been added.
B. Determination
Prep, sample diln, if necessary, to contain ca 5% Me sali-
cylate. Pi pet 5 mL sample or diln into 50 mL ether-pet ether
mixt. (1 + 1) in separator and wash with two 5 mL portions
cold, freshly prepd NaHC0 3 soln. Discard unemulsified aq.
phases. Ext org. layer with two 5 mL portions 5% NaOH soln
followed by two 5 mL portions H 2 0. Let phases sep. 5 min
and drain unemulsified aq. layers into another separator. Wash
combined exts with 10 mL pet ether and drain aq. phase into
another separator. Acidify cautiously with HC1 (litmus paper)
and ext with four 20 mL and one 15 mL portions CHC1 3 . Filter
each ext thru CHCl 3 -moistened plug of cotton into 250 mL
vol. flask. Dil. to vol. with alcohol and transfer 2.00 mL ali-
quot to 100 mL vol. flask.
Dil. to vol. with alcohol and det. A at max. (ca 305 nm).
Calc. as salicylic acid by comparison with A of std soln. Sal-
icylic acid X 1.1016 - Me salicylate.
Refs.: JAOAC 43, 239(1960); 44, 152(1961).
CAS- 119-36-8 (methyl salicylate)
948.29 Phenofphthalein
In Chocolate Drug Preparations
Gravimetric Method
Final Action
A. Reagents
(a) Potassium hydroxide soln. — 5 ± 0.17V.
(b) Iodine soln.—0.5N. Dissolve 12.7 g KI in 10 mL H 2 0,
add 6.35 g I, and when dissolved add 12 mL KOH soln, (a).
Dil. to 100 mL with H 2 0.
(c) Sodium sulfite soln. — Dissolve 12.6 g anhyd. Na 2 S0 3
in H 2 and dil. to 100 mL with H 2 0.
B. Preparation of Alcoholic Extract
Chill sample until hard; then reduce to granules by grating,
shaving, or grinding. Mix thoroly. Accurately weigh amt of
prepd sample contg ca 0.1 g phthln into gooch with thin as-
bestos mat or fritted glass disk. Ext fat with 5,4, and 3 mL
CC1 4 , using slight suction towards end. Place crucible on bell
jar app. Ext phthln from sample with several portions hot al-
cohol, collecting filtrate in 300 mL tall beaker. Wash under-
side of crucible free from phthln with hot alcohol (ca 50 mL
is enough for extn and washings). Evap. combined ale. exts
to dryness on steam bath.
C. Determination
Dissolve residue at room temp, in 1-1.5 mL KOH soln.
(Alk. phthln soln is unstable in air, and phthln should be con-
verted to tetraiodo compd within 1 hr.) Add piece of ice (ca
40 g) and 7-8 mL I soln. Add HC1 dropwise from buret, using
stirring rod, to complete pptn. If ppt and supernate are not
brown, add addnl I soln to ensure excess. Again dissolve ppt
by adding KOH from buret dropwise, with stirring. Wash down
any unreacted phthln adhering to sides of beaker with little
H 2 0. (Soln should now be blue to blue-purple.)
Repeat pptn with acid and resoln with alkali 3 more times,
adding small piece of ice if necessary. Then add 1~L5 mL
Na 2 S0 3 soln to blue alk. soln and filter into 250 mL beaker
thru gooch with thin asbestos mat or coarse fritted glass disk.
Wash crucible several times with H 2 0. Acidify filtrate with
HO, using few mL excess, and heat on steam bath 20-30
min, stirring occasionally. Decant hot supernate thru weighed
gooch (with asbestos mat or medium fritted glass disk). Wash
white to cream-colored ppt in beaker by decantation with hot
H 2 few times. Completely transfer ppt to the gooch and wash
with hot H 2 until filtrate is clear and gives neg. test for CI.
When app. has cooled and ppt has been sucked fairly dry,
wash ppt several times with pet ether, using suction toward
end. Dry tetraiodophenolphthalein to const wt at 110-130°.
Wt ppt x 0.3873 - wt phthln.
Refs.: JAOAC 31, 547(1948); 33, 203(1950).
CAS-8 1-90-3 (phenolphthalein)
942.29 Phenolphthalein
in Drug Emulsions
Gravimetric Method
Final Action
Shake sample well, preferably in mech. shaker, 10 min. Ac-
curately weigh amt of sample contg ca 0.1 g phthln from
weighing buret directly into centrf. bottle. Add 100 mL al-
cohol-ether (1 + 3), stopper bottle, shake vigorously, and then
centrf. until clear. Decant into separator. Wash residue in bot-
tle twice with 10 mL portions solv. mixt., adding these wash-
ings to separator. Dissolve residue in bottle in few mL H 2
and reppt gums with 50 mL solv. mixt. Shake and centrf. as
before, decanting into separator. Wash residue and bottle with
three 10 mL portions solv. mixt. and add these to separator.
Dissolve residue in few mL H 2 and test for complete extn
with NaOH.
Shake exts in separator repeatedly with 25 mL portions ca
0. IN NaOH until phthln is completely removed, as shown by
absence of color. Combine alk. exts in another separator and
acidify soln with dil. H 2 S0 4 (1 + 15).
Ext phthln by shaking acid mixt. repeatedly with 10 mL
portions ether. Test for complete extn with NaOH soln. Com-
bine ether exts in 150 mL beaker, evap. to dryness, and det.
phthln as in 948. 29C, omitting filtration of alk. soln.
Refs.: JAOAC 25, 843(1942); 26, 311(1943).
CAS-8 1-90-3 (phenolphthalein)
923.12* Phenolphthalein in Tablets
Ether Extraction Method
Final Action
Surplus 1972
See 37.138, 12th ed.
930.42* Phenolsulfonates in Drugs
Bromination Method
Final Action
Surplus 1965
See 32.342, 10th ed.
929.12 Thymol in Drug Substance
Titrimetric Method
Final Action
A. Preparation of Solution
Weigh 2 g pulverized thymol, transfer to 500 mL vol. flask,
and add 25 mL 25% NaOH soln. Agitate until thymol is dis-
solved and dil. to vol. at 20° with H?0.
AOAC Official Methods of Analysis (1990)
Analgesics and Antipyretics
553
B. Method I
Transfer 25 mL aliquot thymol soln to 250 mL g-s erlen-
rneyer, add 20 mL hot HC1 (1 + 1), and immediately add 1-
3 mL less than theoretical vol. 0.1N KBr-KBr0 3 , 933.10A(c).
Warm to 70-80°, add 2 drops 0.1% aq. Me orange, and titr.
slowly with KBr-KBr0 3 soln, swirling vigorously after each
addn. When red of Me orange has been bleached, add 2 drops
titrg soln, stopper, shake vigorously 10 sec, add 1 drop Me
orange, and again shake vigorously 10 sec. Continue addn of
KBr-KBr0 3 soln, 2 drops at time, shaking each time until red
disappears. Add 1 drop Me orange, shake vigorously, and if
red does not disappear, repeat alternate addn of 2 drops KBr-
KB1O3 soln and 1 drop Me orange, shaking after each addn
as before until red disappears. Calc. mL KBr-KBr0 3 soln used
to % thymol. 1 mL 0.1JV KBr-KBr0 3 = 0.003756 g thymol.
Reserve mixt. in titrg flask for 929. 12C.
C. Method It
To cooled mixt. from titrn, 929.12B, add 3-5 mL addnl
KBr-KBr0 3 soln. (If sample has not been previously analyzed
by 929. 12B, det. approx. vol, of KBr-KBr0 3 soln to use by
adding 20 mL HC1 (1 + 1) to 25 mL soln, 929.12A, heating
to ca 80°, and titrg slowly with KBr-KBr0 3 soln, while vig-
orously swirling flask, to yellow color maintained 1 min.)
Stopper, shake, add 1 g solid KI, wash sides of flask and stop-
per with H 2 0, and titr. I liberated by excess KBr-KBr0 3 soln
with 0AN Na 2 S 2 3 , using starch soln as indicator. Calc. vol.
Na 2 S 2 3 soln used in terms of KBr-KBr0 3 soln, deduct from
total vol. KBr-KBr0 3 soln added, and calc. to % thymol.
Refs.: JAOAC 12, 296(1929); 14, 330(1931).
CAS-89-83-8 (thymol)
930.43
Thymol In Antiseptics
Titrimetric Method
Final Action
(Caution: See safety notes on distillation, flammable solvents,
and diethyl ether.)
If ale. content is not known, make preliminary alcohol detn.
Transfer 50 mL sample (or aliquot contg 0.05-0.10 g thy-
mol) to Pt or porcelain evapg dish. Add 6-7 mL 50% NaOH
soln, mix well, and carefully dealcoholize by placing dish on
steam bath before elec. fan. Evap. vol. slightly more than amt
of alcohol present. (If >30% alcohol is present, dil. with H 2
to ale. content of 25%. In no case should evapn be carried
>70% of original vol.) Transfer soln to 125 mL separator,
washing out evapg dish with enough H 2 to bring vol. to ca
75 mL.
Ext alk. soln with two 20 mL portions pet ether. Wash com-
bined exts once with 5-10 mL 5% NaOH soln and add wash-
ings to aq. layer. Ext aq. soln contg thymol, together with Na
salts of boric, benzoic, and salicylic acids, with ether as in
960.53A, using 20, 15, 15, 10, and 10 mL. Use 8-10 extns
if prepn contains glycerol. Combine ether exts, transfer to 250
mL g-s erlenmeyer, and add 5 mL recently prepd ale. KOH
soln, 920. 160 A. Evap. most of ether, using steam bath and
elec. fan but do not evap. entirely to dryness. Leave 6-8 mL
residue and add to it 75 mL hot H 2 (80-90°) and 10 mL HC1.
Immediately add 1-3 mL less than theoretical vol. 0. IN KBr-
KBr0 3 , 933.10A(g), swirling constantly. Add 2 drops aq. 0.1%
Me orange and titr. slowly with KBr-KBr0 3 soln as in 929. 12B.
Test for complete extn by shaking aq. layer with two 15-
20 mL portions ether and titrg the thymol, if any, in ether exts.
Add this titrn to that obtained for main ether ext.
If theoretical amt of thymol present is not known, add 2
drops Me orange and titr. slowly, swirling constantly during
addn of KBr-KBr0 3 soln until red color is bleached. Continue
as in 929. 12B, beginning "... add 2 drops titrg soln, stop-
per, shake vigorously ..."
Note: To avoid loss of thymol by volatilization, both evapn
of alcohol and later evapn of ether must be done carefully.
Refs.: JAOAC 13, 332(1930); 14, 330(1931); 15, 418(1932).
CAS-89-83-8 (thymol)
ANALGESICS AND ANTIPYRETICS
970.80 Acetaminophen in Drugs
Spectrophotometric Method
First Action 1970
Final Action 1971
A. Reagents
(a) Bicarbonate-carbonate buffer. — pH 10.1. Weigh 1.0 g
NaHC0 3 and 4.5 g Na 2 C0 3 into 100 mL vol. flask and dil. to
vol. with H 2 0.
(b) Acidic methanol.— 1. mL 0.17V HC1/100 mL MeOH.
Prep, enough to ensure same MeOH is used thruout for std
and sample.
(c) Diatomaceous earth. — See 960. 53B.
(d) Acetaminophen std soln. — 0.008 mg/mL. Accurately
weigh 40 mg acetaminophen std into 100 mL vol. flask. Dil.
to vol. with acidic MeOH and mix well. Transfer 2.0 mL to
100 mL vol. flask and dil. to vol. with acidic MeOH.
B. Preparation of Sample
(a) Sirup.— Transfer 15.0 mL 0. IN NaOH to 25 mL vol.
flask. Dil. to vol. with acetaminophen sirup, avoiding wetting
flask neck above graduation mark while adding sirup, and mix.
Transfer 10.0 mL of this diln to 100 mL vol. flask, dil. to vol.
with H 2 0, and mix.
(b) Tablets. — Det. av. wt /tablet and pulverize. Accurately
weigh portion of powder contg ca 240 mg acetaminophen and
transfer to 250 mL vol. flask. Add 2mL1.0JV NaOH and ca
100 mL H 2 0. Shake, dil. to vol. with H 2 0, and mix.
C. Preparation of Chromatographic Column
Pack fine glass wool plug in base of chromatgc tube (25 x
250 mm) with aid of tamping rod ca 45 cm long and having
disk with diam. ca 1 mm less than tube. To 3.0 g diat. earth,
add 2.0 mL buffer soln and mix until fluffy. Transfer mixt.
to column and tamp gently to compress to uniform mass.
Transfer 2.0 mL sample soln to 100 mL beaker, add 1 drop
HCl, and mix. Add 3.0 g diat, earth, mix thoroly, and transfer
to column. Scrub beaker with 1 g diat. earth and 2 drops H 2 0.
Transfer to column, tamp, and top with fine glass wool pad.
D. Determination
(Caution: See safety notes on distillation, flammable solvents,
and diethyl ether.)
(Use H 2 0-washed solvs thruout.)
Pass 100 mL CHC1 3 thru column and discard eluate. Elute
acetaminophen with 150 mL ether, collecting eluate in 400 mL
beaker. Evap. soln to dryness on steam bath under air stream.
Dissolve residue in acidic MeOH, transfer quant, to 50 mL
vol. flask, and dil. to vol. with same solv. Transfer 10.0 mL
of this soln to 50 mL vol. flask, dil. to vol. with acidic MeOH,
and mix. Scan spectra of sample and std solns from 350 to
240 nm in 1 cm cells, using acidic MeOH as blank.
554
Drugs: Part
AOAC Official Methods of Analysis (1990)
mg Acetaminophen in portion of sirup or tablet taken
- 31.25 x C x (A/A')
where C = mg/mL std soln, and A and A' refer to sample and
std, resp., at max., ca 249 nm.
Ref.: JAOAC 53, 591(1970).
CAS- 103-90-2 (acetaminophen)
987.12 Acetaminophen in Drug Tablets
Liquid Chromatographic Method
First Action 1987
(Applicable to single-component tablets and to multi-compo-
nent tablets contg aspirin and caffeine)
A. Principle
Acetaminophen is detd by reverse phase liq. chromatgy us-
ing MeOH-HOAc mobile phase and UV detention at 280 nm.
B. Apparatus
(a) Liquid chromato graph. — Isocratic system equipped with
detector capable of monitoring A at 280 nm, and sample in-
jection valve with 20 \lL sample loop. Operating conditions:
flow rate ca 2 mL/min; temp, ambient; detector sensitivity
1.28 AUFS.
(b) LC column. — Stainless steel, 300 X 4.6 mm id, packed
with octadecylsilane chemically bonded to 10 fxm porous silica
or ceramic microparticles.
C. Reagents
(a) Solvents. — LC grade MeOH; H 2 0, double distd in glass.
(b) Acetic acid se>/«.— Pipet 7.5 mL HO Ac (AR grade) into
1 L vol. flask, dil. to vol. with H 2 0, and mix.
(c) Mobile phase. — MeOH-HOAc soln (1 +3). Degas with
aid of sonication and vac. pH of this soln should be ca 3.0.
(d) Std preparation. — Accurately weigh ca 60 mg USP
Acetaminophen Ref. Std, previously dried 18 h over silica gel,
and transfer to 100 mL vol. flask. Dissolve and dil. to vol.
with mobile phase, and mix.
D. Sample Preparation
Weigh and finely powder >20 tablets. Transfer portion of
powder, equiv. to ca 60 mg acetaminophen, to 100 mL vol.
flask, add ca 50 mL mobile phase, and sonicate or mech. shake
10 min. Dil. to vol. with mobile phase, and mix. Filter soln
thru microfilter into g-s flask. Discard first portion of filtrate
(ca 10 mL).
E. System Suitability
Make 5 replicate injections of std prepn and record peak
responses (hts or areas). Coeff. of var. (CV) should be <2.0%,
tailing factor should be <2.0, and column efficiency should
be >1000 theoretical plates, all calcd as shown in 985. 49D
and 985.50D.
F. Determination
Make duplicate 20 |iL injections of std prepn and sample
prepn into liq. chromatograph, and measure peak responses
(hts or areas). From av. values, calc. amt acetaminophen, in
mg, in portion of tablets taken for assay as follows:
Acetaminophen, mg in sample weighed = 0.1C x (R/R')
where C = concn of std prepn, |xg/mL, and R and R' = av.
peak responses for sample prepn and std prepn, resp. To verify
that no appreciable change in chromatgc conditions has oc-
curred during course of chromatgc run, at end of series of detns
make dup. injections of std prepn, and evaluate reproducibil-
ity.
Ref.: JAOAC 70, 212(1987).
CAS- 103-90-2 (acetaminophen)
972.49 Acetaminophen and Salicylamide
in Drugs
Spectrophotometric Method
First Action 1972
Final Action 1973
(Applicable in presence of antihistamines, barbiturates, caf-
feine, ascorbic acid, prednisone, and belladonna alkaloids. As-
pirin interferes in salicylamide detn.)
A. Apparatus
(a) Chromatographic tube. — Plain, 250 X 25 mm i.d.
(b) Tamping rod. — See 967.31A(b); use for packing col-
umns.
(c) UV spectrophotometer. — Preferably recording, with
matched 1.0 cm cells.
(d) Infrared spectrophotometer. — With equipment suitable
for prepg KBr disks.
B. Reagents
(a) Tripotassium phosphate . — 20%. Dissolve 10.0 g K 3 P0 4
in H 2 and dil. to 50 mL.
(b) Water-washed chloroform. — Shake equal vols CHC1 3
and H 2 in separator 1 min. Use within 1 day.
(c) Water-washed ethyl acetate. — Shake equal vols EtOAc
and H 2 in separator 1 min. Discard lower phase.
(d) Acetic acid in chloroform,— (J) 10%.— Dil. 10.0 mL
HOAc to 100 mL with CHC1 3 . (2) 1%.— Dil. 10.0 mL 10%
soln to 100 mL with CHCt 3 . Prep, ca 375 mL for each sample
and 225 mL for std solns.
(e) Diatomaceous earth. — See 960. 53B.
(f ) Acetaminophen std soln. — 0.6 mg/100 mL. Accurately
weigh ca 60 mg USP Ref. Std Acetaminophen; dissolve and
dil. with alcohol in 100 mL vol. flask. Dil. 10.0 mL of this
soln to 100 mL with alcohol. Dil. 10.0 mL of second diln to
100 mL with alcohol.
(g) Salicylamide std soln. — 2 mg/100 mL. Accurately weigh
ca 50 mg USP Ref. Std Salicylamide; dissolve and dil. with
1% HOAc-CHCl 3 in 100 mL vol. flask. Dil. 4.0 mL of this
soln to 100 mL with 1% HOAc-CHCl 3 .
C. Preparation of Sample
Det. av. wt of tablets or capsules, reduce to powder passing
No. 60 sieve.
D. Preparation of Columns
Column I. — Place small piece of glass wool in chromatgc
tube. Mix 1.0 g diat. earth, (e), and 0.5 mL HCI (1 + 1) in
beaker until fluffy, and pack in tube. Accurately weigh portion
powd sample contg ca 50 mg acetaminophen into 100 mL beaker
and thoroly wet with 2.0 mL HCI (1 + 1). Add 3.0 g diat.
earth, mix until fluffy, transfer quant, to column, and pack.
Scrub sample beaker with 1.0 g diat. earth and 0.5 mL HCI
(1 + 1), and pack on column. Wipe all utensils and sample
beaker with glass wool and pack on column.
Column II. — Place small piece of glass wool in chromatgc
tube. Mix 3.0 mL 20% K 3 P0 4 and 5.0 g diat. earth until fluffy,
and pack on column. Cover diat. earth with small piece of
glass wool.
AOAC Official Methods of Analysis (1990)
Analgesics and Antipyretics 555
E. Determination
Mount columns so that / elutes into //. Place waste beaker
under column //. Add 100 mL H 2 0- washed CHC1 3 to column
/. After all CHC] 3 has passed thru both columns, sep. col-
umns, rinsing stem of top column with 5 mL H 2 0- washed CHC1 3
into bottom column. Elute column // with addnl 25 mL H 2 0-
washed CHC1 3 . Rinse stem of column // with CHC1 3 after all
C.HCI3 has passed thru. Discard wash CHC1 3 .
Place 250 mL vol. flasks under each column. Elute acet-
aminophen from column / with 100 mL H 2 0-washed EtOAc.
Do not let column run dry until entire 100 mL has passed into
column. Rinse column stem with EtOAc (not H 2 0- washed)
into flask. Add 5 mL alcohol, mix, and dil. to vol. with EtOAc
(not H 2 0-washed). Pipet 15 mL into 50 mL vol. flask and dil.
to vol. with EtOAc (not H 2 0- washed). Pipet 10 mL of this
soln into 100 mL beaker and evap. to dryness with gentle air
current. Do not use heat. Dissolve residue in alcohol and trans-
fer quant, to 100 mL vol. flask with several portions alcohol;
dil. to vol. with alcohol. Det. A of sample and std, (f ), solns
at 248 nm in 1 .0 cm cell against alcohol as ref . If recording
spectrophtr is available, scan soln from 320 to ca 210 nm in
1.0 cm cells against alcohol.
mg Acetaminophen in portion sample taken
- 83.33(A/A') X C
where A and A' refer to sample and std solns, resp., at 248
nm; and C = exact concn of ref. std soln in mg/100 mL final
soln.
Elute salicylamide from column // by passing 10.0 mL 10%
HOAC-CHCI3 thru column, followed by 100 mL 1% HOAc-
CHC1 3 . After complete elution, rinse stem of column into flask
with 1% HOAC-CHCI3 and dil. to vol. with same sol v. Pipet
aliquot contg ca 2 mg salicylamide into 100 mL vol. flask and
dil. to vol. with 1% HOAc-CHCl 3 . Det. A of sample and std,
(g), solns at 308 nm in 1.0 cm cells against 1% HOAc-CHCl 3
as ref. If recording spectrophtr is used, scan solns from 370
to ca 260 nm in 1.0 cm cells against 1% HOAc-CHCl 3 .
mg Salicylamide in portion sample taken
= (250/5) X (A /A') X C
where B = vol. (mL) aliquot taken to contain 2 mg salicyl-
amide; A and A' refer to sample and ref. std solns, resp., at
308 nm; and C = exact concn of ref. std soln in mg/100 mL
final soln.
ft Quantitative Methods— Final Action
(a) Pkenocetin. — (7) Volumetric. See 32.131(a)(1), 10th
ed. (2) Gravimetric. See 32.131(a)(2), 10th ed.
(b) Acetanilid.— See 32.131(b), 10th ed.
916.03* Acetanilid and Caffeine
in Drugs
Final Action
Surplus 1965
See 32.132-32.134, 10th ed.
916.04* Acetanilid, Caffeine,
and Codeine in Drugs
Final Action
Surplus 1965
See 32.135-32.136, 10th ed.
916.05* Acetanilid, Caffeine,
and Quinine in Drugs
Final Action
Surplus 1965
See 32.137-32.139, 10th ed.
916.06* Acetanilid, Caffeine, Quinine,
and Morphine in Drugs
Final Action
Surplus 1965
See 32.140-32.141, 10th ed.
916.07* Acetanilid
and Sodium Salicylate in Drugs
Final Action
Surplus 1965
See 32.142-32.143, 10th ed.
F. identification
Prep. KBr disks of each ingredient from eluates by evapg,
with gentle air current only, aliquot contg ca 1 mg each in-
gredient. Be sure no detectable odor of HO Ac is present from
salicylamide aliquot before prepg KBr disk.
Compare IR spectrum of each with its respective std from
2.0 \xm (5000 cm" 1 ) to 15 fxm (660 cm" 1 ).
Ref.: JAOAC 54, 895(1971).
CAS- 103-90-2 (acetaminophen)
CAS-65-45-2 (salicylamide)
960.59 Aspirin, Phenacetin,
and Caffeine (APC) in Drugs
First Action 1960
Final Action 1961
A. Method I (For Aspirin Only)*
—Surplus 1974
5^36.190, 11th ed.
B. Method Ifr
—Surplus 1974
See 36.191-36.192, 11th ed.
916.02* Acetanilid and Phenacetin
in Drugs
Surplus 1965
A. Qualitative Test for Phenacetin — Procedure
See 32.129, 10th ed.
Chromatographic Method
C. Reagents
(a) Sodium bicarbonate soln. — \M. Dissolve 4.2 g Na-
HCO3 in 48 mL H 2 0.
(b) Washed ether. — Wash USP ether with equal vol. H 2
in separator. Filter thru paper, rejecting first 15 mL. Use within
3 days. Approx. 70 mL required for each sample.
556
Drugs: Part II
AOAC Official Methods of Analysis (1990)
(c) Chloroform,— USF. A against H 2 at 276 nm <0.050.
Use same lot thruout.
(d) Washed chloroform. — Wash and filter CHC1 3 as in (b).
Use within 3 days. Use same lot thruout. Approx. 700 mL is
required for stds and 170 mL for each sample.
(e) Isooctane. — A against H 2 at 286 nm <0.050. Use same
lot thruout.
(f ) Phenacetin std so In. — 7 mg/100 mL. Dissolve 70.0 mg
pure phenacetin in CHC1 3 and dil. to 100 mL with CHC1 3 . Dil.
10 mL aliquot to 100 mL with isooctane.
(g) Caffeine std soln. — 1.4 mg/100 mL. Dissolve 140.0 mg
caffeine in washed CHC1 3 and dil. to 100 mL. Dil. 10 mL
aliquot to 100 mL; dil. 10 mL aliquot of this soln to 100 mL
with washed CHC1 3 .
(h) Aspirin std soln. — 5 mg/100 mL. Dissolve 100.0 mg
aspirin in washed CHC1 3 and dil. to 100 mL. To 5 mL aliquot
add 1.0 mL HOAc and dil. to 100 mL with washed CHC1 3 .
Prep, fresh daily.
(i) Salicylic acid std soln. — 2.5 mg/100 mL. Dissolve 100.0
mg salicylic acid in washed CHC1 3 and dil. to 100 mL. Dil.
25 mL aliquot to 100 mL; to 10 mL aliquot of this soln, add
1.0 mL HOAc and dil. to 100 mL with washed CHC1 3 .
D. Apparatus
See 967.31A.
B. Preparation of Sample
Weigh powd sample contg ca 100 mg aspirin and transfer
to 100 mL vol. flask. Add 60 mL CHC1 3 and shake well. Add
0.2 mL HOAc and dil. to vol. with CHC1 3 .
F. Preparation of Chromatographic Column
Loosely pack small amt of fine glass wool in base of chro-
matgc tube so as to support diat. earth, but not cause irregu-
larity in thickness of diat. earth layer.
To 2.0 g diat. earth, 960.53B, in 100 mL beaker, or glass
mortar, add 2.0 mL H 2 S0 4 (1 -f 9). Mix well with metal spat-
ula. Transfer to chromatgc tube, and with packing rod, com-
press lightly to uniform mass. Mix 2.0 g diat. earth with 2.0
mL \M NaHC0 3 and place in column above acid layer. Wash
column with 15-20 mL washed ether and discard washings.
G. Separation
(Use washed ether and washed CHC1 3 thruout, except for dis-
solving phenacetin residue.)
(a) Phenacetin. — Dil. 5 mL aliquot prepd sample soln with
20 mL ether and pass thru column, receiving eluate in 100 or
150 mL beaker. After soln has passed into adsorbent, wash
with five 5 mL portions ether, letting each portion pass into
adsorbent before adding next. Wash tip of outlet with CHC1 3
and evap. total eluate to dryness by gentle heating on steam
bath with air current. Dissolve phenacetin residue in 5 mL USP
CHCI 3 and dil. with isooctane to 50 mL.
(b) Caffeine. — Immediately after passage of last portion of
ether thru column, replace beaker with 50 mL vol. flask. Pass
48 mL CHCI3 thru column, wash tip with CHC1 3 and dil. eluate
to vol.
(c) Aspirin and salicylic acid. — Immediately replace re-
ceiver with 100 mL vol. flask. Pass soln of 0.5 mL HOAc in
5 mL CHCI3 thru column, followed by 90-92 mL 1% soln of
HOAc in CHC1 3 . Wash tip with CHC1 3 and dil. eluate to vol.
H. Determination
Immediately det. A of acid fraction and of aspirin and sal-
icylic acid std solns at 280 and 310 nm against 1% HOAc in
CHCI3. Det A of phenacetin fraction and std at 286 nm against
isooctane-USP CHC1 3 (9 + 1) and that of caffeine fraction and
std at 276 nm against washed CHC1 3 blank.
Calc. amt of each ingredient in sample. Aspirin and salicylic
acid may be calcd by successive approximations as follows:
Attributing entire A at 310 nm to salicylic acid, use ratio of
salicylic acid std readings at the 2 wavelengths to calc. A due
to salicylic acid at 280 nm, and deduct from total A at 280
nm. Attributing remainder to aspirin, use ratio of aspirin std
readings to calc. A due to aspirin at 310 nm. Deduct this A
from total at 310 nm. Use remainder to calc. amt of salicylic
acid in sample and also to recalc. A due to salicylic acid at
280 nm. Deduct latter from total A at 280 nm and use re-
mainder to calc. amt of aspirin in sample. Alternatively, calc.
these two ingredients by simultaneous equations. Amt of as-
pirin hydrolyzed may be calcd by multiplying amt of salicylic
acid by 1.3044.
Ref.: JAOAC 43, 241(1960).
CAS-50-78-2 (aspirin)
CAS-58-08-2 (caffeine)
CAS-62-44-2 (phenacetin)
940.38* Aspirin, Phenacetin,
and Phenyl Salicylate in Drugs
Final Action
Surplus 1965
See 32.153, 10th ed.
941.22* Aminopyrine, Phenacetin,
and Caffeine in Drugs
Final Action
Surplus 1965
See 32.154, 10th ed.
942.30* Aminopyrine, Phenacetin,
Caffeine, and Phenobarbital in Drugs
Final Action
Surplus 1965
See 32.155, 10th ed.
916.08* Phenacetin and Caffeine
in Drugs
Final Action
Surplus 1970
See 36.202-36.203, 11th ed.
916.09* Phenacetin
and Phenyl Salicylate in Drugs
Final Action
Surplus 1965
A. Acid Hydrolysis Method
See 32.158, 10th ed.
0. Alkaline Hydrolysis Method
See 32.159, 10th ed.
AOAC Official Methods of Analysis (1990)
Analgesics and Antipyretics
557
922.13* Aspirin
Final Action
Surplus 1970
A. Melting Point
See 36.206, 11th ed.
B. Free Salicylic Acid
See 36.207, 11th ed.
C. Total Salicylate
See 36.209(a) and (b)Jlth ed.
D. Combined Acetic Acid
See 36.210, llthed.
E. Double Titration Method
See 37.046-37.047, 13th ed.
955.52 Aspirin and Phenobarbital
in Drugs
Spectrophotometric Method
Final Action 1965
A. Apparatus
(a) Spectrophotometer . — Capable of isolating spectrum of
^2 nm in region 230-300 nm.
(b) Chromatographic tube and tamping rod. — See 967. 31 A.
B. Reagents
(a) Dibasic potassium phosphate soln. — Approx. 2M. Dis-
solve 35 g K9HPO4 in FLO, cool to room temp., and dil. to
100 mL.
(b) Diatomaceous earth. — See 960. 53B.
(c) Washed chloroform.— Wash USP CHC1 3 with V2 vol.
H 2 in separator.
(d) Aspirin std soln. — 5 mg/100 mL. Dissolve 100 mg as-
pirin in CHCI3 and dil. to 100 mL with CHC1 3 . Dil. 5 mL
aliquot to 100 mL with CHC1 3 .
(e) Phenobarbital std soln. — 1 mg/100 mL. Dissolve 100
mg phenobarbital in NH 4 OH (1 + 27) and dil. to 500 mL with
NH4OH (1 + 27). Dil. 5 mL aliquot to 100 mL with NH 4 OH
(1 + 27).
C. Preparation of Sample Solution
Transfer accurately weighed portion of finely ground tablets
contg 60-120 mg phenobarbital to 100 mL vol. flask. Dis-
solve in CHCI3 by shaking vigorously and dil. to vol. with
CHCI3.
D. Preparation of Chromatographic Column
Pack small pledget of glass wool in constricted portion of
stem of tube and place pad of glass wool ca 5 mm thick in
bottom of large portion of tube. Fasten piece of rubber tubing
with attached screw clamp to outlet to limit flow during pack-
ing. Clamp tube in vertical position.
To 10 g diat. earth in mortar add 50 mL CHCl 3 , and mix
with pestle to form slurry. Distribute 10 mL 2M K 2 HP0 4 soln,
(a), over surface of slurry and mix thoroly until homogeneous,
adding more CHC1 3 if necessary. Add this slurry to tube, ca
Vs at time, alternately packing and forming flocculent suspen-
sion by working packing rod up and down. Diat. earth must
be covered with CHCI3 at all times.
After column is packed, remove rubber tube from stem and
rinse stem with CHCI3. Check flow rate of column with CHC1 3
level ca 5 cm above surface of column. Adjust rate of flow to
2-5 mL/min by tightening or loosening glass wool pledget in
constricted portion of stem. When level of solv. just reaches
surface of column, place 100 mL vol. flask under stem.
E Determination of Phenobarbital
Add 5 mL prepd sample soln, 955. 52C, from pipet to side
of tube near diat. earth surface. When level of sample soln
reaches surface of column, add 5 mL washed CHC1 3 , let sink
into column, and repeat with another 5 mL washed CHC1 3 .
After last rinse enters column, add washed CHC1 3 to tube and
keep level of 4-8 cm CHC1 3 above column during elution.
Collect 95 mL eluate in the 100 mL vol. flask. Dil. to vol.
with CHC1 3 , mix, and transfer 20 mL aliquot to 100 mL beaker.
Evap. to dryness on steam bath under air current. Dissolve
residue in NH 4 OH (1 +27) and transfer to 100 mL vol. flask.
Rinse, and dil. to vol. with NH 4 OH (1 + 27). Det. A at max.,
ca 240.5 nm, against blank prepd by passing 5 mL CHC1 3 thru
column as with sample soln.
g Phenobarbital in sample — \0A/a
where a is absorptivity of phenobarbital at 240.5 nm obtained
by dividing A of std phenobarbital soln in 1 cm cell at 240.5
nm by its concn (0.01 g/L).
F. Determination of Aspirin
Dil. 5 mL original sample soln, 955. 52C, to 100 mL with
CHCI3 in vol. flask. Dil. 10 mL aliquot of this soln to 100
mL with CHCI3. Det. A of final diln on spectrophtr at 278 nm
against CHCI3 blank.
g Aspirin in sample = 20A 278 /a 2 78
where a 278 is absorptivity of aspirin at 278 nm obtained by
dividing A of std aspirin soln in 1 cm cell at 278 nm by its
concn (0.05 g/L).
Ref.: J AOAC 38, 635(1955).
CAS-50-78-2 (aspirin)
CAS-50-06-6 (phenobarbital)
938.16* Aspirin and Phenolphthalein
in Tablets
Gravimetric Method
Final Action
Surplus 1977
See 37.054, 13th ed.
923.13
Aminopyrine
Final Action
A. Qualitative Tests
(a) Dissolve 0.01 g sample in 2 mL H 2 and add few drops
of yellow HNO3 (contg HN0 2 ). Purplish blue soln is pro-
duced.
(b) Dissolve 0.01 g sample in 2 mL H 2 and add 1 mL
10% FeCl 3 soln. Purple to violet color develops, which be-
comes red on addn of H 2 S0 4 (1 +9).
(c) Dissolve 0.1 g sample in 2 mL H 2 and add few drops
of 5% AgN0 3 soln. After few sec, purple to violet color is
produced, and on standing, deposit of metallic Ag results (use-
ful for detecting aminopyrine in antipyrine).
(d) Dissolve 0.1-0.2 g sample in 2 mL H 2 0, add 1 or 2
drops 0.2% NaN0 2 soln and few drops of H 2 S0 4 (1 + 9), and
shake few sec. Purplish blue color develops and then gradually
fades, leaving colorless soln. Excess NaN0 2 destroys amino-
pyrine color. On addn of few more drops of NaN0 2 soln and
dil. H 2 S0 4 , yellowish green color remains after purple dis-
558
Drugs: Part II
AOAC Official Methods of Analysis (1990)
appears if antipyrine is present. (Useful for detecting antipy-
rine in presence of aminopyrine.)
Refs.: JAOAC 7, 29(1923); 8, 40(1924); 8, 544(1925).
B. Quantitative Method
(Caution: See safety notes on distillation, toxic solvents, and
chloroform,)
Place 1 g powd sample in 100 mL vol. flask, add 60 mL
IN H 2 S0 4 , and shake several min to ensure complete soln of
aminopyrine. DiJ. to vol. with IN H 2 S0 4 . Filter, if not clear,
thru dry filter, rejecting first part of filtrate. Pipet 20 mL ali-
quot of soln or filtrate into separator; make distinctly alk. with
either NH 4 OH or 5% NaOH; and ext with 20, 15, 10, 10, and
5 mL portions CHCJ 3 . Combine CHC1 3 exts in second sepa-
rator and wash with 2 mL H 2 0. Filter CHC1 3 soln into weighed
beaker thru cotton pledget satd with CHC1 3 . Ext wash H 2
with 5 mL CHC1 3 and add this to combined CHC1 3 exts. Evap.
combined CHC1 3 exts just to dryness on H 2 bath with aid of
air current and dry residue 10 min at 100°. Cool in desiccator,
and weigh as aminopyrine. Identify aminopyrine by its mp,
106.5-109°, and qual. tests, 923. 13A, or microchem. tests,
Table 962.21B.
Refs.: JAOAC 11, 51, 350(1928); 23, 60, 742(1940).
CAS-58-15-1 (aminopyrine)
HYPNOTICS AND SEDATIVES
968.43 AcetylcarbromaB
and Bromisovalum in Drugs
Infrared Spectrophotometric Method
First Action 1968
Final Action 1970
A. Principle
Acetylcarbromal is eluted with heptane-CCl 4 and bromiso-
valum with H 2 0-satd CHC1 3 from diat. earth column. Eluates
are dried; acetylcarbromal residue is dissolved in CC1 4 and
bromisovalum residue in CHC1 3 . Concns are detd by IR spec-
trometry at 5.8 |xm. Identification is made from residues in
KBr disks.
B. Apparatus
(a) Infrared spectrophotometer . — Beckman 1R-5, (replace-
ment model Varian Instruments Carg 2000 Series), or equiv.,
with 1 mm liq. cells.
(b) Chromatographic tube. — See 967. 31 A.
C. Reagents
(a) Diatomaceous earth. — See 960. 53B.
(b) Reference std solns. — 1.00 mg/mL. Completely dis-
solve 50.0 mg acetylcarbromal (mp 109°) in CC1 4 in 50 mL
vol. flask and dil. to vol. with CC1 4 . Completely dissolve 50.0
mg bromisovalum (mp 148-149°) in CHC1 3 in 50 mL vol. flask
and dil. to vol. with CHC1 3 .
D. Column Chromatography
(Caution: See safety notes on distillation, toxic solvents, car-
bon tetrachloride, and chloroform.)
Pack small wad of fine glass wool into bottom of tube. Tho-
roly mix 4 g diat. earth with 5 mL HC1 (2 + 1), transfer to
tube, and tamp to uniform mass with tamping rod.
Det. av. wt/tablet and pulverize. Accurately weigh portion
of powder contg 10-30 mg acetylcarbromal, and mix in beaker
with 1 g diat. earth and 1 mL H 2 0. Transfer to tube, dry-rinse
beaker with small portion diat. earth, and tamp to uniform mass.
Wash beaker with few portions H 2 0-satd CCl 4 -heptane (1 +
1) and pour thru column. Elute acetylcarbromal with CC1 4 -
heptane mixt., collecting 50 mL eluate in beaker. Immedi-
ately, without letting column go dry, elute bromisovalum with
100 mL H 2 0-satd CHC1 3 , collecting eluate in beaker. Evap.
both eluates to complete dryness on steam bath with air cur-
rent.
Dissolve acetylcarbromal residue with several portions of CC1 4
(dried with anhyd. Na 2 S0 4 ) and dil. to concn of 1 mg/mL.
Dissolve bromisovalum residue in several portions of CHC1 3
(dried with anhyd. Na 2 S0 4 ) and dil. to concn of 1 mg/mL.
Using their resp. soivs as ref. solns, det. IR spectrum at 5-7
fxm. For calcn, use A of max. at ca 5.8 |xm, using baseline
technic:
% Compd = (100 x A x C x V)/(A' x HO
where A and A' refer to sample and std solns, resp. , C = concn
of std soln (mg/mL), V = final vol. sample soln, and W ~
mg sample.
B. Identification
Evap. 1 mL each of std and sample soln, and prep. KBr
disk from each of residues, using ca 200 mg KBr. Scan IR
spectra for identification.
Ref.: JAOAC 51, 621(1968).
CAS-77-66-7 (acetylcarbromal)
CAS-496-67-3 (bromisovalum)
943.07
Carbromal in Drugs
Titrimetric Method
Final Action
A. Apparatus
See Fig. 943.07. Consists of 100 mL r-b flask with 24/40
1 inner joint; condenser with jacket ca 130 mm long; and ab-
sorption flask with 2 bulbs. Condenser is equipped with 12/
30 and 24/40 J inner joints. Small dropping funnel is fused
to tube above jacket. Absorption flask has outer 12/30 J joint.
Small springs (not shown) are attached to hooks on joints to
keep app. tightly connected during use.
B. Determination
Place sample calcd to contain 40-60 mg Br in oxidn flask
and dissolve in 2 mL 10% NaOH soln and 8 mL H 2 0. Lu-
bricate joints of app. with H 3 P0 4 and connect flask to con-
denser. Place ca 15 mL 1% N 2 H 4 .H 2 S0 4 soln and 5 mL 10%
NaOH soln in absorption flask and connect to app. (Use <20
mL absorbing soln if app. has smaller absorption bulbs than
indicated.)
Add 5 mL Cr0 3 soln (1 + 1) thru addn tube, wash down
with 2-3 mL H 2 0, and then slowly add 10 mL H 2 S0 4 . If vig-
orous reaction begins, let it subside before heating flask; if
reaction does not begin as acid is added, heat gently with small
flame, but remove flame before reaction becomes too vigor-
ous, otherwise reaction mixt. may foam up into condenser.
When reaction subsides, heat mixt. to boiling. When foaming
subsides, add 5 mL H 2 S0 4 thru dropping funnel, boil 10 min,
add another 5 mL H 2 S0 4 , and boil again 10 min. Drain H 2
from condenser and boil reaction mixt. until 2-3 drops H 2
distil into absorber.
Disconnect absorption flask, wash contents into 500 mL I
flask, and dil. to ca 100 mL with H.O. Add ca 12 mL H 3 P0 4 ,
5 mL 3% KCN soln, and 15 mL 3% KMn0 4 soln, wetting
AOAC Official Methods of Analysis (1990)
Hypnotics and Sedatives 559
S CALE
24/40 $
FIG. 943.07— Bromine apparatus
sides of flask with each reagent as it is added. Stopper flasks
and mix by gentle swirling, wetting entire inside surface. Let
stand >7 min; then add ca 2 g solid FeSO^NH^SO^HaO.
Wash down sides of flask and mix. (Clear, nearly colorless
soln should result.) If any KMn0 4 or Mn0 2 remains, add more
Fe(NH4)2(S04)2.6H20 (2 g excess does no harm).
Add ca 2 g KI and immediately titr. liberated I with 0.05N
Na 2 S 2 3 , using starch indicator. (End point is disappearance
of starch-I color; avoid over-titrn as color of soln remains light
blue.) 1 mL 0.05N Na 2 S 2 3 = 0.001998 g Br or 0.00593 g
carbromal.
Refs.: JAOAC 26, 433(1943); 28, 757(1945); 34, 570(1951).
CAS-77-65-6 (carbromal)
943.08 CarbromaE and Pentobarbital
in Drugs
Gravimetric Method
Final Action
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Transfer 0.5-0.7 g sample to separator, and add 15 mL H 2
and 0.5 mL liV NaOH from pipet. Ext carbromal with at least
five 25 mL portions CHC1 3 , washing each portion in second
separator contg 10 mL H 2 and 2 drops O.IN NaOH. Filter
CHCI3 thru cotton and transfer to tared flask or beaker. Test
for complete extn. Evap. CHCI 3 soln of carbromal nearly but
not quite to dryness on steam bath in air current. Remove con-
tainer and let stand in air to const wt.
Combine aq. solns and proceed as in 925. 59A, beginning
"Acidify to litmus paper . . ." Wt pentobarbital x 1.097 =
wt Na pentobarbital in portion taken for assay. Det. mp of
dried exts. Carbromal melts at 116-119° and pentobarbital at
126-130°.
Refs.: JAOAC 26, 433(1943); 28, 757(1945); 34, 570(1951).
CAS-77-65-6 (carbromal)
CAS-76-74-4 (pentobarbital)
925.59
Barbiturates in Drugs
Gravimetric Method
Final Action
(See also 974.39 for barbital and phenobarbital and 958.14 for
mannitol hexanitrate and phenobarbital.)
A. Method I
(Applicable in absence of stearic acid.)
(Caution; See safety notes on distillation, toxic solvents, and
chloroform.)
Accurately weigh 0.3-0.5 g sample into separator, add 10
mL H 2 0, and shake well. Add 5 mL 0.5N NaOH and shake
again. Acidify to litmus paper with HC1 (1 + 3), added drop-
wise, and add ca 1 mL excess. Ext with successive 40, 30,
20, 20, and 10 mL portions CHC1 3 . Test for complete extn by
shaking with addnl 10 mL sol v. and evapg in sep. beaker.
Combine solv. in second separator and wash with 2 mL H 2
acidified with 1 drop HC1. Filter solv. thru cotton pledget into
small weighed beaker. Evap. on steam bath with aid of air
current, heat 10 min at 80-90°, cool in desiccator, and weigh.
Add 2 or 3 mL anhyd. ether and evap. solv. (Usually 2 treat-
ments with 2 mL each of anhyd. ether are enough to remove
last traces of CHC1 3 and to produce cryst. residue.) Dry at 80-
90°, cool, and weigh. Repeat treatment with anhyd. ether and
evapn to const wt. Det. mp to check purity of residue.
Refs.: JAOAC 8, 47, 510(1925); 25, 799(1942): 26, 101(1943).
B. Method II
(Applicable in presence of stearic acid)
Dissolve residue obtained in 925. 59A in 10 mL alcohol, add
20 mL satd Ba(OH) 2 soln, and stir well. Filter into separator,
and wash residue and filter with two or three 10 mL portions
of the Ba(OH) 2 soln. Acidify filtrate with HC1 (1 + 3) and
proceed as in 925. 59A, beginning "Ext with successive . . ."
Ref.: JAOAC 19, 508(1936).
Barbiturate Drugs
Microscopic Tests
Final Action
See 962.21, Tables 962.21A and B, and 960.57.
955.53* Amobarbital Sodium
and Secobarbital Sodium in Drugs
Final Action
Surplus 1977
See 37.068-37.072, 13th ed.
560
Drugs: Part II
AOAC Official Methods of Analysis (1990)
955.54 Phenobarbital and Aminophylline
in Drugs
Spectrophotometric Method
Final Action
A. Reagents
(a) Dilute ammonium hydroxide soln. — 0.1% NH 3 . Dil. 4
mL NH 4 OH to 1 L with H 2 0.
(b) Phenobarbital std soln. — 10 |jLg/mL. Dissolve 100.0
mg phenobarbital in the dil. NH 4 OH soln in 500 mL vol. flask,
dil. to vol. with the dil. NH 4 OH, and mix. Transfer 5 mL
aliquot to 100 mL vol. flask, dil. to vol. with the dil. NH 4 OH,
and mix.
(c) Theophylline std soln. — 10 |mg/mL. Dissolve 100.0 mg
theophylline in HC1 (1 + 18) in 500 mL vol. flask. Dil. to
vol. with HO (1 + 18) and mix. Transfer 5 mL aliquot to 100
mL vol. flask, dil. to vol. with H 2 0, and mix.
B. Separation of Aminophylline and Phenobarbital
Transfer weighed portion of powd sample contg ca 15 mg
phenobarbital to separator contg 25 mL HC1 (1 + 1). Add 60
mL ether, shake, and let stand to clear. Pass aq. soln succes-
sively thru 2 other separators, each contg 50 mL ether, shake,
and let stand to clear. Transfer ether- washed aq. soln to 500
mL vol. flask. Wash the 3 ether solns successively with three
10 mL portions HC1 (1 + 1) and one 10 mL portion H 2 0, and
add these washes to the vol. flask. Reserve for detn of the-
ophylline.
C. Determination of Phenobarbital
{Caution: See safety notes on distillation, flammable solvents,
and diethyl ether.)
Combine ether solns and evap. to dryness. Dissolve residue
in ca 100 mL of the dil. NH 4 OH and transfer to 200 mL vol.
flask. Dil. to vol. with the dil. NH 4 OH and mix. Filter, if
necessary, transfer 10 mL aliquot to 100 mL vol. flask, dil.
to vol. with the dil. NH 4 OH, and mix. Det. A P at max., ca
240.5 nm, against dil. NH 4 OH. Read this soln same day it is
prepd.
Det. A P of std phenobarbital soln, (b), at same wavelength,
using the dil. NH 4 OH as blank. Calc. a p = A P /cb, where c —
0.01 g/L, andZ? = cell length in cm. Phenobarbital (g/L sam-
ple soln) — A P /a P .
If stearates are present, proceed as above, dissolving residue
in ca 100 mL of the dil. NH 4 OH and dilg to ca 190 mL with
the dil. NH 4 OH. Acidify with HC1, testing with litmus paper.
Dil. to vol. with H 2 0, mix, and filter. Transfer 10 mL aliquot
to 100 mL vol. flask, add 1 drop NH 4 OH (1 + 1), dil. to vol.
with the dil. NH 4 OH, and det. A P at 240.5 nm.
D. Determination of Theophylline
Dil. aq. soln in vol. flask to vol. with H 2 and mix. Trans-
fer aliquot contg 0.5-1.0 mg theophylline to 100 mL vol. flask,
dil. to vol. with H 2 0, and mix. Det. A T at 271 nm against
blank soln contg same vol. HC1. Det. A[ of std theophylline
soln, (c), and calc. a T as in 955. 54C. Theophylline (g/L sam-
ple soln) = A r /a T . Aminophylline, C ]6 H 24 N 10 O 4 .2H 2 O = 1.267
x theophylline.
Ref.: J AOAC 38, 624(1955).
CAS-3 17-34-0 (aminophylline)
CAS-50-06-6 (phenobarbital)
965.43 Phenobarbital and Phenytoin
in Drugs
Spectrophotometric Method
First Action 1965
Final Action 1967
A. Reagents
(a) Water-saturated soln of 15% n-amyl alcohol in CHCl^. —
Sat. 500 mL 15% n-amyl alcohol in CHCJ 3 with 25 mL H 2
and let stand 30 min. Det. suitability of reagent by passing 80
mL thru prepd column followed by 25 mL H 2 0-satd CHCI3,
evapg to dryness on steam bath with air current, dilg to 25.0
mL with alcohol, and reading A in 1 cm cell on recording
spectrophtr from 320 to 250 nm. If (A 258 - A 263 ) <0.190, make
blank correction. Higher A A indicates better grade reagent must
be used.
(b) Acetic acid-chloroform soln. — 1% HO Ac in H 2 0-satd
CHCI3.
B. Preparation of Standards
(a) Phenobarbital std soln. — Weigh and transfer 20 mg
phenobarbital, USP, to 50 mL vol. flask; dissolve and dil. to
vol. with alcohol. Pipet 10 mL into 125 mL g-s erlenmeyer
and evap. to dryness on steam bath with air current (4 mg/
100mL0.WNaOHhasA of ca 1.30 at max., ca 253 nm, in
1 cm cell).
(b) Phenytoin (5 ,5 -Diphenylhydantoin) std soln. — Weigh
and transfer 90 mg diphenylhydantoin (Eastman), to 50 mL
vol. flask; dissolve and dil. to vol. with alcohol. Pipet 10 mL
into 125 mL g-s erlenmeyer and evap. to dryness on steam
bath with air current (18 mg/25 mL alcohol has A of ca 1 .90
at max., ca 258 nm, in 1 cm cell).
C. Preparation of Sample
Accurately weigh powd sample contg ca 90 mg phenytoin
and transfer to 50 mL vol. flask. Add 1.5 mL alcohol, 0.1 mL
HOAc, and ca 25 mL reagent, 965.43A(a). Heat on steam
bath with swirling until CHC1 3 boils. Remove from heat and
swirl 5 min. Heat to boiling as before, remove, and let stand
15 min with frequent agitation. Let cool and dil. to vol. with
reagent (a). (Phenytoin dissolves with difficulty. Turbidity of
soln may persist because of insol. excipients.)
D. Preparation of Column
Use glass tube 25 mm diam. X 15-30 cm long, with stem
plugged with glass wool, and glass tamping rod weighing ca
32 g and having 20-22 mm ram head.
(a) Column packing. — (/) Bottom layer. — Mix 2 g diat.
earth, 960.53B, and 1 mL 12% BaCl 2 soln. (2) Top layer.—
Mix 4 g diat. earth, 960. 53B, and 3 mL satd Na^PQ 4 soln.
(b) Packing technic. — Column must be packed exactly as
follows: Transfer sep. bottom and top packing layers to tube
in 1-2 g portions and tamp 10-15 times with tamping rod after
addn of each portion by dropping rod from 2.5 cm above pack-
ing surface.
Place glass wool pad over diat. earth mixt. and pass 25 mL
reagent (a) thru column at 5-10 mL/min, discarding eluate.
£ Determination
(Caution: See safety notes on distillation, acetic acid, toxic
solvents, and chloroform.)
Place 125 mL g-s erlenmeyer under column and pipet 10
mL sample soln directly over glass wool pad. Let drain into
column, and wash column with three 10 mL portions reagent
(a), letting each drain into column. Add addnl 40 mL reagent
(a). Pass 25 mL H 2 0-satd CHCJ 3 thru column, wash stem with
AOAC Official Methods of Analysis (1990)
Hypnotics and Sedatives
561
CHCI3, and evap. eluate to dryness on steam bath with air
current. (Odor of w-amyl alcohol must be absent.) Residue is
phenytoin.
Place 125 rnL g-s erlenmeyer under column and add to col-
umn 5 mL HOAc in CHC1 3 (1 + 4). Let drain into column.
Add 20 mL reagent, 965.43A(b), let drain into column, and
add 70 mL more. Wash stem with CHC1 3 and evap. eluate to
dryness on steam bath with air current. Residue is phenobar-
bital.
Add 25 mL alcohol by pipet to both sample and std phenyto-
in residues. Stopper and warm with swirling. Let stand, swirl-
ing occasionally, until solid matter is completely dissolved.
Det A of sample and std solns against reagent, 965.43A(a),
on recording spectrophtr from 320 to 250 nm in 1 cm cell.
mg Phenytoin/capsule
= C x (K/W) X (A 258 - A 263 )/(A f 258 - A 263 )
where A 258 and A 263 = max. and min. A of sample soln at ca
258 and 263 nm, resp.; A 258 andA 26 3 = max. and min. A of
std soln at 258 and 263 nm, resp.; C — mg std phenytoin; K
— av. capsule content wt (mg); and W — mg sample. Phenyto-
in x 1.087 — Na phenytoin.
Add 100 mL 0.LV NaOH by pipet to both sample and std
phenobarbital residues. Stopper and shake vigorously 2 min.
Immediately read A of solns on recording spectrophtr from 350
to 230 nm in 1 cm cell.
mg Phenobarbital /capsule = C x (K/W) x (A253M253)
where A 253 and A 253 - A of sample and std solns, resp., at
max., ca 253 nm; C = mg std phenobarbital; K = av, capsule
content wt (mg); W = mg sample.
Ref.: J AOAC 48, 582(1965).
CAS-57-41-0 (diphenylhydantoin)
CAS-50-06-6 (phenobarbital)
949.16 Phenobarbital and Theobromine
in Drugs
Spectrophotometry Method
Final Action
A. Reagents
(a) Theobromine std soln. — 1.00 mg/100 mL. Dissolve 100
mg theobromine in H 2 S0 4 (1 +4), and dil. to 100 mL with
this acid. Transfer 5.0 mL aliquot to 500 mL vol. flask, add
200 mL 5% NaOH soln, and cool to room temp. Dil. to vol.
with H 2 and mix thoroly.
(b) Phenobarbital std soln. — 1.50 mg/100 mL. Dissolve
75.0 mg phenobarbital in CHCI3 and dil. to 100 mL with CHC1 3 .
DiL 10 mL aliquot to 50 mL with CHC1 3 . Transfer 10 mL
aliquot of latter soln to 100 mL vol. flask, dil. to vol. with
CHCI3, and mix.
B. Separation of Theobromine and Phenobarbital
{Caution: See safety notes on distillation, flammable solvents,
and diethyl ether.)
Transfer portion of well mixed sample contg ^15 mg phe-
nobarbital to 125 mL separator, add 15 mL 5% NaOH soln,
and ext with three 30 mL portions CHC1 3 . Wash each CHC1 3
ext with 10 mL 5% NaOH soln in second separator. Discard
CHC1 3 .
Add 30 mL H 2 S0 4 (1 + 4) to alk. mixt. in first separator,
cool thoroly, and shake with 50 mL ether. Transfer aq. layer
contg dissolved theobromine to second separator, cool, and
shake with 40 mL ether. Remove lower phase to third sepa-
rator and wash with another 40 mL portion ether. Repeat extn
thru the 3 separators, using two 40 mL portions H 2 S0 4 (1 +
4) and two 20 mL portions H 2 0. Collect aq. exts in 250 mL
vol. flask, dil. to vol. with H 2 0, and mix. Reserve for theo-
bromine detn.
Filter ether solns thru cotton pledget into beaker, washing
the 3 separators and filter successively with three 5 mL por-
tions ether. Evap. carefully to dryness, and dissolve residue
in CHCI3.
C. Spectrophotometry Determinations
(a) Theobromine.— Pipet aliquot contg 4-8 mg theobro-
mine into 500 mL vol. flask, add 200 mL 5% NaOH soln, and
cool to room temp. Dil. to vol. with H 2 and mix. Det. A at
274 nm of this soln and of std theobromine soln, (a), A', rel-
ative to soln prepd by dilg 10 mL 5% NaOH soln to 25 mL.
Calc. theobromine content of sample.
mg Theobromine in aliquot = 5.0 A /A'
(b) Phenobarbital. — Transfer CH.C.I3 soln to vol. flask and
dil. with CHCI3 to obtain soln contg 20-40 mg phenobarbital/
100 mL. Place 5.0 mL in 100 mL vol.. flask, dil. to vol. with
CHCI3, and mix. Transfer 20 mL aliquot of latter soln to sep-
arator contg 25 mL NH 4 OH (1 + 24). Similarly treat 20 mL
aliquot std phenobarbital soln, (b), and 20 mL portion CHC1 3
as blank. Shake vigorously ^1 min, sep., and discard CHCI3.
Let aq. ext stand 30 min. Det. A at 241 nm of clear aq. solns
of sample, and of std, A', relative to blank, using same cell
for std and sample. Calc. phenobarbital content of sample.
mg Phenobarbital in final aliquot = 0.30A/A'
In presence of salicylates, proceed as in (c):
(c) Phenobarbital in presence of salicylates. — Prep, chro-
matgc column as in 955. 52D, and adjust flow to 2-4 mL/min.
When CHCI3 just stops flowing from tube, pipet 5 mL orig-
inal CHCI3 soln, (b), (equiv. to 1—2 mg phenobarbital) into
tube, and collect eluate in 100 mL vol. flask. As level of CHCl 3
soln reaches top of column, add ca 5 mL CHC1 3 , and repeat
with second CHC1 3 wash. Add enough CHCI3 to keep column
of solv. 2-5 cm high, and collect ca 95 mL eluate. Wash out-
side surface of stem with stream of CHC1 3 and collect wash-
ings in vol. flask. Dil. to vol. with CHC1 3 and mix thoroly.
Det. phenobarbital in eluates as in (b), beginning "Transfer 20
mL aliquot of latter soln ..."
Refs.: JAOAC 34, 566(1951).
CAS-50-06-6 (phenobarbital)
CAS-83-67-0 (theobromine)
972.50 Butabarbitai Sodium in Drugs
Spectrophotometric Method
First Action 1972
Final Action 1973
A. Reagents
(a) Sodium carbonate soln. — \M. Dissolve 10.6 g Na 2 C03
and dil. to 100 mL with H 2 0.
(b) Mixed solvent. — Isooctane-ether (4 + 1). Wash mixed
solv. with equal vol. H 2 0.
(c) Ether. — H 2 0-satd. Use thruout.
(d) Dilute ammonium hydroxide. — Dil. 30 mL NH4OH to
1 L with H 2 0.
(e) Diatomaceous earth. — See 960. 53B.
(f) Dimethylsulf oxide (DMSO). — Spectral grade (Fisher
562
Drugs: Part II
AOAC Official Methods of Analysis (1990)
Scientific Co. D-136; EM Science, OmniSolv, No. MX1456).
(g) Butabarbital std solns. — (/) Stock soln. — 0.1 mg/mL.
Dissolve 20 mg USP Ref. Std in <1 mL MeOH and dil. to
200 mL with dil. NH 4 OH. (2) Working soln.— 0.01 mg/mL,
Dil. 10 mL stock soln to 100 mL with dil. NH 4 OH.
B. Preparation of Sample
Det. av. wt/tablet and pulverize. Transfer accurately weighed
portion contg ca 20 mg butabarbital to 100 mL beaker. Add
1 mL DMSO and 2 drops HO, and swirl to dissolve active
ingredient. Add 1 mL H 2 and 3 g diat. earth, and mix thoroly
until uniform.
C. Preparation of Column
Place small glass wool plug at base of 250 x 25 mm chro-
matgc tube. Transfer uniform mixt. of 4 g diat. earth and 3
mL Na 2 C0 3 soln to tube and tamp to uniform mass. Transfer
sample prepn to column, dry wash beaker with 1 g diat. earth,
transfer wash to column, and tamp.
D. Determination
Pass 75 mL mixed solv. thru column and discard. Elute bu-
tabarbital with 100 mL ether, collecting eluate in 250 mL sep-
arator. Rinse tip of column with ether. Ext eluate with three
50 mL portions dil. NH 4 OH, collect extns in 200 mL vol. flask,
and adjust to vol. with dil. NH 4 OH. Dil. 10.0 mL of this soln
to 100.0 mL with dil. NH 4 OH. Read A of sample and std solns
at max. ca 239 nm against diL NH 4 OH as ref. and calc. Na
butabarbital.
Ref.: JAOAC 55, 152(1972).
CAS- 143-8 1-7 (sodium butabarbital)
972.51 Phenytoin Sodium
in Drug Capsules
Spectrophotometry Method
First Action 1972
Final Action 1973
A. Reagents
(a) Sodium carbonate. — 0.5M. Dissolve 5.3 g Na 2 C0 3 and
dil. to 100 mL.
(b) Mixed solvent. — lsooctane-CHCl 3 (7 + 3). Wash mixed
solv. with equal vol. H 2 0.
(c) Chloroform. — H 2 0-satd. (Caution: See safety notes on
chloroform.)
(d) Acid-alcohol.— U\\. 1 mL HC1 (1 + 99) with 50 mL
alcohol .
(e) Diatomaceous earth. — See 960. 53B.
(f ) Phenytoin std soln. — 0.25 mg/mL. Dissolve 25 mg USP
Ref. Std in 100 mL acid-alcohol.
B. Preparation of Sample
Remove, as completely as possible, contents of capsules and
weigh. Det. av. net contents. Mix, and transfer accurately
weighed portion contg ca 100 mg Na phenytoin to 25 mL vol.
flask. Add 2 mL DMSO, 972.50A(f), and swirl mixt. ca 3
min. Add 4 drops HC1, swirl to mix, and immediately but
cautiously add 0.5M Na 2 C0 3 to vol. Mix, and filter thru rapid
paper.
C. Preparation of Column
Place small glass wool plug at base of 250 x 25 mm chro-
matgc tube. Mix 3 g diat. earth and 2 mL 0.5M Na 2 C0 3 , and
transfer to tube; tamp. Mix 3 g diat. earth and 2.0 mL prepd
sample soln, and transfer to column. Dry- wash beaker with ca
1 g diat. earth, and transfer wash to column. Place pad of glass
wool on top of column.
D. Determination
Pass 75 mL mixed solv. thru column, and discard. Elute
phenytoin with 75 mL CHCJ 3 , collecting eluate in 125 mL
g-s erlenmeyer. Rinse tip of column with CHC1 3 . Evap. solv.
to dryness on steam bath under air current. Dissolve residue
in 25.0 mL acid-alcohol. Det. A of sample and std solns at
max., ca 258 nm, against acid-alcohol as ref. Calc. phenytoin
Na content. Phenytoin x 1.087 = phenytoin Na.
Ref.: JAOAC 55, 170(1972).
CAS-57-41-0 (phenytoin)
968.44 Chloral Hydrate in Drugs
Spectrophotometric Method
First Action 1968
Final Action 1969
A. Principle
Quinaldine ethyl iodide reacts with chloral hydrate to pro-
duce stable blue cyanine dye with A max. at ca 605 nm. Other
poly chlorinated compds do not interfere.
B. Reagents
(a) Quinaldine ethyl iodide soln. — 1.5%. Dissolve 1.5 g
quinaldine ethyl iodide in H 2 and dil. to 100 mL. Filter if
necessary.
(b) 2-Aminoethanol soln. — 0.17V. Dissolve 6.1 g 2-ami-
noethanol in H 2 and dil. to 1 L.
(c) Chloral hydrate std soln.— 100 (xg/mL. Dissolve 0.2500
g chloral hydrate USP in H 2 and dil. to 250 mL. Dil. 10 mL
aliquot to 100 mL with H 2 0.
C. Apparatus
Recording spectrophotometer .
matched 1 cm cells.
-400-800 nm range with
D. Preparation of Sample
(a) Capsules. — Place counted number of capsules contg ca
2.5 g chloral hydrate in g-s 250 mL flask, add 25 mL H 2 0,
stopper, and heat on steam bath with frequent swirling until
dissolved. Cool, and transfer quant, to 250 mL vol. flask with
H 2 0. DiL to vol., mix, and dil. stepwise to ca 100 u^g/mL
with H 2 0.
(b) Solns. — Prep, soln contg ca 100 fxg chloral hydrate/mL
by stepwise diln with H 2 0.
E. Determination
Pipet 10 mL sample soln contg ca I mg chloral hydrate into
100 mL vol. flask and pipet 10 mL std chloral hydrate soln
into second 100 mL vol. flask. Pipet 10 mL H 2 into third
100 mL vol. flask as blank. To each flask add 10 mL quin-
aldine ethyl iodide soln and 60 mL isopropanol, and mix. Add
5 mL 0.1N 2-aminoethanol and dil. to vol. with H 2 0. Place
in H 2 bath 1 hr at 60°. Cool, and record spectra of sample
and std from 400 to 800 nm against blank. Do not exceed 120
nm/min near max. Det A max. at ca 605 nm, using baseline
technic with ca 430 and ca 770 nm as base.
mg Chloral hydrate in sample aliquot
= (net A of sample soln/net A of std soln)
x mg chloral hydrate in 10 mL std soln
Ref.: JAOAC 51, 626(1968).
CAS-302-17-0 (chloral hydrate)
AOAC Official Methods of Analysis (1990)
Hypnotics and Sedatives 563
944.14a (2-lsopropyl-4-Pentenoyl) Urea
in Drugs
Chloroform Extraction
Final Action
Surplus 1965
See 32.237, 10th ed.
932.22* Suifonmethane
or Sulfonethylmethane in Drugs
Ether Extraction
Final Action
Surplus 1965
See 32.238, 10th ed.
970.81 Ethchlorvynol in Drugs
Gas Chromatographic Method
First Action 1970
Final Action 1972
A. Reagents
(a) Ethchlorvynol. — (Caution: See safety notes on distil-
lation and vacuum.) Purify by vac. distn (62° at ca 10 mm) or
assay by titrn as follows: Transfer ca 110 mg ethchlorvynol,
accurately weighed, to 250 mL erlenmeyer contg 50 mL 2.5%
AgN0 3 soln in 70% alcohol. Immediately titr. with 0.057V
NaOH, using 8-10 drops Me red-methylene blue, (e). Perform
blank detn and make any necessary correction. 1 mL 0.05N
NaOH = 7.230 mg ethchlorvynol. (Caution: Protect pure eth-
chlorvynol from excessive exposure to light and air.) Store at
<10° in glass containers with polyethylene or Teflon stopper
liners.
(b) Ethchlorvynol stock soln. — 10 mg/mL. Accurately weigh
ca 0.5 g ethchlorvynol and dissolve in 5 mL alcohol. Transfer
quant, to 50 mL vol. flask with 10 mL alcohol. Dil. to vol.
with H 2 0.
(c) Internal std soln. — 2.0%. Dissolve 2.0 g 1,3-dichloro-
2-propanol in 10 mL alcohol and dil. to 100 mL with H 2 0.
(d) Dichlorodimethylsilane soln. — Dissolve 5 mL dichlo-
rodimethylsilane in 100 mL toluene. (Caution: Dichlorodi-
methylsilane causes severe burns. Vapor is harmful. Avoid
contact with skin, eyes, or clothing. Use effective fume re-
moval device.)
(e) Methyl red-methylene blue mixed indicator. — Dissolve
0.3 g Me red in 60 mL alcohol and dil. to 100 mL with H 2 0.
Dissolve 0.2 g methylene blue in 100 mL 50% alcohol and
add to Me red soln.
B. Apparatus
Gas chromato graph. — With 1.2 m (4') x 4 mm glass col-
umn, packed with Carbowax 20M on 100-120 mesh Gas-Chrom
Q. and H flame ionization detector. Operating conditions: temps
(°) — column 115, detector 190, injection port 200; flow rates
(mL/min) — N 50, H 92, air ca 500. Adjust column temp, to
elute ethchlorvynol in 12-15 min (relative retention time of
internal std is ca 0.8.). Adjust H and air flow rates to give
stable flame and good sensitivity. Adjust electrometer sensi-
tivity so that 12 |ULg ethchlorvynol gives 50-70% deflection.
C. Preparation of GC Column
Carefully wash inside of column and small amt of fine glass
wool with dichlorodimethylsilane soln, rinse with alcohol, and
dry thoroly. Dissolve 5.0 g Carbowax 20M in 100 mL CHCI 3 .
Add Carbowax soln to 10.0 g 100-120 mesh Gas-Chrom Q
in 250 mL filter flask fitted with trap and stopper. Slowly ap-
ply vac. and maintain 5 min. Swirl slurry rapidly and transfer
in small portions to buchner fitted with 9 cm Whatman No. 4
paper. Maintain vac. 5 min after last portion is added; then air
dry coated support 1 hr by spreading on smooth surface. Oven
dry addnl hr at 100°.
Carefully plug column exit with small pad of glass wool.
Apply vac. to exit end and slowly add coated support thru
inlet, tapping very gently to pack firmly. Pack to within 1 cm
of area heated by injection port. Plug with glass wool and con-
dition overnight at 220° with slow N stream.
D. Preparation of Sampie
(a) Capsules (200 — 500 mg). — Place counted number of
capsules contg ca 2.5 g ethchlorvynol in 250 mL vol. flask;
add 75 mL H 2 and 30 mL alcohol, stopper, and heat on steam
bath with frequent swirling until dissolved. Cool and dil. to
vol. with H 2 0.
(b) Capsules (100 mg). — Place 10 capsules in 100 mL vol.
flask, add 50 mL H 2 and 15 mL alcohol, stopper, and heat
on steam bath with frequent swirling until dissolved. Cool and
dil. to vol. with H 2 0.
(c) Solutions. — Prep; soln contg ca 10 mg ethchlorvynol/
mL by stepwise diln with 20% alcohol.
E. Determination
Pipet 10 mL sample soln contg ca 100 mg ethchlorvynol
into 50 mL vol. flask; pipet 10 mL ethchlorvynol stock soln
into second 50 mL vol. flask. Pipet 10 mL internal std soln
into each flask and dil. to vol. with H 2 0.
Rinse 10 pL syringe with 50% alcohol and draw up 1 jjlL
50% alcohol. Draw in 1 |mL air followed by 6 jjlL sample.
Draw in 1 (mL air and note sample vol. Insert needle thru sep-
tum of gas chromatograph, quickly depress plunger, and re-
tract syringe needle. Inject 6 \xL of each soln. Run std before
and after sample. Calc. amt of ethchlorvynol in 10 mL sample
aliquot as follows:
C = C x (X/X r ) x (/'//)
where C and C = mg ethchlorvynol in 10 mL sample aliquot
and std stock soln, resp.; X = area ethchlorvynol peak in sam-
ple cljromatogram; X' = av. area ethchlorvynol peak in std
chromatograms; / = area internal std peak in sample chro-
matogram; and /' = av. area internal std peak in std chro-
matograms.
Ref.: JAOAC 53, 834(1970).
CAS- 113-18-8 (ethchlorvynol)
972.52 Paraldehyde in Drugs
Gas Chromatographic Method
First Action 1972
Final Action 1974
A. Reagents
(a) Paraldehyde std. — USP. Redistil twice and collect only
fraction distg 120.5-123°. Store in amber g-s bottle. (Caution:
Paraldehyde is toxic. Use effective fume removal device.)
(b) Paraldehyde std solns. — (/) Stock soln. — 0.05 mL/mL.
Pipet 5 mL std paraldehyde into 100 mL vol. flask, add 8.0
mL internal std, and dil. to vol. with acetone. (2) Working std
soln. — 0.005 mL/mL. Pipet 5 mL stock soln into 50 mL vol.
flask and dil. to vol. with acetone.
(c) I soamyl alcohol. — (EM Science, No. AX J. 440). Check
purity by injecting 5 u>L into gas chromatograph. If any in-
564
Drugs: Part II
AOAC Official Methods of Analysis (1990)
terfering peaks are present, redistil. (Caution: See safety notes
on distillation.)
(d) Internal std. — Dil. isoamyl alcohol, (c), with equal vol.
acetone.
B. Apparatus
(a) Gas chromatographic column. — 2% cyclohexane di-
methanol succinate (HIEFF-8BP) plus 20% Carbowax 20M on
80-100 mesh Diatoport S (Hewlett-Packard Co., Avondale,
PA 19311) or Chromosorb W (HP). Prep, as follows; Weigh
500 mg HIEFF-8BP plus 5.0 g Carbowax 20M into 500 mJL
Morton flask. Add 200 mL CHC1 3 and, if necessary, heat on
steam bath with swirling to dissolve liq. phase. Add 20 g 80-
100 mesh Diatoport S (or Chromosorb W (HP)) and evap. CHC1 3
under reduced pressure on rotating evaporator. Remove last
traces CHC1 3 in 100° oven. Pack 1.8 m (6') x 4 mm id glass
column and condition column 16 hr at 190° with N flow of 60
mL/min before use.
(b) Gas chromato graph. — Packard Model 7800 (replaced
by 5700) with flame ionization detector and Hewlett-Packard
Model 3370A (replaced by 3380) electronic integrator, orequivs.
GC conditions: temps (°) — column 110, injection port 125,
detector 125; flow rates (mL/min) — N carrier gas 60, air 600,
H 60; sensitivity 1 X 10™ 8 amp; and chart speed 26"/ nr -
C. Preparation of Sample
Pipet amt elixir contg 2.5 mL paraldehyde into 50 mL vol.
flask. Add 4.0 mL internal std and dil. to vol. with acetone.
Pipet 5 mL this soln into 50 mL vol. flask and dil. to vol.
with acetone.
D. Determination
Inject ca 4 (xL std and sample solns into gas chromatograph
operated as in 972.52B(b). Make >:3 injections of sample and
std solns and take av.
Calc. amt paraldehyde in sample as follows:
% Paraldehyde = (RjRd x C x DF
where /? x = ratio of sample to internal std peak area in sample
soln, R s = ratio of std to internal std peak area in std soln, C
= concn of std (%, v/v), and DF = sample diln factor.
Since GC peaks for paraldehyde are narrow, symmetrical,
and well defined, peak hts may be used in place of peak area.
Ref.: J AOAC 55, 166(1972).
CAS-123-63-7 (paraldehyde)
968.45 Phenaglycodol in Drugs
Infrared Spectroscopic Method
First Action 1968
Final Action 1970
A. Apparatus
Recording infrared spectrophotometer. — With two 1.0 mm
liq. cells with NaCl windows (preferably matched) and KBr
disk holder.
B. Reagents
(a) Phenaglycodol std.
(b) Carbon disulfide. — Spectral grade.
(c) Cotton. — Wash thoroly with CHC1 3 and dry.
C. Determination
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Det. av. wt/tablet or capsule, pulverize, and sieve to obtain
uniform sample. Accurately weigh sample contg ca 200 mg
phenaglycodol and transfer to 125 mL separator with 50 mL
CHC1 3 . Add 15 mL 0.5N NaOH, shake 1 min, and filter sepd
CHCI3 layer thru cotton into 150 mL beaker. Ext alk. soln with
two addn.1 25 mL portions CHC1 3 , filtering each sepd CHC1 3
layer into beaker. Evap. combined exts just to dryness, using
gentle current of air, at temp. <50°. Dissolve residue in CS 2 ,
transfer quant, to 50 mL vol. flask, and dil. to vol. with CS 2 .
Accurately weigh ca 200 mg std phenaglycodol, transfer to
125 mL separator with 50 mL CHC1 3 , and ext as above.
Det. baseline A of sample and std solns against CS 2 at ca
9.85 |xm. Draw baseline between minima at ca 9.75 jxm and
ca 10.0 |xm. Calc. phenaglycodol content of sample.
Prep. KBr disk by grinding 2 mg residue and 200 mg spec-
troscopic grade KBr in Mullite mortar and press in die with
hydraulic press. Record spectrum at 2-15 jxm and compare
with spectrum of extd std residue to det. identity of sample.
Ref.: JAOAC 51, 631(1968).
CAS-79-93-6 (phenaglycodol)
968.46 Meprobamate in Drugs
Infrared Spectroscopic Method
First Action 1968
Final Action 1970
A. Apparatus
(a) Spectrophotometer. — Recording IR spectrophtr, effec-
tive over 0.75-3.5 \xm range, with 1 cm matching near-IR
silica cells. Peak at ca 2.91 jxm for meprobamate must be re-
solved.
(b) Chromatographic tubes. — Glass, 20 X 300 mm.
B. Reagents
(a) Alcohol-free chloroform. — Thruout detn use only CHC1 3
prepd daily as follows: Ext alcohol by passing CHC1 3 succes-
sively thru three 500 mL separators, each contg 50-75 mL
H 2 0. Pack 2 chromatgc tubes half-full with alumina (80-200
mesh, Fisher No. A -540, or equiv.) activated by heating 2 hr
at 300°. Mount one column above other and pass CHC1 3 thru
both columns. Pass <500 mL CHC1 3 at one time. If more C.HO3
is needed, repeat purification with fresh alumina.
(b) Meprobamate std soln. — 0.5 mg/mL. Accurately weigh
ca 25 mg USP Meprobamate Ref. Std and transfer to 50 mL
vol. flask. Dissolve in and dil. to vol. with CHCl 3 . Absorp-
tivity should be ca 1.0 if 2.91 jxm peak is properly resolved.
C. Preparation of Sample
Finely pulverize tablets, accurately weigh portion contg ca
50 mg meprobamate, and transfer to dry 100 mL vol. flask.
Add 50 mL CHC1 3 , shake 15-20 min, and dil. to vol. with
CHCI3. Filter soln thru dry Whatman No. 1 paper, or equiv.
Discard first 20-25 mL and collect remainder in dry g-s er-
lenmeyer.
D. Determination
Zero instrument at 2.914 |xm with CHC1 3 in both cells. Scan
sample and std solns against CHCJ 3 between 3.000 and 2.790
ixm. Measure baseline A values at max., ca 2.91 |xm, from
straight line drawn between minima at ca 2.980 and 2.875.
mg Meprobamate /tablet = A sampte x(C/A & J x 100 x (T/W)
where A refers to baseline values; C = mg meprobamate/mL
std soln; T and W = av. wt/tablet and sample wt, resp., in
mg.
AOAC Official Methods of Analysis (1990)
Anticoagulants 565
Ref.: JAOAC 51, 616(1968).
CAS-57-53-4 (meprobamate)
ANTICOAGULANTS
973.72 Acenocoumarol, Dicumarol,
Phenprocoumon, Warfarin Potassium,
and Warfarin Sodium in Drugs
Automated Spectrophotometry Method
First Action 1973
Final Action 1975
A. Principle
Basic soln of drug is acidified and extd with CHC1 3 or CHC1 3 -
pyrimidine-propylene glycol (for dicumarol), and A of extd
material is read in flowceM at 308 nm.
B. Apparatus
(a) Automatic analyzer. — AutoAnalyzer with following
modules (Technicon Instruments Corp.): Sampler II with 20/
hr (2:1) cam; proportioning pump I; manifold (Fig. 973.72).
(b) Filter. — Fill 50 X 5 mm id glass tubing completely, but
loosely, with glass wool.
(c) Spectrophotometer. — Double-beam spectrophtr which
records A at fixed wavelength, with 10 mm flowcell (Thomas
Scientific) (2 mm flowcell for dicumarol).
(d) Ultrasonic generator. — Model II, 150 watt (Heat Sys-
tems-Ultrasonic, Inc., 38 E Mall, Plainview, NY 11803).
C. Reagents
(a) Chloroform. — H 2 0- washed and filtered thru paper. Prep,
fresh daily. (Use in all except dicoumarol detn.)
(b) Chloroform-pyridine-propylene glycol soln. — Mix 50
mL pyridine with 50 mL propylene glycol and diL to 1 L with
CHC1 3 . Use in dicumarol assay.
(c) Acenocoumarol std soln. — 0.08 mg/mL. Accurately
weigh ca 20 mg acenocoumarol std in 250 mL vol. flask. Dis-
solve in ca 100 mL 0.0 1W NaOH with aid of ultrasonic gen-
erator and dil. to vol. with O.OW NaOH.
(d) Dicumarol (bishydroxycoumarin) std soln. — 0.25 mg/
mL. Accurately weigh ca 25 mg USP Ref. Std Bishydroxy-
coumarin in 100 mL vol. flask. Dissolve in ca 50 mL 0.01 N
NaOH with aid of ultrasonic generator and dil. to vol. with
same sol v. Prep, fresh daily.
(e) Phenprocoumon std soln. — 0.12 mg/mL. Accurately
weigh ca 30 mg USP Ref. Std Phenprocoumon in 250 mL vol.
flask. Dissolve in ca 100 mL 0.01 AT NaOH. with aid of ultra-
sonic generator and dil. to vol. with 0.017V NaOH.
(f) Warfarin std soln. — 0.1 mg/mL. Accurately weigh ca
25 mg USP Ref. Std Warfarin into 250 mL vol. flask, and
dissolve and dil. to vol. with 0.0 1A/ NaOH.
D. Preparation of Sample
Disintegrate individual tablet or capsule or disperse weighed
composite in accurately measured vol. 0.017V NaOH to give
drug concn (mg/mL) as follows: acenocoumarol 0.08, dicu-
marol 0.25, phenprocoumon 0. 12, Na or K warfarin 0. 10. Use
ultrasonic generator ca 10 min to assure tablet disintegration.
Let suspension stand 1.5 hr with occasional mixing.
E. Analytical System
Sample is withdrawn, segmented with air, and acidified with
0.2N H2SO4. Solv., (a) or (b), is added, mixed in beaded coil,
and phases are sepd in BO fitting. Org. phase contg extd drug
is debubbled, and A of soln at 308 nm is measured in 10 mm
flowcell (2 mm flowcell for dicumarol).
F. Start-Up
Pump alcohol thru solv. line 10 min; then pump solv. thru
line 5 min. Place remaining tubes in their resp. solns and let
system equilibrate 20-30 min. Calibrate spectrophtr at 2 or 3
Afl Transmission Tubing
0.065" A
Coil, Delay (Settling}
5.5 Turns 2.4mm id
To Weir Box -*-
Waste
UV Recorder
Spectrophotometer
| Upper lo q o) SAMPLER 2
■ Level l - nl
Lower" ^<po
Level S Tube Si
s?\ ml/min
—(2) I 0.01A/NaOh
^ - 0.42T
0.2/VI-hSO,
RATE (per hour) 20 2:1
Organic Solvent
T ~ Tygon. S = Solvaflex.A = Actdftex
FIG. 973.72— Flow diagram for automated analysis for acenocoumarol, dicumarol, phenprocoumon, potassium warfarin, and
sodium warfarin
566
Drugs: Part II
AOAC Official Methods of Analysis (1990)
absorbances. Adjust mask in ref. compartment of spectrophtr
to set desired baseline.
G. Shut-Down
Place acid, base, and sampling lines in H 2 0, leave solv. line
in its reservoir, and pump 5 min. Remove acid, base, and sam-
pling lines from H 2 and continue pumping 5 min to purge
system of H 2 0. Place solv. line in alcohol and pump 5 min.
Remove line and pump system dry.
H. Determination
Fill sample cups in following order: 4 cups std soln, 5 cups
sample soln, 1 cup std soln, 5 cups sample soln, etc. Place 2
cups std soln at end of each run. (Extra cups of std solns at
start and end of sampling pattern are used to overcome carry-
over effect in transitions from wash soln to std soln and vice
versa. Three extra cups at beginning and 1 extra cup at end
should suffice, but det. exact number needed for equilibrium
by experiment. System should give uniform response for at
least final pair of extra std cups before sample pattern is started.)
Start Sampler II. After last cup has been sampled, let system
operate until steady baseline is obtained. Draw tangent to ini-
tial and final baselines. Subtract baseline to det. net A and A'
for each sample and std peak, resp. Discard values for first 3
and last std peaks and calc. av. std A' .
mg warfarin Na (or K) in portion taken =
1.071 (or 1.124) x {A/ A') X C x D
where 1 .071 and 1,124 = ratios of MW of Na and K warfarin
to warfarin, resp.; C — concn of std in mg/mL, and D — diln
factor.
mg Acenocoumarin, dicumarol, or phenprocoumon in
portion taken = (A /A') x C x D.
Refs.: JAOAC 56, 692(1973); 58, 80(1975).
CAS- 152-72-7 (acenocoumarol)
CAS-66-76-2 (dicumarol)
CAS-435-97-2 (phenprocoumon)
CAS-2610-86-8 (potassium warfarin)
CAS- 129-06-6 (sodium warfarin)
CAS-81-81-2 (warfarin)
988.24 Dicumarol, Phenprocoumon,
and Warfarin Sodium
in Drug Tablets
Liquid Chromatographic Method
First Action 1988
A. Principle
Coumarin anticoagulants dicumarol, phenprocoumon, and
warfarin Na are identified and quant, detd in tablets by reverse
phase LC on C 18 column with tetrahydrofuran-MeOH-H 2 0-
HOAc mobile phase, and photometric detection at 311 nm.
B. Apparatus
(a) Liquid chromatograph. — Equipped with DuPont Model
8800 solv. pump, variable wavelength detector, and strip chart
recorder (E.I. DuPont de Nemours & Co.), or equiv.; and
Rheodyne Model 7125 injection valve with 20 |xL sample loop
(Rheodyne Inc.), or equiv. Operating conditions: column temp,
ambient; solv. flow rate 1.5 mL/min; detector wavelength 311
nm; detector attenuation 16 AUFS; recorder range 1 mV; chart
speed 0.5 cm/min.
(b) Chromatographic column. — Stainless steel, 30 cm X
3.9 mm id, packed with 10 fxm imBondapak Ci 8 (Waters As-
sociates, Inc.), or equiv.
C. Reagents
(a) Solvents. — LC grade MeOH and reagent grade glacial
HOAc (Fisher Scientific Co.); tetrahydrofuran (Mallinckrodt,
Inc.); and H 2 double distd in glass.
(b) Mobile phase.— Tetrahydrofuran-MeOH-H 2 0-HOAc
(35 + 10 +65 + 0.1 v/v/v/v). Filter thru 0.45 |xm mem-
brane and degas under vac.
(c) Dicumarol std soln. — 0.05 mg/mL. Accurately weigh
ca 25 mg USP Ref. Std Dicumarol into 100 mL vol. flask,
dissolve in and dil. to vol. with 0.0 IN NaOH, and mix. Pipet
5 mL of this soln into 25 mL vol. flask, dil. to vol. with mo-
bile phase, and mix.
(d) Warfarin Na std soln. — 0.2 mg/mL. Accurately weigh
ca 10 mg USP Ref. Std Warfarin Na into 50 mL vol. flask,
and dissolve in mobile phase. Dil. to vol. with mobile phase,
and mix.
(e) Phenprocoumon std soln. — 0.12 mg/mL. Accurately
weigh ca 3 mg USP Ref. Std Phenprocoumon into 25 mL vol.
flask, and dissolve in mobile phase. Dil. to vol. with mobile
phase, and mix.
D. Preparation of Sample
(a) Tablet composites. — Weigh and finely powder >20 tab-
lets. Transfer accurately weighed amt of powder to suitable
vol. flask and quant, dissolve in mobile phase with aid of ul-
trasonic bath. Dil. to vol. with mobile phase to prep, soln contg
ca 0. 1 2 mg/mL of phenprocoumon or 0.2 mg/mL of warfarin
Na. For dicumarol samples, first dissolve powder in 0.01N
NaOH with aid of ultrasonic bath to obtain soln contg 0.25
mg/mL; then quant, dil. 5.0 mL aliquot of soln with mobile
phase to final dicumarol concn of ca 0.05 mg/mL. Filter all
sample prepns prior to injection into LC system.
(b) Single tablets. — Place 1 powdered tablet in suitable vol.
flask, and proceed as described for tablet composites.
B. Determination
Equilibrate system with mobile phase at 1.5 mL/min until
baseline is steady. Use sampling valve to inject measured vol.
of std soln into LC system. Adjust injection vol. and operating
parameters so std soln gives peak ht ca 60% full scale. Under
these conditions, 3 replicate injections of a std soln should give
RSD <3% and tailing factor <2.0. Make alternate injections
of equal vols of std and sample solns. Measure peak responses
in sample and std solns.
F. Calculations
Calc. amt coumarin anticoagulant in sample as follows:
Tablet composite sample:
mg/tablet = (H/H') x (W /D') x (D/W) x A
Single tablet sample:
mg/tablet - {H/H') x (W /D') X D
where H and H' = peak responses of sample and std solns,
resp.; W and W = mg sample and std taken, resp.; D and D'
— diln factors for sample and std solns, resp.; and A - av.
tablet wt, mg. To calc. amt warfarin Na in either tablet com-
posites or individual tablets, use 1.071 as multiplier in above
equations (1.071 = ratio of MW of warfarin Na/MW of war-
farin) .
Ref.: JAOAC 70, 834(1987).
CAS-66-76-2 (dicumarol)
CAS-435-97-2 (phenprocoumon)
CAS-81-81-2 (warfarin)
CAS- 129-06-6 (warfarin Na)
AOAC Official Methods of Analysis (1990)
Sulfonamides
567
971.38 Menadione Sodium Bisulfite
in Drugs
Spectrophotometric Method
First Action 1971
Final Action 1973
(Applicable to injections)
A. Principle
Aq. menadione. NaHS0 3 soln is mixed with diat. earth and
placed in chromatgc column over lower layer of HCl-diat. earth.
Excipients are eluted with CHC1 3 and menadione with NH 3 -
CHC1 3 . Excess NH 3 which could decompose menadione is
neutzd by acidic lower layer. Menadione is detd by UV spec-
trophotometry.
B. Reagents
(a) Chloroform. — Use H 2 0- washed CHCl 3 thruout.
(b) Ammoniacal chloroform. — Mix 1 part NH 4 OH with 25
parts CHCI3 as needed.
(c) Menadione std soln. — 50 u>g/mL. Dissolve 50 mg USP
Menadione Ref. Std in CHC1 3 and dil. to 100 mL with CHC1 3 .
DiL 10 mL aliquot to 100 mL with CHC1 3 .
C. Determination
Mix 1 mL HC1 (1+3) with 1.5 g diat. earth, 960.53B, and
pack into chromatgc tube, 967. 31 A. Dil. aq. sample soln to
contain ca 5 mg menadione. NaHS0 3 ,3H 2 0/mL, mix 2.0 mL
this soln with 3 g diat. earth in beaker, and pack into tube.
Dry-wash beaker with 1 g diat. earth, add to tube, tamp until
compressed, and overlay with piece of glass wool used to wipe
beaker.
Wash column with 100 mL CHC1 3 and discard CHC1 3 . Wash
tip of column with few mL CHC1 3 . With 100 mL vol. flask
as receiver, add 5 mL NH 4 OH-CHCl 3 , let sink into diat. earth,
and elute menadione with 90 mL CHC1 3 . Rinse tip of column
with CHCI3 and dil. to vol. with CHC1 3 . Scan soln and std
soln from 280 to 400 nm against CHC1 3 and det. A at max.,
ca 334 nm.
mg Menadione. NaHS03.3H 2 0/mL dild assay soln
= 0.05 x 1.918 x C x (A/A f )
where 1.918 is factor to convert menadione to menadi-
one. NaHS0 3 .3H 2 0; C = jxg menadione/mL std soln; and A
and A' refer to sample and std solns, resp.
Ref.: JAOAC54, 593(1971).
CAS- 130-37-0 (menadione sodium bisulfite)
C. Reagents
(a) Mobile phase. — 52/48 ratio of aq./org. phases: (1)
Aqueous. — Acetic acid~H 2 (1 + 99). (2) Organic. — LC grade
CH 3 CN.
(b) Chlorpropamide std soln. — Transfer ca 50 mg, accu-
rately weighed, USP Chlorpropamide Ref. Std to 100 mL vol.
flask and dissolve in mobile phase. Dil. quant, to final concn
of ca 0.05 mg/mL in mobile phase.
(c) Resolution soln. —Chlorpropamide + />chlorobenzene-
sulfonamide (PCBS) (ca 0.05 mg/mL of each) in mobile phase.
D. Preparation of Sample
Transfer accurately weighed portion of finely ground tablets
equiv. to 45-55 mg chlorpropamide to 100 mL vol. flask. Add
ca 70-80 mL mobile phase and shake thoroly 6-8 min (or
sonicate 3-4 min) and dil. to vol. with mobile phase. Dil.
quant, to final concn ca 0.05 mg/mL in mobile phase. Filter
portion thru 0.45 (xm filter for LC analysis.
E. System Suitability
Set mobile phase at flow rate ca 1.5 mL/min. Retention
time for chlorpropamide should not be <4.0 min. Adjust flow
rate and /or solv. ratio (do not exceed 50% CH 3 CN) for desired
retention time. Column should conform to following perform-
ance parameters: theoretical plates (n) not <1500; tailing fac-
tor (T) not >L5; resolution (/?) between chlorpropamide and
PCBS not <2.0. Relative std deviation for 4 consecutive std
injections should be <2.0%.
F. Determination
Make 20 u,L injections of std and samples. Det. peak re-
sponses (area or ht) obtained and calc. amt of chlorpropamide:
Chlorpropamide, mg/tab. = (r/r r ) x (C/W) xDFx ATW
where r and r' - responses for sample and std, resp.; C =
concn of chlorpropamide std soln, mg/mL; W = sample wt,
g; DF — diln factor for sample, mL; ATW = av. tablet wt,
g/tab.
Ref.: JAOAC 69, 519(1986).
CAS-94-20-2 (chlorpropamide)
SULFONAMIDES
964.26* Sulfonamide Mixtures in Drugs
Paper Chromatographic Method
First Action 1964
Surplus 1980
986.37 Chlorpropamide in Drug Tablets
Liquid Chromatographic Method
First Action 1986
A. Principle
Chlorpropamide is dissolved in mobile phase and detd by
liq. chromatgy with UV detection at 240 nm.
B. Apparatus
(a) Liquid chromato graph. — Equipped with sampling valve
capable of introducing 20 pJL injections, UV detector capable
of operating at 240 nm, and recorder/integrator.
(b) Column. — Zorbax ODS, 5-6 |xm diam. spherical par-
ticles, 4.6 mm x 25 cm (E.I. Dupont, or equiv.).
(c) Filters. — Millipore type HVLP, 0.45 p,m porosity (Mil-
lipore Corp.), or equiv.
See 37.152-37.156, 13th ed.
973.73 Trisulfapyrimidines in Drugs
Spectrophotometric Method
First Action 1973
Final Action 1974
Total Trisulfapyrimidines
A. Principle
Total trisulfapyrimidines in sample are detd by coupling with
AM-naphthyl ethylenediamine.2HCl (NED), recording spectra
of samples and stds between 660 and 480 nm. Individual sul-
fonamides are sepd by TLC and their ratios detd spectrophtric
after coupling with NED.
568
Drugs: Part II
AOAC Official Methods of Analysis (1 990)
B. Reagents
(a) Ammonia-rnethanol soln. — Dil. 5 mL NH 4 OH to 100
mL with MeOH.
(b) Sulfamerazine std soln. — Approx. 6 (xg/mL acid soln.
Accurately weigh calcd amt USP Ref. Std Sulfamerazine, pre-
viously dried, and dissolve in NH 4 OH-MeOH soln; dil. quant.
and stepwise with MeOH to obtain soln contg ca 120 fxg/mL.
Transfer 5.0 mL to 100 mL vol. flask and dil. to vol. with
0.12N HC1 (10 mL HC1 dild to 1 L). Acidic soln is stable >1
month.
(c) Dilute ammonia soln. — Dil. 400 mL NH 4 OH to 1 L with
H 2 0.
(d) N~l-Naphthyl ethylenediamine dihydrochloride (NED)
soln. — 0.1%. Prep, fresh before use.
C. Preparation of Sample
(a) Tablets. — Accurately weigh finely powd portion contg
ca 180 mg total sulfonamides and transfer to 50 mL vol. flask,
using 10 mL dil. NH 4 OH, (c). Let stand ca 15 min, mixing
occasionally, dil. to vol. with MeOH, and centrf. portion to
clarify (Soln I). Dil. 5.0 mL clarified soln to 250 mL with
H 2 0; dil. 4.0 mL of this soln to 50 mL with H 2 (Soln II).
(b) Suspensions.— Shake in original container to ensure ho-
mogeneity, let stand long enough for entrapped air to rise, and
invert carefully just before removing portion for weighing. Det.
sp gr by weighing 100 mL in tared 100 mL vol. flask. Thoroly
mix and weigh portion contg 180 mg total sulfonamides and
proceed as in (a).
D. Determination
Pipet 5.0 mL aliquots sulfamerazine std soln and prepd Soln
II into sep. 10 mL vol. flasks. Add 1.0 mL HC1 (1 + 1) to
each flask, mix, and cool. (Solns must be at room temp, for
quant, results.) Add 1.0 mL 0.1% NoN0 2 , mix well, and let
stand 2 min. Add 1.0 mL 0.5% NH 4 sulfamate, and mix. After
2 min, add 1.0 mL NED soln, (d). Mix and adjust to vol. with
H 2 0. Record spectra of samples and stds against H 2 between
660 and 480 nm (peak ca 545 nm) within 15-60 min. Correct
A by subtracting A at 660 nm from peak A at ca 545 nm.
mg Total sulfapyrimidines in sample = (A /A') X 31.25C
where A and A' = corrected A of dild assay soln and sulfa-
merazine std soln, resp., and C — |xg sulfamerazine/mL std
soln.
Ratio of Sulfadiazine Sulfamerazine .Sulfamethazine
E. Reagents and Apparatus
(a) Chromatographic identification standards. — Prep. sep.
solns of USP Ref. Std Sulfadiazine, Sulfamerazine, and Sul-
famethazine in NH 4 OH-MeOH soln, 973.73B(a), to contain
ca 1 mg/mL each.
(b) Developing solvent.— CHCl v MeOH-NH 4 OH (30 + 12
+ 1).
(c) Thin layer plate. — 20 x 20 cm, coated 0.25 mm thick
with silica gel GF (Brinkmann Instruments, Inc.). Divide into
2 approx. equal parts by scraping thin vertical line thru coat-
ing.
F. Thin Layer Chromatography
Line suitable chromatgc tank with blotting paper. Wet bot-
tom of tank and paper with developing sol v., seal tank, and
let equilibrate 30 min. Apply ca 50 |xL centrfd Soln I to start-
ing line of thin layer plate in streak ca 8 cm long (not to extend
within 1 cm of plate edge or center line), using N stream. (It
is not necessary to spot accurately measured vol.) On other
half of plate, spot sep. 10 jjlL chromatgc identification stds,
evenly spaced, under N stream. Develop plate in tank equil-
ibrated 0.5 hr, letting solv. migrate 10-15 cm above starting
line. Air dry plate, locate bands under shortwave UV light,
and circle with stylus. Remove silica gel from each band by
scraping onto glazed weighing paper, and transfer to sep. 50
mL g-s centrf. tubes. Add 10.0 mL 1W NaOH to each tube,
shake 3 min, and centrf. Transfer 5.0 mL aliquots of each
supemate to 10 mL vol. flasks. Add 1.0 mL HO (1 + I) to
each flask, mix, and cool. (Soln must be at room temp, for
quant, results.) Develop color and record spectra as in 973.73D.
G. Calculations
Calc. fraction of each sulfapyrimidine in total sulfapyrimi-
dines as follows:
Sulfadiazine fraction = 0.947A d /T
Sulfamerazine fraction - A T (T
Sulfamethazine fraction = 1.053A m /T
where A d , A r , and A m = corrected A of the sulfadiazine, sul-
famerazine, and sulfamethazine bands, resp., and T - 0.947A t)
+ A r + 1.053A m .
Ref.: J AOAC 56, 689(1973).
CAS-68-35-9 (sulfadiazine)
CAS- 127-79-7 (sulfamerazine)
CAS-57-68-1 (sulfamethazine)
954.15 Sulfadiazine and Sulfamerazine
in Drugs
Spectrophotometry Method
Final Action 1962
A. Reagents
(a) Citrate buffer soln. — Dissolve 37 g Na 3 C 6 H 5 7 .2H 2
in H 2 0, add 32 mL HCl, and dil. to 250 mL with H 2 0.
(b) 2-Thiobarbituric acid (TEA) soln. — Recrystallize acid
twice from H 2 0. Dissolve 5 g recrystd acid in 20 mL IN NaOH
dild with 500 mL H 2 0. Add 250 mL citrate buffer soln and
adjust to pH 2.0. Reagent is stable when stored in g-s bottle
in refrigerator.
B. Determination
(a) Sulfadiazine. — To powd sample contg ca 0.1 g mixed
sulfonamides, add 50 mL IN HC1. Shake intermittently 10 min,
filter if necessary, and dil. filtrate and washings to 100 mL
with H 2 0. To 5 mL aliquot, add 7.5 mL 17V HC1 and dil. to
100 mL. Designate this soln (contg ca 5 mg mixed sulfon-
amides/100 mLO.W HC1) as Soln X. To 1.0 mL aliquot Soln
X in g-s test tube, add 10.0 mL TBA soln, stopper, and heat
1 hr at 100°. Weigh tube before and after heating, and com-
pensate for any loss by addn of H 2 0. Similarly treat 1.0 mL
std contg 25 fig sulfadiazine in 0.1/V HC1 and blank contg
0.17V HCl. Det. A of sample and of std, A', at 532 nm against
blank.
mg Sulfadiazine in sample taken = 50 A /A'
(b) Sulfamerazine. — Det. A T of Soln X y and A& and Am of
solns contg 5.0 mg pure sulfadiazine and sulfamerazine, resp.,
in 100 mL 0. IN HCl, at 305 nm against 0. 17V HCl blank. Then
A of Soln X due to sulfadiazine (A D ) = A' D x (mg sulfadiazine
in Soln X/5.0), and A due to sulfamerazine (A M ) = A T — A D .
mg Sulfamerazine in sample taken — 100 A M /A' M
Ref.: J AOAC 37, 697(1954).
AOAC Official Methods of Analysis (1990)
Sulfonamides 569
CAS-68-35-9 (sulfadiazine)
CAS- 127-79-7 (sulfamerazine)
964.27 Sulfadiazine in Presence
of Other Sulfonamide Drugs
Final Action 1965
Det. sulfadiazine as in 954.15B(a) from soln prepd to con-
tain ca 25 jxg sulfadiazine /mL 0.1N HC1.
Refs.: JAOAC 47, 194, 474(1964).
CAS-68-35-9 (sulfadiazine)
939.1 7* Sulfanilamide in Drugs
Hydrolysis Method
Final Action
Surplus 1970
See 36.483, 11th ed.
985.48 Sulfamethoxazole in Drug Tablets
Liquid Chromatographic Method
First Action 1985
Final Action 1987
A. Principle
Sulfamethoxazole is extd with MeOH, sulfamerazine is added
as internal std, and compds are detd by liq. chromatgy on nor-
mal phase silica column with isooctane-CH 2 Cl 2 -2-propanol-
CH 3 CN-HOAc (70 + 25 + 5 + 5 + 0.5) mobile phase and
UV detector set at 254 nm.
B. Apparatus and Reagents
(a) Liquid chromatography — Isocratic system operated at
room temp., with UV detector set at 254 nm, and strip chart
recorder or computing integrator.
(b) Analytical column. — Stainless steel, 300 x 3.9 (id) mm,
packed with 5-10 (xm particle size, uncoated silica (ixPorasil,
Waters Associates, or equiv.). At mobile phase flow rate of
2.0 mL/min, approx. retention times of sulfamethoxazole and
sulfamerazine are 3 and 5 min, resp.
(c) Mobile phase . — Isooctane-CH 2 Cl 2 -2-propanol-CH 3 CN~
HOAc (70 + 25 + 5 + 5 + 0.5).
(d) Internal std soln. — Dissolve USP Sulfamerazine Ref.
Std in MeOH in vol. flask to give soln contg ca 2.0 mg/mL.
(e) Sulfamethoxazole std soln. — Dissolve accurately weighed
amt of USP Sulfamethoxazole Ref. Std in MeOH in vol. flask
to obtain soln contg ca 5.0 mg/mL.
C. Preparation of Sample Solution
Weigh and finely powder >20 tablets. Accurately weigh
portion of powder equiv. to ca 500 mg sulfamethoxazole and
transfer to 100 mL vol. flask. Dissolve in 70 mL ca 60° MeOH.
Cool soln to room temp., dil. to vol. with MeOH, mix, and
filter.
D. Determination
Transfer 2.0 mL each of sample soln and std soln to indi-
vidual 100 mL vol. flasks contg 5.0 mL internal std soln, add
20 mL CH 2 C1 2 , dil. to vol. with mobile phase, and mix.
Using suitable micro syringe or sampling valve, test system
suitability by making 5 replicate injections (between 10 and
20 uX) of std mixt. If necessary, adjust injection vol. and flow
rate to give std peak ht of ca 60% AUFS. In properly func-
tioning system, resolution factor between std and internal std
peaks is >2.5, and CV for 5 replicate injections is <3.0% for
peak ht ratios of std to internal std.
Introduce equal vols (between 10 and 20 mX) of sample soln
and std soln into liq. chromatograph operated at room temp.
For each injection, calc. response ratio (ht of sulfamethoxazole
peak to ht of internal std peak).
Sulfamethoxazole, mg/tab. = 100 C X (R/R f ) x (T/W)
where C = concn, mg/mL, of USP Sulfamethoxazole Ref.
Std in std soln; R and R f = ratios for sample and std solns,
resp.; T = av. tablet wt, g; and W - sample wt, g.
Ref.: JAOAC 68, 88(1985).
CAS-723-46-6 (sulfamethoxazole)
983.29 Sulfisoxazole in Drug Tablets,
Solutions, and Ointments
Liquid Chromatographic Method
First Action 1983
Final Action 1986
A. Principle
Sulfisoxazole content of tablets, solns, and ointments is detd
by reverse phase LC using ternary aq. mobile phase, UV de-
tection at 254 nm, and sulfadimethoxine as internal std.
B. Apparatus
(a) Liquid chromatograph. — DuPont Model 841 sol v. pump
equipped with 254 nm detector (E. I. duPont de Nemours and
Co.), 10 |xL injection valve (Valco Instruments Co., Inc., PO
Box 55603, Houston, TX 77255), and Model 3380A integra-
tor (Hewlett-Packard). Equiv. LC system and strip chart re-
corder may be used.
(b) Chromatographic column. ^Stainless steel, 30 cm X
3.9 mm id, packed with 10 \xm p,Bondapak C )8 (Waters As-
sociates, Inc.) or equiv. meeting appropriate HPLC system
suitability requirements.
C. Reagents
(a) Solvents. — UV grade MeOH and n-heptane (Fisher Sci-
entific Co., or equiv.), and acetonitrile (Burdick & Jackson
Laboratories, Inc., or equiv.).
(b) Mobile phase. — Acetonitrile-acetic acid-H 2 (30 + 1
+ 69); flow rate 2.0 mL/min. Retention times: sulfisoxazole,
ca 4 min; internal std, ca 5.5 min. Vary ratio of acetonitrile
to H 2 to meet PLC system suitability requirements. Increased
acetonitrile decreases retention time.
(c) Internal std soln. — Dissolve 80 mg USP Ref. Std Sul-
fadimethoxine in MeOH and dil. to 100 mL with MeOH.
(d) Sulfisoxazole std soln (5.0 rng/JOO mL). — Transfer 100
mg accurately weighed USP Ref. Std Sulfisoxazole (previ-
ously dried 2 h at 105°) to 100 mL vol. flask and dil. to vol.
with MeOH. Transfer 5.0 mL aliquot to 100 mL vol. flask
contg 5.0 mL internal std soln, dil. to vol. with mobile phase,
and mix.
D. Sample Preparation
(a) Tablets. — Det. av. wt and grind to pass No. 60 sieve.
Transfer accurately weighed portion of powd. equiv. to 500
mg sulfisoxazole to 100 mL vol. flask, add 25 mL MeOH,
stopper, mix on mech. shaker 30 min, dil. to vol. with MeOH,
mix, and filter. Transfer 20.0 mL aliquot of filtrate to 100 mL
vol. flask and dil. to vol. with MeOH. Transfer 5.0 mL aliquot
to 100 mL vol. flask contg 5.0 mL internal std soln, dil. to
vol. with mobile phase, and mix.
(b) Liquids (injections and ophthalmic solns). — Accurately
570
Drugs: Part II
AOAC Official Methods of Analysis (1990)
transfer voL of dosage form contg ca 200 mg sulfisoxazole to
200 mL vol. flask and dil. to vol. with MeOH. Transfer 5.0
mL aliquot to 100 mL vol. flask contg 5.0 mL internal std
soln, dil. to vol. with mobile phase, and mix.
(c) Ointments. — Transfer accurately weighed amt of sam-
ple (5) contg ca 50 mg sulfisoxazole to 125 mL separator contg
50 mL n- heptane, shake to disperse ointment, and ext with
three 25 mL portions of MeOH-H 2 (2 + 1), passing each
ext consecutively thru second 125 mL separator contg 50 mL
/7-heptane. Collect exts in 100 mL vol. flask and dil. to vol.
with MeOH. Transfer 10.0 mL aliquot to 100 mL vol. flask
contg 5.0 mL internal std soln, dil. to vol. with mobile phase,
and mix.
£. Determination
Equilibrate column with mobile phase at flow rate of 2 mL/
min. Make 3 replicate injections of sulfisoxazole std soln. Us-
ing either peak area or peak ht measurements, for each injec-
tion det. response ratio of sulfisoxazole to internal std. In suit-
able system, capacity factor, k' , for sulfisoxazole should be
1.0-3.0; resolution factor, R, for sulfisoxazole peak and in-
ternal std peak should be >1 .5; and coefficient of variation of
response ratio for 3 replicate injections each of sample and
sulfisoxazole std working solns should be <2.0%. When chro-
matgc requirements are met, make alternate injections of std
and sample solns, and det. response ratio for each. Retention
times for sulfisoxazole and internal std must be same for sam-
ple and std soln injections.
F. Calculations
Calc. content of sulfisoxazole in dosage form as follows:
Tablets: mg/tablet = (RR/RR') x C x (T/S) X 100
Solns: mg/mL = (RR/RR') X (C/V) X 40
Ointments: mg/g - (RR/RR f ) x (C/S) x 10
where RR and RR f — response ratio of sample and std solns,
resp.; C = amt sulfisoxazole in 100 mL final std soln, mg; T
= av. tablet wt, g; 5 = sample wt, g; and V = vol. soln taken,
mL.
Ref.: J AOAC 66, 1182(1983).
CAS- 127-69-5 (sulfisoxazole)
THIAZIDES
976.33 Bendroflumethiazide in Drugs
Spectrophotometric Method
First Action 1976
Final Action 1977
A. Principle
Bendroflumethiazide is eluted from 0.1M Na 2 C0 3 -diat. earth
column with HOAc-CHCI 3 and measured directly by UV spec-
trophotometry.
B. Apparatus
(a) Chromatographic tube and tamping rod, — See 967.31 A.
(b) Diatomaceous earth. — See 960. 53B.
C. Reagents
(a) Sodium carbonate soln. — 0.1M. Dissolve 10.6 g Na 2 C0 3
in H 2 0, dil. to 1 L with H 2 0, and mix.
(b) Bendroflumethiazide std solns. — (/) Stock soln. — 0.4
mg/mL. Accurately weigh ca 20 mg USP Bendroflumethia-
zide Ref. Std into small beaker, add 5 mL DMSO, and mix
with glass rod until dissolved. Transfer quant, to 50 mL vol.
flask with MeOH and dil. to vol. with MeOH. (2) Working
soln. — 0.012 mg/mL. Transfer 3.0 mL stock soln to 100 mL
vol. flask contg 2 mL MeOH and 2 drops HC1. Dil. to vol.
with CHC1 3 .
D. Preparation of Sample
Finely powder tablets to pass No. 60 sieve. Transfer portion
contg 15-20 mg bendroflumethiazide to 50 mL vol. flask. Add
5 mL DMSO, wetting entire sample. (Caution: DMSO can be
harmful. Avoid skin contact by wearing heavy rubber gloves.
Use effective fume removal device.) Let stand 10 min with
frequent mixing. Dil. to vol. with 0. \M Na 2 C0 3 . Filter portion
thru paper, discarding first few mL.
E. Preparation of Columns
(a) Lower layer. — Mix 2 g diat. earth with 1 mL 0.1 A/
Na 2 C0 3 in 150 mL beaker, transfer to tube, and tamp to uni-
form mass.
(b) Upper layer. — Mix 4 g diat. earth with 3.0 mL sample
soln, transfer to tube, and tamp. Dry- wash flask contg sample
mixt. with 1 g diat. earth and 2-3 drops H 2 0; transfer to col-
umn and tamp. Top with glass wool pad.
F. Determination
(Use H 2 0- washed solvs thruout. Caution: See safety notes on
flammable solvents and isooctane.)
Let 75 mL isooctane elute thru column and discard eluate.
Use 100 mL vol. flask contg 2 drops HC1 in 5 mL MeOH as
receiver, and elute column with 90 mL CHCl^-HOAc (98 +
2). Dil. to vol. with CHC1 3 .
Det. A of sample and working std solns at max., ca 271 nm
(a = 48.8), in 1 cm cells with spectrophtr against CHCl 3 -HOAc
(98 + 2) as ref.
G. Identification
Macerate portion finely powd tablets contg 4-5 mg bendro-
flumethiazide with H 2 0. Transfer to 125 mL separator, using
small portions H 2 0, and dil. to ca 15 mL. Ext with three 20
mL portions CHC1 3 , collecting CHC1 3 in 100 mL beaker. Evap.
to dryness on steam bath with aid of air current. Dissolve res-
idue in small vol. MeOH and evap. to dryness. Compare IR
spectrum in KBr matrix of residue with that of ref. std pre-
viously recrystd from alcohol.
Ref.: J AOAC 59, 90(1976).
CAS-73-48-3 (bendroflumethiazide)
985.46 Methyldopa, Methyldopa-
Hydrochlorothiazide, or Methyldopa-
Chlorothiazide in Drug Tablets
Liquid Chromatographic Method
First Action 1985
Final Action 1987
A. Principle
Methyldopa, chlorothiazide, and hydrochlorothiazide are detd
by comparison with stds, using liq. chromatgy with UV de-
tection and theobromine internal std.
B. Apparatus
(a) Liquid chromato graph. — Isothermal, isocratic pump
system, photometric detector capable of monitoring A at 280
nm, suitable recorder, and 20 jxL injection loop.
(b) Chromatographic column.— 300 X 3.9 mm id, contg
jxBondapak C 18 , 10 jxm particle size (Waters Associates).
AOAC Official Methods of Analysis (1990)
Thiazides
571
(c) Filter system. — 0.45 |xm vac. filter app. (Millipore
Corp.).
C. Reagents
(a) Mobile phase. — Mix 96 mL 3% HOAc with 4 mL
MeOH, both either LC or reagent grade. Filter thru filter app.,
(c), before use.
(b) Stock std soln. — Transfer ca 50 mg anhyd. USP Meth-
yldopa Ref. Std, accurately weighed, to 100 mL vol. flask.
Add accurately weighed amt of USP Hydrochlorothiazide Ref.
Std or USP Chlorothiazide Ref. Std, in same ratio with USP
Methyldopa Ref. Std as that in sample tablets. Add 70 mL
MeOH-H 2 (1 + 1) and mix by sonication with occasional
swirling to dissolve. Let flask and contents cool to ambient
temp. Dil. to vol. with MeOH-H 2 (1 + I). Mix thoroly and
filter thru 0.45 jxm membrane filter, (c), discarding first 5 mL
filtrate.
(c) Internal std so In. — Weigh ca 22 mg theobromine into
100 mL vol. flask. Add ca 80 mL mobile phase. Warm gently
on steam bath with occasional swirling to dissolve. Cool, dil.
to vol. with mobile phase, and mix.
(d) Methyldopa or methyldopa-chlorothiazide std soln. —
Pipet 4.0 mL stock std soln and 5.0 mL internal std soln into
25 mL vol. flask. Dil. to vol. with mobile phase, and mix.
(e) Methyldopa-hydrochlorothiazide std soln. — Pipet 10.0
mL stock std soln and 3.0 mL internal std soln into 25 mL
vol. flask. Dil. to vol. with mobile phase, and mix. Do not
use methyldopa-hydrochlorothiazide std soln >2 days old.
D. Preparation of Samples
(a) Methyldopa and methyldopa-chlorothiazide tablets. —
Weigh and finely powder >20 tablets. Weigh portion of pow-
der equiv. to 125 mg methyldopa and transfer to 250 mL vol.
flask. Add 170 mL MeOH-H 2 (1. + 1) and mix by sonication
with occasional swirling for at least the length of time needed
to dissolve corresponding stds. Dil. to vol. with MeOH-H 2
(1 + 1) and mix thoroly. Filter thru 0.45 jxm membrane filter,
(c), discarding first 5 mL filtrate. Transfer 4.0 mL filtrate to
25 mL vol. flask. Pipet 5.0 mL internal std soln into flask and
mix. Dil. to vol. with mobile phase and mix.
(b) Methyldopa-hydrochlorothiazide tablets. — Follow pro-
cedure in (a), except transfer 10.0 mL sample filtrate and 3.0
mL internal std soln to 25 mL vol. flask.
(c) Methyldopa drug substance. — Accurately weigh portion
of sample equiv. to ca 125 mg methyldopa, and transfer to
250 mL vol. flask. Follow procedure in (a), starting with "Add
170mLMeOH-H 2 O (1 + ])... ."
£ Suitability Test and Determination
Equilibrate LC column with mobile phase at 1.5 mL/min.
Inject 20 jjlL std soln. Methyldopa, chlorothiazide, and hy-
drochlorothiazide peaks elute at ca 4, 6, and 7 min, resp. Re-
tention time of theobromine internal std should be ^9 min.
Resolution factor between methyldopa and chlorothiazide and
between hydrochlorothiazide and theobromine should be >3.5.
Calc. resolution factor, R, as follows:
R - [2(t 2 - t x ))/(W 2 + IV!)
where t 2 and /, = retention times of the 2 components, and
W 2 and W, ~ corresponding widths of bases of peaks, obtained
by extrapolating the relatively straight sides of the peaks to the
baseline.
Change flow rate or percentage of MeOH in mobile phase
slightly if necessary. Adjust detector sensitivity so that peaks
are ca 35-99% AUFS. Change detector sensitivity between
methyldopa and hydrochlorothiazide peaks in combination
samples. If necessary, adjust vol. of internal std soln added to
sample soln and std soln to obtain satisfactory peak response
for internal std. Make replicate injections of each std soln and
compare peak responses between injections to det. reproduc-
ibility of system. In suitable system, relative std deviation, S Ti
for 5 replicate injections is <2.0%. Calc. S r as follows:
- *) 2 "
n =
1 _
-1V2
100
S„ % =
X
where x- y — an individual determination of ratio of peak re-
sponse of substance to peak response of internal std, and x =
mean of set of n measurements.
Proceed with sample analysis by injecting 20 \xL each of
sample soln and corresponding std soln.
F. Calculations
Using peak response ratios (R and R') relative to internal
std, calc. mg drug per tablet from:
mg/tablet - (R/R') x C x (D/W) x T
where R and R' — peak response ratios for sample soln and
std soln relative to internal std, resp.; C = concn of std soln,
mg/mL; W = wt of sample taken, mg; D = sample diln; and
T = av. tablet wt, mg.
Ref.: JAOAC 67, 1118(1984).
CAS-58-94-6 (chlorothiazide)
CAS-58-93-5 (hydrochlorothiazide)
CAS-555-30-6 (methyldopa)
971.39 Methyldopa
and Chlorothiazide Combination in Drugs
Ion Exchange Method
First Action 1971
Final Action 1974
A. Reagents and Apparatus
(a) Ion exchange resin.— AG 50W-X4, 100-200 mesh, H' +
form (Bio-Rad Laboratories). Strongly acidic nuclear sulfonic
groups on polystyrene lattice.
(b) Acidic methanol. — Add 1 drop HC1 to 500 mL anhyd.
MeOH. (Dissoln of thiazides is retarded by >2 drops.)
(c) Methanolic hydrochloric acid. — \N. DiL 42 mL HC1 to
500 mL with anhyd. MeOH.
(d) Ion exchange tube. — 150 X 12 mm id, with replaceable
coarse fritted glass disk, Teflon stopcock, and Buna-N "O"
ring seal (Kontes Glass Co., No. K-422280, or equiv.).
(e) Methyldopa std soln. — 30 |ULg/mL. Accurately weigh ca
3 mg std, previously dried at 105° overnight, and dissolve in
100.0 mL W methanolic HC1.
(f) Chlorothiazide std soln. — 10 jxg/mL. Accurately weigh
ca 1 mg std, previously dried 1 hr at 105°, and dissolve, with
heat if necessary, in 100.0 mL acidic MeOH.
B. Preparation of Column
Prep, slurry of 2 g resin with 20-25 mL anhyd. MeOH and
transfer to tube with stopcock closed and contg plug of glass
wool under fritted disk. Let resin settle by gravity; then top
with small pledget of glass wool. Column need not be tamped.
Drain solv., wash column with several portions anhyd. MeOH,
and discard all washings. Prevent column from drying before
use by maintaining head of 2-3 mL MeOH or H 2 0.
When resin is being used for first time and on completion
of sepns, wash thoroly with 15-20 mL HCl-MeOH (1 + 1)
to recondition resin. With stopcock closed and glass wool re-
moved, stir resin to obtain slurry, let settle, and drain. Repeat
572
Drugs: Part
AOAC Official Methods of Analysis (1990)
twice. Finally, wash resin first with H 2 until excess acid is
removed and then with several portions anhyd. MeOH. Store
under H 2 0.
C. Preparation of Sample
Det. av. wt/tablet and pulverize to pass No. 80 sieve. Ac-
curately weigh amt contg ca 25 mg chlorothiazide into 50 mL
beaker. Add 15 mL acidic MeOH and heat carefully on steam
bath ca 30 min to dissolve active ingredients. Cool, transfer
to 50 mL vol. flask, and dil. to vol. with acidic MeOH.
D. Determination
(a) Chlorothiazide. — Collect eluate in 100 mL vol. flask at
rate of 2-3 drops/sec. Transfer 10.0 mL sample soln to prepd
column. After sample soln has entered column, rinse down
sides with three 2 mL portions anhyd. MeOH, letting each
portion sink into resin completely before next addn. Do not
agitate column mech. or by addn of solv. Complete elution
with 50 mL anhyd. MeOH. Rinse column tip with acidic MeOH.
Add 1 drop HC1 to eluate and dil. to vol. with anhyd. MeOH.
Further dil. 5.0 mL to 25.0 mL with acidic MeOH. Scan sam-
ple and std solns between 235 and 360 nm against acidic MeOH.
Calc. chlorothiazide from A at max., ca 277 nm. Proceed im-
mediately to methyldopa detn before column dries.
(b) Methyldopa. — Place another 100 mL vol. flask under
column after elution in (a). Elute with 50 mL 1/V methanol ic
HC1 at 2~3 drops/sec. Rinse column tip with IN methanolic
HC1. Dil. to vol. with IN methanolic HC1 and further dil. to
concn of ca 30 |xg methyldopa/mL. Scan sample and std solns
between 230 and 360 nm against IN methanolic HO. Calc.
methyldopa from A at max., ca 280 nm.
Ref.: JAOAC 54, 603(1971).
CAS-58-94-6 (chlorothiazide)
CAS-555-30-6 (methyldopa)
973.74 Thiazide Drugs
Spectrophotometric Method
First Action 1973
Final Action 1974
(Applicable to benzthiazide, chlorothiazide, methyclothiazide,
hydrochlorothiazide, and hydroflumethiazide.)
A. Principle
Benzthiazide, hydrochlorothiazide, or hydroflumethiazide is
eluted from 0.2/V NaOH-diat. earth column with HO Ac-ethyl
ether, extd into 0.2/V NaOH, and detd by UV spectrophotome-
try. Chlorothiazide is eluted from 0.2M K 2 HP0 4 -diat. earth
column with HOAc-ethyl ether, extd into 0.2/V HC1, and detd
by UV spectrophotometry. Methyclothiazide is eluted from 0. IM
NaHC0 3 -diat. earth column with CHC1 3 and measured directly
by UV spectrophotometry.
B. Apparatus and Reagents
(a) Chromatographic tube and tamping rod. — See 967.31 A.
(b) Diatomaceous earth. — See 960. 53B.
(c) Dipotassium phosphate solns. — 0.2 and AM, 34.85 and
17.43 g K 2 HP0 4 /L, resp.
(d) Benzthiazide, hydrochlorothiazide, and hydroflumethia-
zide std solns. — Prep, with ether-satd 0.2/V NaOH. (J)
Benzthiazide.— 3.0 mg USP Ref. Std/200 mL. (2) Hydro-
chlorothiazide. —2.0 mg USP Ref. Std/ 200 mL. (J) Hydro-
flumethiazide.— 2.0 mg USP Ref. Std/200 mL.
(e) Chlorothiazide std solns. — ( / ) Stock soln. — 1.4 mg/mL.
Accurately weigh ca 70 mg USP Chlorothiazide Ref. Std into
small beaker, add 2 mL dimethylsulf oxide (DMSO), and mix
with glass rod until dissolved. Transfer quant, to 50 mL vol.
flask, using 0.2M K 2 HP0 4 , and dil. to vol. with same solv.
(2) Working soln. — 0.014 mg/mL. Dil. 2.0 mL stock soln to
200 mL with 0.2/V HC1.
(f ) Methyclothiazide std solns. — (J) Stock soln. — 0,2 mg/
mL. Accurately weigh 20 mg USP Methyclothiazide Ref. Std
into 100 mL vol. flask and dil. to vol. with MeOH. (2) Work-
ing soln. — 0.01 mg/mL. Dil. 10 mL stock soln to 200 mL
with CHC1 3 .
C. Preparation of Sample
Finely powder to pass No. 60 sieve.
(a) Benzthiazide, hydrochlorothiazide, or hydroflumethia-
zide. — Transfer portion contg 75 mg benzthiazide or 50 mg
hydrochlorothiazide or hydroflumethiazide to 50 mL vol. flask,
using 0.27V NaOH. Shake to dissolve completely and dil. to
vol.
(b) Chlorothiazide. — Transfer portion contg ca 70 mg to
small beaker and add 2.0 mL DMSO. Mix thoroly 2-3 min
with glass rod to dissolve completely. Transfer to 50 mL vol.
flask, using 0.2M K 2 HP0 4 , and dil. to vol. with same solv.
Mix thoroly.
(c) Methyclothiazide. — Transfer portion contg ca 2 mg to
150 mL beaker. Add 2 mL MeOH and mix thoroly. Add 2
mL 0.1M NaHC0 3 and mix.
£?. Preparation of Columns
(a) Benzthiazide, chlorothiazide, hydrochlorothiazide, or
hydroflumethiazide . — (I) Lower layer. — Mix 2 g diat. earth
with 1 mL 0.2/V NaOH (1 mL 0AM K 2 HP0 4 for chlorothia-
zide) in 150 mL beaker, transfer to tube, and tamp to uniform
mass.
(2) Upper layer. — Mix 3 g diat. earth with 2 mL sample
soln, transfer to tube, and tamp. Dry-wash flask contg sample
mixt. with 1 g diat. earth and 2-3 drops H 2 0; transfer to col-
umn and tamp. Top with glass wool pad.
(b) Methyclothiazide. — (./) Lower layer. — Mix 3 g diat. earth
with 2 mL 0. \M NaHC0 3 in 150 mL beaker, transfer to tube,
and tamp to uniform mass.
(2) Upper layer. — Proceed as in (a)(2), except use 4 g diat.
earth.
E. Determination
(Use H 2 0-satd solvs thruout.)
(a) Benzthiazide, hydrochlorothiazide, and hydroflumethia-
zide. — Pass 50 mL CHCI3, followed by 50 mL ether, thru col-
umn; discard eluate. Using 250 mL separator as receiver, elute
column with 0.1 mL HOAc in 100 mL ether. Wash tip of
column with ether. Add 65 mL isooctane to eluate and ext org.
phase with three 50 mL portions 0.2/V NaOH; combine NaOH
soln in 200 mL vol. flask and dil. to vol.
Det. A of sample and std solns in 1 cm cells with spectrophtr
against 0.2/V NaOH as ref.
(b) Chlorothiazide . — Proceed as in (a), except use 0.25 mL
HOAc in 100 mL ether, 50 mL isooctane, and 0.2N HC1 in-
stead of NaOH. Use 0.2/V HC1 as ref. solv.
(c) Methyclothiazide. — Pass 75 mL isooctane-ether (9+1)
thru column; discard eluate. Use 200 mL vol. flask as receiver
and elute column with 100 mL CHC1 3 . Wash tip of column
with ether. Add 10 mL MeOH and dil. to vol. with CHC1 3 .
Use CHCI3 as ref. solv.
Wavelength of max. A, and a of individual compds are as
follows:
AOAC Official Methods of Analysis (1990)
Sulfur-Containing Drugs 573
Compd
X Max., nm
Absorptivity
Benzthiazide
Chlorothiazide
Methyclothiazide
Hydrochlorothiazide
Hydroflumethiazide
295
29.6
278
32.4
268
51.8
273
49.1
273
45.4
F. identification
(a) Benzthiazide, hydrochlorothiazide, and hydroflumethia-
zide. — Acidify portion sample soln with \N HC1 and ext with
50 mL ether. Evap. ether to dryness, add 5 mL alcohol, and
evap. again. Compare IR spectrum in KBr matrix of residue
with that of ref. std previously recrystd from alcohol.
(b) Chlorothiazide. — Transfer 5 mL prepd soln, 973.74C(b),
to 125 mL separator, add 10 mL H 2 0, acidify with IN HC1,
and ext with 75 mL ether. Evap. ether to dryness. Add 5 mL
alcohol to residue and evap. to dryness. Compare IR spectrum
in KBr matrix of residue from 400 to 600 cm -1 with that of
ref. std previously recrystd from alcohol.
(c) Methyclothiazide. — Transfer portion sample contg ca 4
mg active ingredient to 125 mL separator, add 20 mL O.lAf
NaHC0 3 , and ext with ca 75 mL ether. Proceed as in (b).
Ref.: JAOAC 55, 161(1972); 56, 677(1973).
CAS-91-33-8 (benzthiazide)
CAS-58-94-6 (chlorothiazide)
CAS-58-93-5 (hydrochlorothiazide)
CAS- 135-09-1 (hydroflumethiazide)
CAS- 135-07-9 (methyclothiazide)
974.41 Polythiazide in Drugs
Spectrophotometry Method
First Action 1974
Final Action 1976
(Applicable to formulations contg vanillin)
A. Principle
Vanillin, which interferes in method, is condensed thru al-
dehyde group with primary amine group of sulfanilic acid to
form strongly polar and H 2 0-sol. Schiffs base, which is re-
tained in aq. immobile phase of column. Less polar polythia-
zide is eluted with mobile phase, ether-isooctane, and detd by
UV spectrophotometry.
B. Apparatus and Reagents
(Use H 2 0- washed solvs thruout.)
(a) Chromatographic tube and tamping rod. — See 967.31 A.
(b) Diatomaceous earth. — See 960. 53B.
(c) Dilute ammonium hydroxide. — IN. Dil. 17 mL NH 4 OH
to 250 mL with H 2 0.
(d) Ammonium sulfanilate soln. — 6%. Dissolve 6.0 g sul-
fanilic acid in \N NH 4 OH and dil. to 100 mL with IN NH 4 OH.
(e) Polythiazide std soln. — 10 jxg/mL. Accurately weigh
ca 100 mg polythiazide, transfer to 100 mL vol. flask, and dil.
to vol. with MeOH. Further dil. 10 mL of this soln to 100 mL
with MeOH and 10 mL dild soln to 100 mL with MeOH.
C. Preparation of Sample
Accurately weigh sample contg ca 1 mg polythiazide and
transfer to 150 mL beaker. Add 0.25 mL dimethylsulfoxide
(DMSO) and mix thoroly to wet entire sample. Let stand 3-
4 min.
D. Preparation of Column
(a) Lower layer. — Mix 6 g diat. earth, (b), and 5 mL NH 4
sulfanilate soln in 150 mL beaker, transfer to tube, and tamp
to uniform mass.
(b) Upper layer. — Add 4 mL NH 4 sulfanilate soln to sam-
ple soln, and mix. Add 4 g diat. earth, mix, transfer to tube,
and tamp to uniform mass. Dry-wash beaker with 1 g diat.
earth and few drops H 2 0, transfer to tube, and tamp. Top with
glass wool pad.
E. Determination
Pass 100 mL isooctane thru column; discard eluate. Elute
polythiazide with 100 mL isooctane-ether (1 + 1), receiving
eluate in 250 mL beaker. Immediately evap. eluate to dryness.
Dissolve residue in small amt MeOH and transfer quant, to
100 mL vol. flask. Dil. to vol. with MeOH. Filter thru glass
wool, discarding first 20 mL. Det. A of sample and std solns
against MeOH in 1 cm cell at max., ca 268 nm.
Ref.: JAOAC 57, 716(1974).
CAS-346-18-9 (polythiazide)
OTHER SULFUR-CONTAINING DRUGS
967.32 Methimazole in Drugs
Infrared Spectrophotometry Method
First Action 1967
Final Action 1977
{Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
A. Apparatus
(a) Chromatographic tube. — 200 X 22 mm.
(b) Spectrophotometer . — Recording double-beam IR spec-
trophtr, with 2 mm cells and NaCl windows.
B. Reagents
(a) Diatomaceous earth.
(b) Methimazole std,
not in use.
See 960.53B.
Store in desiccator over P 2 O s when
C. Column Chromatography
Transfer amt of freshly ground tablet mixt. contg 10 mg
methimazole to 100 mL beaker, add 3 mL H 2 0, and mix tho-
roly to wet sample. Add 4 g diat. earth and mix thoroly. Trans-
fer in 2 equal portions to chromatgc tube contg pledget of glass
wool and pack tightly. Rinse beaker with 0.5 g diat. earth and
add to column; top with glass wool pad. Rinse beaker with
150 mL H 2 0-washed isooctane (redistd) and add rinses to col-
umn. Let last drops of isooctane drain from column before
proceeding. Discard isooctane eluate.
Rinse beaker with three 5 mL portions H 2 0-washed CHC1 3
and add rinses to column. Collect eluate. Elute methimazole
with 200 mL H 2 0- washed CHC1 3 , maintaining sol v. head <75
mm (3") during elution. Combine CHC1 3 eluates, and evap. at
ca 40-60° with air stream to ca 10 mL, washing down sides
of beaker with small portions CHC1 3 during evapn. Do not heat
excessively, since methimazole may oxidize. Quant, transfer
cone, to 30 mL beaker with several small portions CHC1 3 . Evap.
solv. at ca 30-40° under air stream. (Make certain all traces
of isooctane are removed.) Dry residue in vac. over anhyd.
P 2 O s 30 min. (If necessary, store residue over desiccant in
dark; methimazole oxidizes on standing.)
574 Drugs: Part II
AOAC Official Methods of Analysis (1990)
D. Determination
Add 5 mL CS 2 to residue in beaker, cover with watch glass,
and warm to dissolve. Cool and quant, transfer soln to 10 mL
vol. flask with CS 2 . Repeat with two 2 mL portions CS 2 , cool,
transfer to flask, and dil. to vol.
Prep, std soln methimazole in CS 2 , with warming, to con-
tain exactly 1.00 mg/mL. Record quant. IR spectra of sample
and std solns between 7.6 and 8.4 |xm in 2 mm NaCl cells.
Measure baseline A values of 7.83 jutm max., using minima at
7.7 and 8.3 |xm.
mg Methimazole/tablet -Ax (C /A') x 10 x (T/W)
where A and A' = baseline values for sample and std, resp.;
C - mg/mL std soln; T and W = av. wt /tablet and sample
wt, resp., in mg.
Identify samples by comparing IR spectra of quant, solns
with spectrum of std over 2-15 |xm, using CS 2 as blank.
Ref.: J AOAC 50, 674(1967).
CAS-60-56-0 (methimazole)
952.27 Propylthiouracil in Drugs
Spectrophotometric Method
Final Action
Start and complete detn on same day.
Transfer accurately weighed sample contg ca 150 mg pro-
pylthiouracil to 200 mL vol. flask, and transfer 150.0 mg pure
propylthiouracil to another 200 mL vol. flask as std. To each
flask add 150 mL NH 4 OH (1 + 13), washing down necks.
Shake flasks moderately and continuously 1 min to dissolve
propylthiouracil. Dil. each to vol. with NH 4 OH (1 + 13) and
mix.
Filter sample soln, discarding first 25 mL filtrate. Dil. 20
mL aliquot clear filtrate to 200 mL with H 2 in vol. flask (or
25 mL aliquot to 250 mL) and mix. Dil. 20 mL aliquot of this
soln to 200 mL in vol. flask (or 25 mL aliquot to 250 mL)
and mix. Prep, same double diln of std soln to obtain final
concn of 0.0075 mg/mL.
Det. A of final solns of std and sample against H 2 blank
in silica cells in spectrophtr at 234 nm. Apply cell corrections
unless same cell is used for both std and sample. Calc. pro-
pylthiouracil content of sample.
Ref.: J AOAC 35, 572(1952).
CAS-5 1-52-5 (propylthiouracil)
948.30*
Thiouracil in Drugs
Bromination Method
First Action
Surplus 1965
uct. Aliquot is treated with Cul to form colored cupric die-
thyldithiocarbamate which is measured at ca 428 nm.
B. Reagents
(a) Sodium hydroxide soln. — 1M. Dissolve 8 g NaOH in
and dil. to 200 mL with H 2 0.
(b) Cuprous iodide. — No. C-465 (Fisher Scientific Co.), or
equiv. Grind with mortar and pestle to fine powder.
(c) Disulfiram ref. std soln. — Approx. 50 |xg/mL CH 2 C1 2 .
Accurately weigh NF Disulfiram Ref. Std and dissolve in
CH 2 C1 2 . Dil. quant, and stepwise with CH 2 C1 2 to desired concn.
(d) TLC std soln.— 0.5 mg disulfiram/mL CH 2 C1 2 .
(e) 7XC developing solvent. — 1% HO Ac in toluene.
(f ) Thin layer sheets. —Sheet A.— 20 X 20 cm, coated 0.25
mm thick with alumina with fluorescent indicator (Analtech,
Inc., Product No. 4011, or equiv.). Sheet B.— 20 x 20 cm,
coated 0.25 mm thick with silica gel with fluorescent indicator
(Analtech, Inc., Product No. 2611, or equiv.).
C. Determination
Det. av. tablet wt. Accurately weigh portion of pulverized
sample composite contg ca 100 mg disulfiram and transfer to
200 mL vol. flask. Add CH 2 C1 2 , mix thoroly, and dil. to vol.
with CH 2 CI 2 . Transfer 10.0 mL aliquot to 125 mL separator,
and add 15 mL CH 2 C1 2 and 10 mL 1M NaOH. Shake 1 min
and transfer CH 2 C1 2 layer to 100 mL vol. flask thru compact
pledget of absorbent cotton. Ext aq. layer with second 25 mL
portion of CH 2 C1 2 , filter CH 2 C1 2 layer thru same cotton plug,
and combine with first ext. Dil. ext to vol. with CH 2 C1 2 and
mix.
Transfer 10.0 mL CH 2 C1 2 ext and 10.0 mL ref. std soln to
sep. 25 mL g-s flasks. To each flask add ca 50 mg powdered
Cul. Shake flasks and let stand 1 h. Quant, filter each soln
into sep. 50 mL vol. flasks thru tight pledget of absorbent cot-
ton previously wet with CH 2 C1 2 . Dil. to vol. with CH 2 C1 2 . Scan
sample and std solns from 600 to 325 nm, against CH?C1 2 blank.
Det. max. A at ca 428 nm, and calc. amt disulfiram as follows:
mg Disulfiram /tablet = (A /A') x C X 200 x (D/W)
where A and A' = values for sample and std, resp., C ~ mg
disulfiram in 10 mL ref. std soln; D — av. tablet wt (g); W —
wt sample (g); 200 = diln factor.
D. TLC Confirmatory Test
Line suitable chromatgc tank with filter paper and add de-
veloping solv. Apply sep. spots of 5 |xL of original 200 mL
sample soln and 5 |xL TLC std soln to either TLC sheet. De-
velop 10-15 cm above starting line, and air dry. Locate and
mark spots under shortwave UV light. R f of sample and std
spots should be ca 0.60 and 0.25 for alumina and silica gel,
resp.
Ref.: J AOAC 64, 554(1981).
CAS-97-77-8 (disulfiram)
See 32.394, 10th ed.
981.25 Disulfiram in Drug Tablets
Colorimetric Method
First Action 1981
Final Action 1983
(Caution: See safety notes on sodium hydroxide.)
A. Principle
Disulfiram is extd with CH 2 C1 2 and washed with NaOH soln
to eliminate diethyldithiocarbamic acid decomposition prod-
968.47 Phenothiazine in Drugs
Gas Chromatographic Method
First Action 1968
Final Action 1982
A. Reagents and Apparatus
(All CHC1 3 solns must be protected from light and assay must
be completed within 8 hr.)
(a) Phenothiazine std soln. —Dissolve phenothiazine in 10
parts toluene with heat. Add 0.1 g activated charcoal for each
AOAC Official Methods of Analysis (1990)
Sulfur-Containing Drugs
575
4 g phenothiazine. Boil 10 min under reflux and filter while
hot thru heated filter. Cool soln, and collect phenothiazine
crystals on buchner or fritted glass filter. Dry crystals at 100°
and then in vac. desiccator contg paraffin chips. Repeat re-
crystn, if necessary, until mp is 184-185°. Dissolve 100.0 mg
purified phenothiazine in CHC1 3 in 50 mL vol. flask and dil.
to vol. with CHC1 3 .
(b) Internal std soln. — Dissolve 125 mg promethazine. HC1
in CHCI3 in 25 mL vol. flask and dil. to vol. with CHC1 3 .
(c) Chromatographic column. — Slurry 20 g Gas-Chrom Q,
100-120 mesh, with 100 mL CHC1 3 in 500 mL r-b flask. Add,
with stirring, 1.0 g Apiezon L dissolved in 50 mL CHC1 3 .
Evap. to dryness in 70° H 2 bath, using rotary vac. evapo-
rator. Apply vac. (ca 50 cm Hg) to one end of 1 .2 m (4') glass
column (4 mm id) and, with gentle tapping only, fill tube with
coated support. Condition column by heating 48 hr at 240°
with N flow of ca 10 mL/min.
(d) Gas chromatography — Any gas chromatograph with H
flame ionization detector capable of using specified column.
B. Determination
Grind representative sample portion to pass No. 60 sieve.
Accurately weigh sample contg ca 200 mg phenothiazine and
transfer to 100 mL vol. flask. Add 80 mL CHC1 3 and shake
vigorously until phenothiazine is completely dissolved (ca 20
min). Dil. to vol. with CHC1 3 , mix thoroly, and let stand 15
min. Pipet 5 mL aliquots of clear supernate and phenothiazine
std soln into sep. 25 mL g-s erlenmeyers. Pipet 4 mL aliquots
of promethazine. HCl internal std soln into each flask.
About 1 hr before initial injection, adjust app. to following
temps: column 215°, detector 230°, injector 230°. Set N carrier
gas flow rate to give phenothiazine retention time of ca 8 min
(ca 20 psig regulator outlet pressure). Retention time of inter-
nal std will be ca 17 min. Inject similar vol. of sample and
std soln contg ca 10 u,g phenothiazine, using sensitivity setting
that gives 70-90% of full-scale deflection.
% Phenothiazine in original sample
= (M/W)(P U /P a ) x (P h /P p ) x 200
where M — mg phenothiazine used to prep, std soln, W — mg
sample, P u — phenothiazine sample soln peak area, P. A - pro-
methazine. HO sample soln peak area, P h - prometha-
zine. HC1 std soln peak area, and P p — phenothiazine std soln
peak area.
Refs.: JAOAC 49, 857(1966); 50, 682(1967); 51, 273(1968).
CAS-92-84-2 (phenothiazine)
985.47 Allopurinol in Drug Tablets
Liquid Chromatographic Method
First Action 1985
Final Action 1987
A. Principle
Allopurinol content is detd by Jiq . chromatgy on reverse phase
column with aq. ammonium phosphate mobile phase, UV de-
tection, and hypoxanthine internal std.
B. Apparatus
(a) Liquid chromatograph. — Model 950 sol v. pump with
Model 970A variable wavelength detector capable of moni-
toring A at 254 nm (Tracor Instruments, Inc., replacement
models 951 and 971, resp.), 20 |jlL injection valve (Valco In-
struments, Inc., PO Box 55603, Houston, TX 77255) and suit-
able strip chart recorder, or equiv. LC system,
(b) Chromatographic column. — Stainless steel, 300 x 3.9
mm id, packed with uJBondapak 10 |xm (Waters Associates,
Inc.) or equiv. column meeting LC system suitability require-
ments.
C. Reagents
(a) Ammonium phosphate.— Monobasic, FW 1 15.031 (Fisher
Scientific Co.), or equiv.
(b) Hypoxanthine . — Reagent grade (Fisher Scientific Co.),
or equiv.
(c) Mobile phase. — Prep. 0.05M ammonium phosphate
(monobasic) soln, 5.75 g (NH 4 )H 2 P0 4 /L H 2 0. Degas under
vac. or by ultrasonic treatment 5-10 min. (Do not leave mo-
bile phase in column overnight; after draining mobile phase,
flush entire system >20 min with H 2 0, followed by MeOH
for 20 min.)
(d) Internal std preparation. — Accurately weigh ca 50 mg
hypoxanthine and transfer to 50 mL vol. flask. Add 10 mL
0. IN NaOH and shake mech. 10 min, or until completely dis-
solved. Dil. with H 2 to vol., and mix. Prep, this soln fresh
daily .
(e) Std preparation. — Accurately weigh ca 50 mg USP Al-
lopurinol Ref. Std and transfer to 50 mL vol. flask. Add 10
mL 0.1N NaOH and shake mech. 10 min. Dil. with H 2 to
vol., and mix. Prep, this soln fresh daily. Transfer 4.0 mL of
this soln to 200 mL vol. flask, add 2.0 mL internal std prepn,
dil. with mobile phase to vol., and mix.
D. Sample Preparation
Weigh and finely powder >20 tablets. Accurately weigh
portion of powder equiv. to ca 50 mg allopurinol, and transfer
to 50 mL vol. flask. Add 10 mL O.IN NaOH, shake mech.
10 min, dil. with H 2 to vol., and mix. From this point, pro-
ceed with detn without delay. Filter portion of soln thru suit-
able paper or 0.45 |xm membrane filter, discarding first 10 mL
filtrate. Transfer 4.0 mL filtrate to 200 mL vol. flask, add 2.0
mL internal std prepn, dil. with mobile phase to vol. , and mix.
E. Suitability Test and Determination
Inject equal vols (10-20 jmL) of sample prepn and std prepn
into liq. chromatograph by means of sampling valve or high
pressure microsyringe. Operate chromatograph at ambient temp,
while pumping mobile phase at 1.5 mL/min. Adjust detector
sensitivity so that peak response for allopurinol and hypoxan-
thine is 40-60% AUFS. In suitable chromatogram, lowest and
highest peak response ratios of 3 successive injections of std
prepn do not differ >3.0%, and resolution factor, R, for sam-
ple peak and internal std peak is >5. Retention times are hy-
poxanthine 7 min, allopurinol 12 min.
Calc. resolution factor, R, as follows:
R = [2fe - t ] )]/(W 2 + W { )
where t 2 and t } — retention times of the 2 components, and
W 2 and W] - corresponding widths of bases of peaks, obtained
by extrapolating the relatively straight sides of the peaks to the
baseline.
Standard peak response ratio, R\ is ratio of std allopurinol
peak to internal std peak.
F. Calculation
Calc. amt of allopurinol in dosage form, using response ra-
tios based on either peak hts or peak areas, according to fol-
lowing equation:
Allopurinol, mg/tablet - (/?//?') X (W /W) X T
where R and R f - response ratios of allopurinol peak to in-
ternal std peak for sample and std, resp.; W and W = mg
taken for sample and std prepns, resp.; and T = av. tablet wt,
mg.
576 Drugs: Part II
AOAC Official Methods of Analysis (1990)
Ref.: JAOAC67, 1121(1984).
CAS-3 15-30-0 (allopurinol)
986.36 Primidone in Drug Tablets
Liquid Chromatographic Method
First Action 1986
Final Action 1988
A. Principle
Sample is dissolved in mobile phase, filtered, injected into
liq. chromatgc system, and quantitated by comparison with ex-
ternal std.
B. Reagents
(a) Solvents.— LC grade H 2 and MeOH (J.T. Baker Inc.,
Nos. 4218 and 9093, resp.).
(b) LC mobile phase. — In suitable flask, combine 500 mL
H 2 and 500 mL MeOH; stir mag. Filter thru 0.4 jxm mem-
brane filter wetted with MeOH. Place in ultrasonic bath 10 min
to deaerate.
(c) Std soln.— Accurately weigh 50 mg USP Ref. Std Prim-
idone, previously dried 2 h at 105°, and transfer to 50 mL vol.
tlask. Add 35 mL mobile phase, place in ultrasonic bath 15
min, cool, and dil. to vol. with mobile phase. Place in ultra-
sonic bath for addnl 15 min and cool. Soln is stable 1 week.
C. Apparatus
(a) Liquid chromato graph. — System equipped with injec-
tor, solv. delivery system, and UV detector. Operating con-
ditions: flow rate 1.0 mL/min; 254 nm detector, 0.2 AUFS;
temp, ambient; 20 (xL injection.
(b) LC column. — Macherey-Nagel Nucleosil C-8, 10 |xm
particle size, 25 cm X 3.2 mm, or equiv.
(c) Recorder. — 10 mV with 0.5 cm /min chart speed.
(d) Membrane filters. — Nylon-66 pore size 0.45 u-m (Rainin
Instrument Co., Mack Rd, Woburn, MA 01801), or equiv.
D. Preparation of Samples
Det. av. wt of 20 tablets and grind to pass No. 60 sieve.
Transfer accurately weighed portion of powder equiv, to 50
mg primidone to 50 mL vol. flask. Add 35 mL mobile phase,
place in ultrasonic bath 15 min, cool, and dil. to vol. with
mobile phase. Place in ultrasonic bath addnl 15 min and cool.
Filter soln thru 0.45 |xm membrane filter and use as sample
prepn.
E. Determination
Equilibrate system with column in instrument and mobile
phase set at 1.0 mL/min. Inject 20 jjlL std soln and adjust flow
rate and sensitivity so that peak response is ca 45% full scale,
with retention time ca 3 min. In suitable system, coefficient
of variation (CV) of peak responses of 5 replicate injections
is <2.0%. Proceed with sample analysis, using 20 jxL injec-
tions for each std and sample soln.
F. Calculation
Det. peak responses of std and sample peaks and calc. amt
of primidone in tablets.
Primidone, mg/tablet = (R/R f ) x 50 x C x (T/W)
where R and R f = peak response of sample and std solns,
resp.; C = concn of primidone std soln in mg/mL; T = av.
tablet wt in mg; and W = sample wt in mg.
Ref.: JAOAC 68, 85(1985).
CAS-125-33-7 (primidone)
988.23 Diethylpropion Hydrochloride
in Drug Substance and Tablets
Liquid Chromatographic Method
First Action 1988
A. Principle
DEPH content of drug substance and tablets is detd by re-
verse phase liq. chromatgy using anthracene as internal std,
UV detection at 254 nm, and MeOH-H 2 0-phosphate mobile
phase. Purity is confirmed by UV, LC, and spot tests.
B. Apparatus
(a) Liquid chromato graph. — Equipped with 30 cm X 4 mm
id stainless steel column packed with ODS bonded to micro-
particulate silica; UV detector; and suitable integrator. Oper-
ating conditions: column temp, ambient; mobile phase flow
rate ca 1.5 mL/min; vol. injected 20 jjlL; detector wavelength
254 nm.
(b) UV-visible scanning spectrophotometer.
C. Reagents
Use deionized, purified H 2 and anal, reagent grade chem-
icals unless otherwise specified.
(a) Mobile phase.— Dissolve 0.40 g KH 2 P0 4 and 2.26 g
anhyd. Na 2 HP0 4 in H 2 to make 1000 mL. Mix 1 part of this
soln with 4 parts of MeOH, and filter thru suitable membrane
of <1 jxm porosity. pH of resulting soln should be ca 7.7.
Degas soln by sparging with He. Make adjustments, if nec-
essary, to obtained desired retention times and resolution.
(b) HiP0 4 soln.—{\ in 2000). Dil. 1 mL H 3 P0 4 (85%) to
2 L with H 2 0.
(c) Internal std soln. — Dissolve 20 mg anthracene in 500
mL MeOH. Store in air-tight container.
(d) DEPH std soln. — Dry USP Diethylpropion Hydrochlo-
ride Ref. Std over silica gel >4 h. Accurately weigh ca 25 mg
dried std and transfer to 200 mL vol. flask contg 40 mL H 3 P0 4
soln. Pipet 40.0 mL internal std soln into tlask, dil. to vol.
with MeOH, and mix.
D. Chromatographic System Suitability Test
Let chromatgc system equilibrate and inject replicate 20 |xL
portions of DEPH std soln. Typical retention times for diethyl-
propion and anthracene are ca 6 and 9 min, resp., at flow rate
between 1.0 and 2.0 mL/min. In suitable system, resolution,
R, between these peaks is >3.0 and relative std dev. (RSD)
for ratios of peak responses, R s , for 6 replicate injections of
DEPH std soln is <1.0%.
E. Sample Preparation
(a) Bulk drug assay soln. — Dry DEPH bulk drug over silica
gel ^4 h. Accurately weigh ca 25 mg dried sample and trans-
fer to 200 mL vol. flask contg 40 mL H 3 P0 4 soln. Pipet 40.0
mL internal std soln into flask, dil. to vol. with MeOH, and
mix.
(b) Tablet assay soln.— Weigh and finely powder >20 DEPH
tablets. Into 200 mL vol. flask, transfer accurately weighed
amt of powder equiv. to ca 25 mg DEPH. Add 40 mL H 3 P0 4
soln and shake mech. >15 min. Transfer 40.0 mL internal std
soln to flask, dil. to vol. with MeOH, and mix. Centrifuge
portion of soln, and filter supernate thru suitable membrane
filter ^1 [xm porosity.
F. Determination
(a) Bulk drug. — Sep. inject equal vols (ca 20 (xL) of DEPH
std soln and bulk drug assay soln in duplicate into LC system,
record chromatograms, and measure response for major peaks.
ReL retention times are ca 0.7 for diethylpropion and 1.0 for
AOAC Official Methods of Analysis (1990)
Sulfur-Containing Drugs 577
anthracene. Calc. amt DEPH, mg, in portion of DEPH drug
substance taken as follows:
mg DEPH - F x C x (RJR,)
where F = vol. sample soln/1000 jig/mg = 200 mL/1000
|xg/mg = 0.2; and where C = concn, |xg/mL, of USP DEPH
Ref. Std in DEPH std soln, and R u and R s are ratios of peak
responses of diethylpropion to anthracene obtained from bulk
drug assay soln and DEPH std soln, resp.
(b) Tablets. — Use tablet assay soln and follow procedure
for bulk drug detn. Calc. amt DEPH, mg/tablet, as follows:
DEPH, mg/tablet - {[0.2C X (7? u /R s )]/mg sample} x W
where W = av. tablet wt, mg.
G. Identification Tests
(a) Bulk drug.—XJV spectrum of 1 in 100 000 soln in 0.1N
HC1 exhibits max. and min. at same wavelengths as that of
similar prepn of USP DEPH Ref. Std concomitantly measured,
and molar a values (anhyd. basis) at wavelength max. at ca
253 nm do not differ by >3.0%.
(b) Tablets. — Chromatogram of tablet assay soln obtained
as directed for tablet assay exhibits major peak for diethylpro-
pion, with retention time that corresponds with that exhibited
in chromatogram of DEPH std soln, both relative to internal
std.
H. Chromatographic Purity
(a) Test preparations.- — Test prepn A. — Dissolve 250 mg
bulk drug in 20 mL H 3 P0 4 soln in 100 mL vol. flask, dil. to
vol. with MeOH, and mix. Test prepn B. — Transfer 1.0 mL
test prepn A to 100 mL vol. flask, dil. to vol. with 4: 1 mixt.
of MeOH and H 3 P0 4 soln, and mix.
(b) LC system. — Use system described in Apparatus.
(c) Procedure. — Inject 20 |ulL test prepn B into LC system,
adjust detector output to obtain peak ht for diethylpropion
> 40% but <100% full scale deflection on chart, adjust in-
Common and Chemical Names of Drugs in this Chapter
Common Name
tegrator sensitivity accordingly, and det. area of diethylpro-
pion peak. Inject 20 jxL test prepn A, chromatograph for total
time equal to 3 times retention time of diethylpropion, and det.
sum of areas of impurity peaks between sol v. front peak and
diethylpropion peak. Perform sol v. blank detn by injecting 20
jxL of 4: 1 mixt. of MeOH and H 3 P0 4 soln. Calc. % chromatgc
impurities by formula:
% impurities = 100 (r A - r s )/[100r B + (r A - r s )]
where r A = sum of areas of impurity peaks in chromatogram
obtained from test prepn A, r s = sum of areas of peaks in
chromatogram obtained from solv. blank, and r B = area of
diethylpropion peak in chromatogram obtained from test prepn
B. Impurities found should be <0.5%.
/. Secondary Amines
(a) Acetaldehyde test soln. — Mix 4 mL acetaldehyde, 3 mL
alcohol, and 1 mL H 2 0. Prep, soln fresh.
(b) Procedure. — Dissolve 100 mg bulk drug in 2 mL CH 2 C1 2
in centrf. tube. Transfer to second tube 2 mL std soln of dieth-
ylamine-HCl (DEAH) (dried 2 h at 105° before using) in CH 2 C1 2 ,
which has known concn of 250 |jLg/mL. Treat each soln as
follows: Ext with 2 mL buffer soln contg 5.7 g Na 2 C0 3 and
3.0 g NaHCO 3 /100 mL H 2 0. Centrifuge, if necessary, to clar-
ify upper phase, and immediately transfer 0.5 mL of soln to
spot plate. Immediately add 2 drops of acetaldehyde test soln,
and then, in rapid succession, add 1 drop of Na nitroferri-
cyanide soln (1 in 100) to each spot. Immediately and simul-
taneously, briefly stir both spots to mix reagents. Any blue
color formed within 3 min by bulk drug soln is not visibly
more intense than that of DEAH std soln (<0.5% of secondary
amines as DEAH). Note: Failure of DEPH std to form blue
color has been shown to be due to decomposed acetaldehyde.
Ref.: Pharm. Forum Sept. -Oct. 1985, p. 791.
CAS-90-84-6 (diethylpropion)
CAS- 134-80-5 (diethylpropion HC1)
Chemical Name
Acenocoumarol
Acetaminophen
Acetanilide
Acetylcarbromal
Allopurinol
Aminophylline
Aminopyrine
p-Aminosalicylic Acid
Amobarbital
Aspirin
Bendroflumethiazide
Benzthiazide
Bromisovalum
Butabarbital sodium
Caffeine
Carbromal
Chloral hydrate
Chlorothiazide
Chlorpropamide
Codeine
Dicumarol
Diethylpropion Hydrochloride
Diphenylhydantoin
Disulfiram
Ethchlorvynol
Guaiacol
Guaiafenesin
Hexylresorcinol
Hydrochlorothiazide
Hydroflumethiazide
Isoniazid
Mandelic Acid
4-Hydroxy-3-[1-(4-nitrophenyl)-3-oxybutyl]-2H-1-benzopyran-2-one
/V-(4-Hydroxyphenyl)acetamide
A/-Phenylacetamide
1-Acetyl-3-(a-bromo-a-ethylbutyryl)urea
1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
3,7-Dihydro-1 ,3-dimethyl-1 H-purine-2,6-dione with 1 ,2-ethanediamine
4-Dimethylamino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one
4-Amino-2-hydroxybenzoic acid
5-Ethyl-5-(3-methylbutyl)-2,4,6(1H,3H,5H)-pyrimidinetrione
2- (Acetyloxy) benzoic acid
3,4-Dihydro-3-(phenylmethyl)-6-(trifluoromethyl)-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 , 1 -dioxide
6-Chloro~3-[[(phenylmethyl)thio]-methyl]-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 , 1 -dioxide
2-Bromo-3-methylbutyrylurea
5-Ethyl-5-(1-methylpropyl)-2,4,6(1H,3H,5H)-pyrimidinetrione
3,7-Dihydro-1 ,3,7-trimethyl-1 H-purine-2,6-dione
2-Bromo-2-ethylbutyrylurea
2,2,2-Trichloro-1 , 1 -ethanediol
6-Chloro-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 , 1 -dioxide
1-[(p-Chlorophenyl)sulfonyl]-3-propylurea
(5a, 6a)-7,8-Didehydro-4,5-epoxy-3-methoxy-17-methylmorphinan-6-ol
3,3'-Methylenebis[4-hydroxy-2H-1-benzopyran-2-one]
2-(Diethylamino)propiophenone hydrochloride
See phenytoin
Tetraethylthioperoxydicarbonic diamide
1 -Chloro-3-ethyl-1 -penten-4-yn-3-ol
o-Methoxyphenol
3-(2-Methoxyphenoxy)-1,2-propanediol
4-Hexyl-1 ,3-benzenediol
6-Chloro-3,4-dihydro-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 ,1 -dioxide
3,4-Dihydro-6-(trifluoromethyl)-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 , 1 -dioxide
4-Pyridinecarboxylic acid, hydrazide
a-Hydroxybenzeneacetic acid
(Continued)
578
Drugs: Part II
AOAC Official Methods of Analysis (1990)
Common and Chemical Names of Drugs in this Chapter (Continued)
Common Name
Chemical Name
Menadione Sodium Bisulfite
Meprobamate
Methimazole
Methyclothiazide
Methyldopa
Methyl salicylate
Morphine
Oxyquinoline sulfate
Paraldehyde
Pentobarbital
Phenacetin
Phenagiycodol
Phenobarbital
Phenolphthalein
Phenothiazine
Phenprocoumon
Phenyl salicylate
Phenytoin
Polythiazide
Primidone
Propylthiouracil
Quinine
Salicylamide
Salicylic Acid
Secobarbital sodium
Sodium salicylate
Sulfadiazine
Sulfamerazine
Sulfamethazine
Sulfamethoxazole
Sulfanilamide
Sulfisoxazole
Sulfonethyl methane
Suifonmethane
Theobromine
2-Thiouracil
Thymol
Warfarin potassium
Warfarin sodium
1,2,3,4-Tetrahydro-2-methyl-1 ( 4-dioxo-2-naphthalenesu!fonic acid sodium salt
2-Methyl-2-propyl-1 ,3-propanediol dicarbamate
1 ,3-Dihydro-1 -methyl-2H-imidazole-2-thione
6-Chloro-3-(chloromethyl)-3,4-dihydro-2-methyl-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 ,1 -dioxide
3-Hydroxy-a-methyl-i_-tyrosine
2-Hydroxybenzoic acid methyl ester
7,8-Didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol
8-Quinolinol sulfate (2:1) (salt)
2,4,6-Trimethy!-1 ,3,5-trioxane
5-Ethyl-5-{1 -methylbutyl)-2,4,6(1 H,3H,5H)-pyrimidinetrione
/V-(4-Ethoxyphenyt)acetamide
2-(p-Chlorophenyl)-3-methyl-2,3-butanediol
5-Ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione
3,3-Bis(4-hydroxyphenyl)-1(3H)-isobenzofuranone
Thiodiphenylamine
4-Hydroxy-3-(1-phenylpropyl)-2H-1-benzopyran-2-one
2-Hydroxybenzoic acid phenyl ester
5,5-Diphenyl-2,4-imidazolidinedione
6-Chloro-3,4-dihydro-2-methyl-3-[[(2,2,2-trifluoroethyl)thio]methyl]-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1 -dioxide
5-Ethyldihydro-5-phenyl-4,6(1H,5H)pyrimidinedione
2,3-Dihydro-6-propyl-2-thioxQ-4(1H)-pyrimidinone
6'-Methoxycinchonan-9-ol
o-Hydroxybenzamide
2-Hydroxybenzoic acid
5-(1 -Methylbutyl)-5-(2-propenyl)-2,4,6(1 /-/,3H,5H)-pyrimidinetrione monosodium salt
2-Hydroxybenzoic acid monosodium salt
4-Amino-W-2-pyrimidinyl-benzenesulfonamide
4-Amino-A/-(4-methyl-2-pyrimidinyl)-benzenesulfonamide
4-Amino-A/-{4,6-dimethyl-2-pyrimidinyl)-benzenesulfonamide
W 1 -(5-Methyl-3-isoxazolyl)sulfanilamide
p-Aminobenzenesulfonamide
4-Amino-A/-(3,4-dimethyl-5-isoxazolyl)-benzenesulfonamide
2,2-Bis(ethylsulfonyl)butane
2,2-Bis(ethylsulfonyl)propane
3,7-Dihydro-3,7-dimethyl-1 H-purine-2,6-dione
2,3-Dihydro-2-thioxo-4(1H)-pyrimidinone
5-Methyl-2-(1-methylethyl)phenol
4-Hydroxy-3-(3-oxo-1 -phenylbutyl)-2H-1 -benzopyran-2-one potassium salt
4-Hydroxy-3-(3-oxo-1-pheny1butyl)-2H-1-benzopyran-2-one sodium salt
Source: USAN and the USP Dictionary of Drug Names (1983; 1989) U.S. Pharmacopeia! Convention, Rockville, MD.
20. Drugs: Part III
Edward Smith, Associate Chapter Editor
Food and Drug Administration
961.18
OPIUM ALKALOIDS
Opium Alkaloid Drugs
First Action 1961
Final Action 1965
A. Microchemical Tests
See 930.40 and Table 980.34, and 960.57.
B. General Titration Method
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Det. av. wt/tablet or other unit and grind to fine powder.
Accurately weigh sample equiv. to 100-200 mg alkaloid and
transfer to separator with ca 20 mL H 2 0. Add 1 mL H 2 S0 4
(1+9) and ext with three 25 mL portions CHC1 3 . (Extn from
acid soln is not necessary in absence of CHCl 3 -soL acidic or
neut. components.) Add ca 1 mL NH 4 OH (use excess solid
NaHC0 3 for apomorphine or physostigmine) and ext with four
25 mL portions CHC1 3 (use CH 2 C1 2 for ephedrine and CHC1 3 -
isopropanol (4 + 1) for morphine). Use correspondingly larger
vols of solv. if larger vols aq. soln are required, as in case of
sirups or with excessive amts of excipients. Check alky of soln
after first extn by touching indicator paper to stopper. If not
distinctly alk., add addnl NH 4 OH. Check for complete extn
by evapg 1 mL final ext to dryness; if more than trace of res-
idue remains, ext with addnl portions solv. Filter ext thru plug
of cotton or fine glass wool, previously wet with CHC1 3 , into
200 mL erlenmeyer. Complete detn by either of following
methods (C must be used for arecoline and cocaine).
C. Determination
Evap. combined exts on steam bath with air current to ca
10 mL. Add measured excess 0.02/V H 2 S0 4 and continue evapn
to remove solv. Cool, add Me red, and titr. excess acid with
0.02N NaOH.
D. Alternative Determination
Evap. combined exts on steam bath with air current to dry-
ness. Dissolve residue in ca 2 mL MeOH, heating if necessary.
Add Me red, and titr. with 0.02N H 2 S0 4 to faint pink. If al-
kaloid is not completely dissolved, heat gently to complete
soln. Add ca 40 mL freshly boiled, cooled H 2 0, and complete
titrn.
E. Titration Factors
See Table 961.18 for titrn factors.
Ref.: JAOAC 44, 293(1961).
Apomorphine in Drug Tablets
See 961.18B
Codeine in Drug Tablets
See 961.18B.
961.19 Codeine in Presence
of Antihistamines in Drugs
Chromatographic Method
First Action 1961
Final Action 1965
(Applicable to sirups contg codeine with pyrilamine, metha-
pyrilene, prophenpyridamine, and similar antihistamines)
A. Apparatus
See 967.31A.
B. Reagents
(a) Triethylamine.— If blank A, 961. 19D, is >0.010, pu-
rify as follows: Reflux 100 mL Et 3 N with 20 mL H 2 and 2
g Na hydrosulfite >8 hr. Wash with four or five 20 mL por-
tions H 2 0, dry by either distg into Dean-Stark trap or by salt-
ing-out with anhyd. K 2 C0 3 , and then distil, collecting first 75
mL. Store over anhyd. Na 2 C0 3 or K 2 CO v (Caution: See safety
notes on distillation, toxic solvents, and triethyl amine.)
(b) Codeine std soln. — Accurately weigh ca 100 mg co-
deine sulfate.5H 2 (or other salt), dissolve in MeOH, transfer
to 100 mL vol. flask, and diL to vol. with MeOH. 1 mL =
0.8067 mg codeine. H 2 0.
(c) Chloroform. — Use CHC1 3 satd with H 2 thruout.
(d) Diatomaceous earth. — See 960. 53B.
C. Preparation of Sample
Prep, following 3 columns (columns II and III need not be
quant.):
(a) Column J. — Pipet 2.0 mL sample, draining thoroly, into
small beaker. Add 0.5 mL IN NaOH and 3 g diat. earth. Mix
thoroly and transfer quant, to tube, 967.31A(a). Dry-wash
beaker with small portion diat. earth and few drops H 2 0, and
add to tube. Tamp column firmly with tamping rod, and press
pad of glass wool on top.
(b) Column //.—Mix 3 g diat. earth and 2 mL l/V HN0 3 ,
and prep, column as in (a).
(c) Column III.— Mix 3 g diat. earth and 2 mL IN H 2 S0 4 ,
and prep, column as in (a).
D. Determination
Arrange columns so that effluent from / flows into // and
then into ///. Pass 100 mL CHC1 3 over columns. Discard Col-
umn / which retains excipients.
Pass 50 mL CHC1 3 thru Column // (which retains antihis-
tamine) onto ///; then pass 25 mL CHC1 3 over ///. Remove
Column //.
To recover codeine, place 50 mL vol. flask contg 10 mL
MeOH and 1 mL HC1 under Column ///. Pass 5 mL CHC1 3
contg 1 mL Et 3 N over column followed by 32 mL 1% Et 3 N
in CHC1 3 . Dil. to vol. with CHC1 3 and det. A at 287 nm against
CHCt 3 . (Film of Et 3 N.HCl may adhere to walls of cells. Rinse
cells carefully with H 2 and alcohol; then wipe clean before
use.) Correct for blank A of mixt. of 10 mL MeOH, 1 mL
Et 3 N, and 1 mL HC1 dild to 50 mL with CHC1 3 . Also det. A'
of std prepd by dilg 5 mL std soln contg 1 mg codeine
salt/mL MeOH, to 50 mL with CHC1 3 and 5 drops HC1.
579
580
Drugs: Part
AOAC Official Methods of Analysis (1990)
Table 961.18 Titration Factors for Alkaloids
Alkaloid
Formula
mg/mL 0.02/V
H 2 S0 4
Apomorphine hydrochloride
C 17 H 17 0aN.HCI. , / 2 H 2
6.25
Arecoline hydrobromide
C 8 H 13 2 N.HBr
4.72
Atropine
C 17 H 23 N0 3
5.79
Atropine sulfate
(C 17 H 23 N0 3 ) 2 .H 2 S0 4 .H 2
6.95
Cocaine hydrochloride
C 17 H 21 4 N.HCI
6.80
Codeine sulfate
(C 18 H 21 3 N) 2 .H 2 S0 4 .5H 2
7.87
Codeine phosphate
C 18 H 21 03N.H 3 P04.iy<;H 2
8.49
Emetine hydrochloride
C 29 H 40 O 4 N2.2HCI
5.54
Ephedrine
C 10 H 15 ON
3.30
Ephedrine hydrochloride
C 10 H 15 ON.HCI
4.03
Ephedrine sulfate
(C 10 H 15 ON) 2 .H 2 SO 4
4.29
Ethylmorphine hydrochloride
C, 9 H 23 3 N.HCI.2H 2
7.72
Homatropine hydrobromide
C l6 H 21 3 N.HBr
7.13
Homatropine hydrochloride
C 16 H 21 3 N.HCI
6.24
Hydrocodone hydrochloride
C ie H 21 3 N.HCI.H 2
7.08
Hydrocodone bitartrate
C 18 H 21 03N.C 4 H 6 6 .2V 2 H 2
9.89
Morphine hydrochloride
C 17 H 19 3 N.HCI.3H 2
7.52
Morphine sulfate
(C 17 H 19 3 N) 2 .H 2 S0 4 .5H 2
7.59
Physostigmine salicylate
Ci5H 21 2 N 3 .C 7 H 6 3
8.27
Physostigmine sulfate
(C 15 H 21 2 N 3 } 2 .H 2 S04
6.49
Pilocarpine hydrochloride
CnH 16 2 N 2 .HCI
4.89
Pilocarpine nitrate
CnH^OsNa.HNOs
5.43
Procaine hydrochloride
C 13 H 20 O 2 N 2 .HCI
5.46
Strychnine
C 2 irl 22 2 N2
6.69
Strychnine sulfate
(C 21 H 22 2 N 2 ) 2 .H 2 S0 4 .5H 2
8.57
Strychnine nitrate
C 21 H 22 2 N 2 .HN0 3
7.95
mg Codeine salt in sample = 5 AC /A'
where A and A' refer to corrected A of sampJe and std, resp.,
and C = mg codeine salt/mL std soln.
Ref.: J AOAC 44, 285(1961).
CAS-76-57-3 (codeine)
CAS-52-28-8 (codeine phosphate)
CAS-1420-53-7 (codeine sulfate)
965.44 Codeine and Terpin Hydrate
in Drug Elixirs
Spectrophotometric Method
First Action 1965
Final Action 1966
A. Reagents
(a) Color reagent.— Either Folin-Denis reagent, 952.03A(a),
or phosphotungstic- phosphomolybdic acid reagent prepd as
follows: To 100 g pure Na tungstate and 20 g phosphomo-
lybdic acid (free from nitrates and NH 4 salts), add 100 g H 3 P0 4
and 700 mL H 2 0. Boil over free flame 1.5-2 hr, cool, filter
if necessary, and dil. to 1 L with H 2 0. Equiv. amt of pure
molybdic acid may be substituted for phosphomolybdic acid.
(b) Terpin hydrate std soln. — Accurately weigh ca 80 mg
terpin hydrate, add 2 mL HOAc, and stir until terpin hydrate
is almost dissolved. Add 10 mL alcohol, stir, and transfer to
100 mL vol. flask. Rinse dish with three 10 mL portions al-
cohol. Finally rinse few times with H 2 and dil. to vol. with
H 2 0. Soln keeps indefinitely.
(c) Codeine std soln. —See 961.19B(b).
(d) Water-saturated ether. — Add 100 mL H 2 to 200 mL
ether in separator, shake, let stand 30 min, and discard H 2 0.
(e) Acidified water-saturated chloroform. — Sat. 300 mL
CHCI3 with H 2 0. After 30 min standing, transfer CHC1 3 to
flask contg 3 mL HOAc.
(f) Diatomaceous earth. — See 960. 53B.
B. Determination of Terpin Hydrate
Pipet 5 mL sample into distg flask and add 100 mL satd
NaCl soln, 35 mL alcohol, 2 mL HOAc, and 10 mL H 2 0.
Distil, collecting 100 mL distillate.
Pipet 5 mL color reagent into 50 mL vol. flask. Cool under
running H 2 while slowly adding 5 mL H 2 S0 4 . Let mixt. come
to room temp, and then add exactly 2 mL sample distillate.
Place flask in boiling H 2 20 min. Cool under H 2 to room
temp, and dil. to vol. with dil. alcohol (1 + 3). Shake every
few min until soln is clear (10-15 min). (If soln fails to clear,
phosphomolybdic acid used to prep, color reagent is unsatis-
factory.)
Let stand 0.5 hr and det. A at 725 nm against reagent blank
prepd without sample. Det. A' of std soln prepd simulta-
neously with sample, beginning "Pipet 5 mL color reagent ..."
Terpin hydrate (g/ 100 mL elixir) - A X C X 20/ A'; where
C ~ g terpin hydrate /1 00 mL std soln.
C. Determination of Codeine
Pipet 5 mL sample into 100 mL beaker, add 0.5 g p-tolu-
enesulfonic acid, and stir with glass rod. Add 6 g diat. earth,
mix to fluffy mass, and transfer to tube, 967.31A(a), contg
plug of glass wool at base. Tamp firmly, and cover with glass
wool. Pass H 2 0-satd ether over column and discard ether (Col-
umn/).
Mix 2 g diat. earth and 1 mL IN NaHC0 3 (8.4 g/100 mL).
Add to second tube (//), tamp, and cover with glass wool.
Mount Column / over Column // and place 100 mL vol. flask
contg 10 mL MeOH and 4 drops HC1 under//. Add in 4 equal
portions enough acidified H 2 0-satd CHC1 3 to Column / to fill
vol. flask to mark. Completely drain each portion before add-
ing next.
Prep, std codeine soln contg 10 mL std soln, (c), and 2
drops HC1 dild to 50 mL with H 2 0-satd CHCI 3 .
Det. A and A' at 287 nm of sample and std solns, resp.,
against mixt. of 10 mL MeOH and 2 drops HC1 dild to 50 mL
with H 2 0-satd CHC1 3 .
Codeine. H 2 0, mg/100 mL elixir = A x C x 20 /A', where
C = mg codeine.H 2 in 100 mL std soln.
AOAC Official Methods of Analysis (1990)
Opium Alkaloids 581
Refs.: JAOAC 42, 459(1959); 48, 607(1965).
CAS-76-57-3 (codeine)
CAS-6059-47-8 (codeine mono hydrate)
CAS-2451-01-6 (terpin hydrate)
972.53 Codeine in APC Drug Tablets
Chromatographic Method
First Action 1972
Final Action 1973
(Wash all ether with H 2 and wash 0. 17V H 2 S0 4 with ether
before use in prepn of reagents and in detn.)
A. Reagents
(a) Phosphate-citrate buffer.— pH 5.1. See 970.78A (d).
(b) Di-(2-ethylhexyl) phosphoric acid (DEHP) soln. — Prep.
daily 1% soln in ether. (Caution: Avoid contact with skin.)
(c) Codeine std soln. — 120 |ULg codeine salt/mL 0. \N H 2 S0 4 .
Prep, fresh daily from phosphate or sulfate salt. Dissolve 60
mg codeine salt in 0. \N H 2 S0 4 and dil. to 50 mL. Dil. 10 mL
aliquot of this soln to 100 mL with 0. IN H 2 S0 4 .
B. Determination
Grind sample to pass No. 60 sieve.
Pack small glass wool plug in base of 200 x 22 mm id
chromatgc tube. Mix 2 g diat. earth, 960.53B, and 1 mL pH
5.1 buffer, transfer to tube, and tamp. Accurately weigh ground
sample contg 12 mg codeine for 0.5 and 1 grain tablets, 6 mg
for 0.25 and 0.125 grain tablets, and 2,5 mg for 1 mg tablets
into 100 mL beaker. Add 2.0 mL pH 5.1 buffer and mix to
smooth suspension. Add 3 g diat. earth and mix well. Quant,
transfer mixt. to tube, above pH 5.1 layer, with aid of scoop-
type spatula. Wipe beaker and spatula with glass wool, add to
tube, and tamp.
Add 50 mL ether to sample beaker, swirl, and transfer to
column. Elute with 4 addnl 50 mL portions ether, letting each
portion pass into column before adding next. Rinse tip of col-
umn with 2-3 mL CHC1 3 . Discard rinse and eluate which con-
tain aspirin, acetophenetidin, and caffeine (APC).
Wash column with 50 mL ether and discard eluate. Rinse
tip of column with 2-3 mL CHC1 3 and dry tip with tissue pa-
per. Place 125 mL separator under column and elute codeine
with 50 mL 1% DEHP soln. After soln passes into column,
elute with 50 mL ether. Rinse column tip with 5-10 mL ether
into separator. Ext with three 10 mL portions 0.1JV H 2 S0 4 ,
collecting exts in vol. flask (100 mL flask for 1 and 0.5 grain
codeine, 50 mL flask for 0.25 and 0.125 grain codeine), and
dil. to vol. with 0. \N H 2 S0 4 . (For tablets contg 1 mg codeine,
use 10, 10, and 4 mL 0. IN H 2 S0 4 and dil. to 25 mL.) Det.
A of sample and std solns at max., ca 284 nm, against 0AN
H 2 S0 4 . Calc. codeine salt content of tablets.
Ref.: JAOAC 55, 142(1972).
CAS-76-57-3 (codeine)
CAS-52-28-8 (codeine phosphate)
CAS- 1420-53-7 (codeine sulfate)
967.33 Hydrocodone (Dihydrocodeinone)
in Drugs
First Action 1967
Final Action 1968
A. General Method
See 961. 18B.
In Presence of Antihistamines
(Caution: See safety notes on distillation, toxic solvents and
chloroform.)
B. Apparatus and Reagents
(a) Chromatographic tubes and tamping rod. — See 967. 31 A.
(b) Column I. — Thoroly mix 4 g acid-washed diat. earth,
960. 53B, and 3 mL ca 2N HC1. Transfer to tube and tamp to
uniform mass, using gentle pressure.
(c) Column II. — Mix and tamp layers as in (b). (I) Lower
layer. — 2 g diat. earth and 1 mL IN NaHC0 3 . (2) Upper
layer. — 4 g diat. earth and 3 mL 6% succinic acid.
(d) Equilibrated sulfuric acid. — Thoroly shake \N H 2 S0 4
with small vol. H 2 0-satd CHC1 3 .
(e) Hydrocodone std soln. — 175 |xg hydrocodone bitar-
trate/mL. Dissolve 17.5 mg hydrocodone bitartrate in
equilibrated H 2 S0 4 in 100 mL vol. flask and dil. to vol. with
equilibrated H 2 S0 4 . Shake ca 20 mL std soln with ca 75 mL
H 2 0-satd CHCI3. Det A' of aq. phase from 360 to 250 nm.
C. Determination
Mount Column / directly over Column //. Transfer 10.0 mL
sample (or vol. contg ca 3-4 mg hydrocodone bitartrate) to
separator. Add 5 mL H 2 and 1 mL ca IN NaOH, and ext
with four 30 mL portions of CHC1 3 . Pass each ext thru col-
umns; let individual exts drain completely into both columns.
Wash with 50 mL H 2 0-satd CHC1 3 . Discard Column /. Wash
Column II with addnl 100 mL H 2 0-satd CHC1 3 . Discard eluate.
Add mixt. of 3.5 g diat. earth and 3 mL NH 4 OH to Column
//, directly onto packing. Tamp. Pass 150 mL H 2 0-satd CHC1 3
thru column. Evap. eluate to ca 75 mL or until NH 3 is com-
pletely removed (test vapors with moistened indicator paper).
Quant, transfer to separator contg 20.0 mL equilibrated H 2 S0 4
and shake thoroly. Det. A of aq. phase from 360 to 250 nm,
using max., ca 282 nm, for calcn.
mg Hydrocodone bitartrate in mL sample taken
= (A/A r ) x C X V
where A and A' refer to sample and std, resp., C - mg hy-
drocodone bitartrate/mL in std, and V = vol. H 2 S0 4 (20 mL).
Refs.: JAOAC 50, 655(1967); 51, 494(1968).
CAS- 143-7 1-5 (hydrocodone bitartrate)
Morphine in Drug Sirups and Tablets
Final Action
See 961. 18B
Codeine, Acetanilid, and Caffeine in Drugs
See 916.04*.
Ethylmorphine (Dionine) in Drug Sirups
See 961. 18B.
970.82 Morphine in Opium and Paregoric
Chromatographic Method
First Action 1970
Final Action 1972
(Caution: See safety notes on distillation, flammable solvents,
toxic solvents, triethylamine, diethyl ether, and chloroform.)
582
Drugs: Part
AOAC Official Methods of Analysis (1990)
A. Apparatus
(a) Chromatographic tubes. — See 967. 31 A.
(b) Diatomaceous earth. — See 960. 53B.
B. Reagents
(a) Triethylamlne. — Purified as in 961.19B(a).
(b) Morphine std soln. — 0.08 mg anhyd. morphine /mL.
Accurately weigh morphine base or salt equiv. to 4 mg anhyd.
morphine into 50 mL vol. flask. Add 10 mL MeOH, 1 mL
HC1, and 1 mL Et 3 N, and dil. to vol. with CHC1 3 . Alterna-
tively, prep, stock soln by dissolving accurately weighed std
equiv. to ca 40 mg anhyd. morphine in 0.5 mL Et 3 N in 100
mL vol. flask, and dil. to vol, with MeOH. Pipet 10 mL of
this stock soln into 50 mL vol. flask, add 1 mL Et 3 N and 1
mL HC1, and dil. to vol. with H 2 0-satd CHC1 3 .
(c) Citrate buffer. — 0.1M, pH 4.4. Mix equal vols 0.1M
Na citrate (2.94 g Na->C 6 H,O 7 .H 2 O/100 mL) with 0.1M citric
acid (2.10 g H 3 C 6 H 5 O 7 .H 2 O/100 mL).
C. Preparation of Sample
(a) Opium. — Accurately weigh ca 2 g opium into 100 mL
vol. flask. Add 20 mL dimethyl sulfoxide (DMSO) and heat
in beaker of boiling H 2 or in steam bath ca 15 min. Swirl
gently to dissolve, keeping opium particles in contact with
DMSO and not letting particles remain on walls. Inspect soln
carefully. If undissolved material remains, continue heating.
Small amt insol. material, such as fine leaf fragments, sandlike
particles, and gelatinous particles, may remain undissolved;
add more DMSO, if necessary. Cool, add H 2 to ca 90 mL,
and mix. Let soln reach room temp., dil. to vol. with H 2 0,
and mix. (If foaming occurs on mixing, use 1 drop ether or
alcohol to dispel foam.)
If sample is in pieces too large to fit in neck of vol. flask,
accurately weigh into 250 mL beaker, add 20 mL DMSO, and
heat in boiling H 2 or steam bath. Use stirring rod to disperse
sample while heating. Decant into 100 mL vol. flask. If un-
dissolved opium remains in beaker, heat with addnl 3 mL por-
tions DMSO as needed until soln is complete as possible (DMSO
concn in final soln can vary over wide range without adverse
effect.) DiJ. to vol. with H 2 as above.
Filter prepd soln thru paper, rejecting first 20 mL filtrate.
Use 2 mL aliquot for prepn of Column /.
(b) Paregoric. — Evap. 10.0 mL paregoric, contg ca 4 mg
morphine, to ca 2 mL on steam bath under stream of air. If
evapn continues beyond 2 mL, dil. to 2 mL with H 2 0. Cool
soln to room temp, and then use for prepn of Column /.
D. Preparation of Columns
(a) Column I. — (I) Lower layer. — Mix 3 g diat. earth and
2 mL citrate buffer; transfer to tube and tamp as in 961.19C.
(2) Upper layer. — Add 0.5 mL citrate buffer to 2.0 mL aliquot
of sample ext, 970.82C(a) or (b). Add 3 g diat. earth, mix,
and transfer to tube. Dry-wash beaker with 1 g diat. earth and
add to column; tamp and add glass wool pad.
(b) Column II. — Mix 3 g diat. earth and 2 mL 1 .0M K 2 HP0 4
(17.42 g/100 mL); transfer to tube, tamp, and add glass wool
pad.
(c) Column III. — Mix 3 g diat. earth and 2 mL 0.5Af NaOH;
transfer to tube, tamp, and add glass wool pad.
E. Determination
(Use H 2 0-satd solvs thruout. Rinse each column tip with CHC1 3
before discarding columns or changing receivers.)
Pass 100 mL ether, followed by 100 mL CHC1 3 , thru Col-
umn /. Discard eluates. Mount Columns // and HI in series
below Column /. Pass thru columns 5 mL 20% (v/v) Et 3 N in
CHC1 3 , followed by four 10 mL portions 1% Et 3 N in CHC1 3 .
Let each portion pass thru completely before next addn. Con-
tinue elution without delay. Discard Column /, and pass three
5 mL portions 1% Et 3 N in CHC1 3 thru remaining columns.
Discard Column //. Wash Column /// successively with 10 mL
1% Et 3 N in CHCI3, 50 mL CHC1 3 , 2 mL 10% HO Ac in CHC1 3 ,
and 50 mL 1% HO Ac in CHC1 3 . Discard all eluates.
Place as receiver under Column /// 50 mL vol. flask contg
10 mL MeOH and 1 mL HC1. (Remove metal leashes from
vol. flasks to prevent contamination during transfer to cells.)
Elute column with 5 mL 20% Et 3 N in CHC1 3 , followed by 33
mL 1% Et 3 N in CHC1 3 . Dil. eluate to vol. with CHC1 3 .
Scan spectrum of eluate and morphine std from 360 to 255
nm, using CHC1 3 as ref. (Film of Et 3 N.HCl may adhere to
walls of cells. Rinse cells carefully with H 2 and alcohol; then
wipe clear before scanning.) Correct A at max., ca 285 nm,
by extrapolating baseline from 340 to 310 nm to this wave-
length.
mg Anhyd. morphine in aliquot taken = (W x A/A') X f
where W ~ mg morphine in std soln, A and A f = corrected
A of sample and std, resp., and/ = factor to convert wt std
to its equiv. in anhyd. morphine (if hydrated morphine or mor-
phine salt is used as std).
Refs.: JAOAC 51, 1315(1968); 53, 603(1970).
CAS-57-27-2 (morphine)
989.08 Morphine Sulfate
in Bulk Drug and Injections
Liquid Chromatographic Method
First Action 1989
Method Performance:
s r - 0.91; s R = 1.41; RSD r = 0.9%; RSD R = 1.4%
Injection, 2 mg/mL:
s f = 0.027; s R = 0.035; RSD r - 1.3%; RSD R = 1.7%
Injection, 8 mg/mL:
s r - 0.050; s R - 0.173; RSD r = 0.6%; RSD R = 2.1%
Injection, 10 mg/mL:
s r = 0.061; s R - 0.185; RSD r = 0.6%; RSD R = 1 .8%
A. Principle
Bulk drug and injection samples are prepd by direct diln in
modified LC mobile sol v. Morphine sulfate is quantitated and
preservative phenol is identified by UV detection at 284 nm.
Degradation product pseudomorphine and contaminant 2-mer-
captobenzothiazole (2-MCBT) are detected and identified at
230 nm.
B. Apparatus
(Equiv. app. may by substitued.)
(a) Liquid chromato graph. — Equipped with injection valve
with 20 (xL sample loop, solv. delivery system, recording in-
tegrator, and variable wavelength UV detector capable of
monitoring at 230 and 284 nm. Operating conditions: flow rate
1.5-2.0 mL/min (morphine sulfate should be eluted between
5 and 8 min).; temp, ambient; injection vol. 20 u>L. (System
must meet system suitability requirements.)
(b) LC column. — Stainless steel, 30 cm x 3.9 mm id, packed
with ixBondapak C ]8 , 10 |xm (Waters Associates, Inc.). Use
of different C 18 column may require addn of amine modifier
to meet system suitability requirements.
AOAC Official Methods of Analysis (1990)
Tropane Alkaloids
583
(c) Membrane filters. — Nylon 66, pore size 0.45 (xm, 25
and 47 mm diam.
C. Reagents
(Protect all morphine sulfate solns from direct light.)
(a) Methanol. — LC grade.
(b) Acetic acid. — Anal, grade.
(c) 1 -Heptane sulfonic acid N a salt. — Monohydrate. >98%
(Eastman Kodak Co., or equiv.).
(d) Morphine sulfate reference std. — USP Morphine Sul-
fate (Pentahydrate) Ref. Std. Do not dry before use.
(e) Phenol. — Crystals, anal, grade.
(f ) Pseudomorphine — Prep, as follows: Add 10 g morphine
to hot soln of 2.0 g KOH in 1 L H 2 0, and let mixt. cool to
room temp. Then add soln of 11.58 g K 3 Fe (CN) 6 in 400 mL
H 2 during 50 min, with stirring, and continue stirring addnl
30 min. Collect solid matter and stir with hot MeOH to remove
morphine (1 g). Dissolve MeOH-insol. material (8.4 g) in coned
NH 3 soln, dil. with H 2 to 700 mL, and boil mixt. 7.7 g pseu-
domorphine is deposited as almost colorless rods that decom-
pose at ca 330°. A maxima are at 231 and 261 nm in 0.1N
H 2 S0 4 . (From Bentley, K.W., & Dyke, S.F. J. Chem. Soc.
2574(1959).)
(g) 2-Mercaptobenzothiazole (2-MCBT).— 9%% (Aldrich
Chemical Co.).
(h) Mobile solvent. — Mix 240 mL MeOH with 720 mL
0.005M 1-heptanesulfonic acid Na salt monohydrate in H 2
and add 10 mL HOAc. Pass soln thru 0.45 ixm filter and degas
before use. Adjust MeOH or 1-heptanesulfonic acid Na salt
soln content so that system meets suitability test requirements.
(i) Dilution solvent. — Mobile solv. (h) prepd without 1-
heptanesulfonic acid Na salt.
D. Preparation of Standard Solutions
(a) Morphine sulfate std soln. — Dissolve accurately weighed
amt USP Morphine Sulfate Ref. Std in diln solv. to prep, soln
contg ca 0.24 mg morphine sulfate/mL.
(b) Morphine sulfate-phenol std soln. — Dissolve accu-
rately weighed amts USP Morphine Sulfate Ref. Std and phenol
in diln solv. to prep, soln contg ca 0.24 mg morphine sulfate
and ca 0.15 mg phenol/mL.
(c) Phenol std solns. — Stock soln. — About 2.0 mg phenol/
mL H 2 0. Working soln. — Add diln solv. to stock soln to prep,
soln contg ca 0.15 mg phenol/mL.
(d) Pseudomorphine std solns. — Stock soln. — 0.2—0.3 mg
pseudomorphine/mL diln solv. Working soln. — Add diln solv.
to stock soln to prep, soln contg ca 20 |mg pseudomorphine/
mL.
(e) 2-MCBT std solns.— About 80 ^g 2-MCBT/mL MeOH.
Working soln. — Add diln solv. to stock soln to prep, soln contg
ca 5-6 jxg 2-MCBT/mL.
(f) Mixed std soln. — Prep, soln in diln solv. to contain
morphine sulfate, phenol, pseudomorphine, and 2-MCBT at
conens approx. equal to individual working std solns.
E. Preparation of Samples
(a) Bulk drug. — Accurately weigh ca 100 mg bulk drug into
25 ml vol. flask and dil. to vol. with diln solv. Dil. soln with
diln solv. to final concn of ca 0.24 mg morphine sulfate/mL.
(b) Injections. — Dil. accurately measured vol. morphine
sulfate injection with diln solv. to final concn of ca 0.24 mg
morphine sulfate/mL.
F. Determination
Filter all solns thru 0.45 jjliti filter before injection.
Let LC system equilibrate 1 h with mobile solv. flow rate
ca 1 .5 mL/min. Set wavelength at 284 nm. Inject 20 jxL mor-
phine sulfate-phenol std soln (b). Coeff. of var. (CV) of mor-
phine sulfate peak area for 5 replicate injections should be <2%.
Resolution factor for phenol-morphine sulfate pair should be
>2. Tailing factor at 5% peak ht for morphine sulfate peak
should be <2. Proceed with analysis by alternating duplicate
20 |jlL injections of samples and morphine sulfate std soln (a).
If contaminants such as 2-MCBT and pseudomorphine are
suspected in sample, det. identity by using stds (c)-(f). Detect
phenol and morphine sulfate and quantitate latter at 284 nm.
Detect 2-MCBT and pseudomorphine at 230 nm. Approx. re-
tention items are 10-20 min for 2-MCBT and 20-45 min for
pseudomorphine .
G. Calculations
Calc. results as follows;
Morphine sulfate bulk drug, % purity
- (R/R f )x (W'/W) x (D'/D) x 100
Morphine sulfate, mg/mL injection
- (R/R') xW'x (D'/D)
where R and R' = av. peak areas for duplicate injections of
sample and std, resp.; W and W = wt of std and sample, resp.;
and D' and D = diln of std and sample, resp.
Ref.: JAOAC71, 1046(1988).
CAS-621 1-15-0 (morphine sulfate, pentahydrate)
TROPANE ALKALOIDS
958.16* Atropine in Drug Tablets
Infrared Spectroscopic Method
Final Action 1965
Surplus 1983
See 38.028, 14th ed.
932.23* Belladonna
and Stramonium Alkaloids in Drug Ointments
Final Action
Surplus 1965
See 32.037 and 32.038, 10th ed.
Homotropine in Drug Tablets
Final Action
See 961.18B.
973.75 Benztropine Mesylate in Drugs
Spectrophotometric Method
First Action 1973
Final Action 1975
(Not applicable in presence of compds reacting with bromo-
phenol blue, e.g., quaternary ammonium compds)
A. Principle
Benztropine is extd from acid soln by bromophenol blue-
CHC1 3 soln, forming dye complex with max. A at ca 410 nm.
B. Reagents and Apparatus
(a) Dye soln. — Weigh 100 mg reagent bromophenol blue
into 1 L vol. flask, add ca 750 mL CHC1 3 , stir mech. 10 min
584
Drugs: Part
AOAC Official Methods of Analysis (1990)
to dissolve, and dil. to vol. with CHC1 3 . Filter thru small pad
of glass wool. Dil. 50 mL to 500 mL with CHC1 3 . Prep, fresh
daily.
(b) Benztropine mesylate std soln. — 1 mg/100 mL. Weigh
100 mg USP Ref. Std into 100 mL vol. flask and dissolve and
dil. to 100 mL with 0.2N H 2 S0 4 . Dil. 10 mL aliquot to 100
mL with 0.27V H 2 S0 4 and further diL 10 mL dild soln to 100
mL with 0.27V H 2 S0 4 . Prep, fresh daily.
(c) Spectrophotometer . — Recording, with 5 cm matched
cells.
C. Preparation of Sample
(a) Tablets. — Transfer accurately weighed ground portion
contg ca 1 mg benztropine mesylate to 100 mL vol. flask,
using ca 70 mL 0.27V H 2 S0 4 . Shake mech. 15 min and filter
thru Whatman No. 541 paper wetted with 0.27V H 2 S0 4 into
100 mL vol. flask. Rinse flask and filter with three 5 mL por-
tions 0.2/V H 2 S0 4 , rinse filter with several small portions 0.27V
H 2 S0 4 , adding rinses to soln, and dil. to vol. with 0.27V H 2 S0 4 .
(b) Injections. — Transfer aliquot contg ca 1 mg benztropine
mesylate to 100 mL vol. flask and dil. to vol. with 0.27V H 2 S0 4 .
D. Determination
Perform detn on same day sample and std solns are prepd.
Place 25 mL each sample soln and std soln and 0.27V H 2 S0 4
for blank into sep. 250 mL separators and treat similarly. Add
50 mL dye soln and shake vigorously 1 min. Let sep. and drain
lower layer into 125 mL separator contg 25 mL 0.27V H 2 S0 4 .
Wash by inverting 5 times and let stand ca 20 min. Filter lower
CHC1 3 layer thru glass wool wetted with CHC1 3 into 100 mL
vol. flask, covering funnel with watch glass. Re-ext aq. soln
in 250 mL separator with 50 mL dye soln, shake vigorously
1 min, drain into same 125 mL separator, and wash and filter
as before, re wetting glass wool with CHC1 3 if necessary. DiL
to vol. with CHC.I3, mix, and place in dark 40 min.
Record spectra of std and sample solns against blank in
matched 5 cm cells, and det. A at max., ca 410 nm.
mg Benztropine mesylate/100 mL — (A/A f ) x C
where A and A' refer to sample and std solns, resp; and C =
concn std soln in mg/100 mL.
Ref.: J AOAC 56, 681(1973).
CAS- 132- 17-2 (benztropine mesylate)
927.12* Mydriatic and Myotic Drugs
Cat-Eye Bioassay Method
Final Action
Surplus 1972
See 36.084-36.088, 12th ed.
XANTHINE ALKALOIDS
Caffeine in Drugs
(a) Microchemical tests. — See Table 960.56.
(b) With acetanilid.— See 916.03*.
(c) With acetanilid and codeine. — See 916.04*.
(d) With acetanilid and quinine. — See 916.05*.
(e) With acetanilid, morphine, and quinine. — See 916.06*.
(f) With phenacetin.— See 916.08*.
(g) With phenacetin and aminopyrine. — See 941.22*.
(h) With phenacetin, aminopyrine, and phenobarhital. — See
942.30*.
(i) With phenacetin and aspirin. — See 960.59*.
(j) With phenacetin, aspirin, and codeine. — See 972.53.
(k) With effervescent potassium bromide. — See 980. 35E*.
936.1 8 Theobromine
in Theobromine-Calcium Salicylate Drugs
Final Action
A Method I
Dry ca 0,5 g sample at 110° to const wt. Weigh 0.2 g dried
substance into g-s 100 mL vol. flask, add 2 mL HO Ac, and
warm on steam bath. Add 10 mL boiling H 2 and shake until
dissolved, adding more boiling H 2 if necessary. Cool soln to
room temp. (Soln should be clear or nearly so.) Add 50 mL
0AN I, 20 mL satd NaCl soln, and 2 mL HC1. Shake well
and dil. to vol. with H 2 0. Shake again and let stand overnight.
Filter, discarding first 10 mL filtrate. Titr. 50 mL filtrate with
0.17V Na 2 S 2 3 , using starch soln, as indicator (mix ca 2 g finely
powd. potato starch with cold H 2 to thin paste; add ca 200
mL boiling H 2 0, stirring const., and immediately discontinue
heating; add ca 1 mL Hg, shake, and let soln stand over the
Hg). 1 mL 0.LV I - 0.00450 g theobromine, C 7 H 8 2 N 4 .
Ref.: J AOAC 19, 534(1936).
Method II
B. Indicator
Phenol red indicator. — Triturate 0.1 g phenol red in agate
mortar with 15 mL 0.027V NaOH until dissolved and dil. soln
to 200 mL with recently boiled H 2 0.
C. Determination
Weigh 0.5 g powd tablets, 0.4 g powder, or 0.2 g theo-
bromine alkaloid into 300 mL beaker and add 100 mL H 2 0.
Warm moderately over flame and add 15 mL ca 0. \N H 2 S0 4 .
Heat to boiling to ensure complete soln and to remove C0 2 .
Cool to room temp. Add 1.5 mL phenol red indicator and make
slightly alk. with ca 0.17V NaOH (violet-red); then titr. care-
fully to acid reaction with 0.17V H 2 S0 4 (yellow). To this soln
add 25 mL (an excess) neut. 0.17V AgN0 3 , 941.18, and im-
mediately titr. liberated HN0 3 with 0. IN NaOH to distinct vi-
olet-red. Cautiously titr. dropwise with const stirring near end
point. 1 mL O.IN NaOH = 0.01802 g C 7 H 8 2 N 4 .
Refs.: JAOAC 21, 555(1938); 22, 729(1939).
CAS-83-67-0 (theobromine)
937.14* Theophylline in Drugs
Gravimetric Method
Final Action
Surplus 1974
(Applicable to solns and tablets)
See 38.101, 12th ed.
IPECAC ALKALOIDS
Emetine Hydrochloride in Drug Tablets
Final Action
See 961. 18B.
AOAC Official Methods of Analysis (1990)
Ipecac Alkaloids
585
971.40 Ipecac Alkaloid in Drugs
Chromatographic Method
First Action 1971
Final Action 1973
(Applicable to sirup, fluidextract, and powd prepns)
(Caution: See safety notes on distillation, triethylamine,
diethyl ether, chloroform, and isooctane.)
A. Principle
Principal ipecac alkaloids, phenolic cephaeline and its Me
ether emetine, constitute over 90% of total alkaloids of ipecac.
They occur in ratios varying from 3:1 to 1:3 in the several
species and constitute total of ca 2-3% of wt of root. Minor
alkaloids, mainly psychotrine, o-methyl psychotrine, and eme-
tamine, are closely related structurally to emetine, differing
principally by presence of addnl double bonds, which affect
their UV spectra.
Four-column system isolates emetine and cephaeline from
ipecac sirup, fluidext, and powd root. Prepn of sample itself,
made alk. with NaHC0 3 , constitutes immobile phase in first
column. Ether eluate of this column, contg total alkaloids to-
gether with other ether extractives from sample, is passed thru
phosphate buffer column IN with respect to chloride ion. Al-
kaloids are retained in column while nonalkaloidal extractives
are partly washed thru column. Major purification is achieved
in next step in which mixt. of CHC1 3 and ether selectively
removes emetine and cephaeline (with perhaps trace amts of
other alkaloids) from phosphate column, and carries them onto
pH 4.0 column, on which they are retained. Purification
achieved in this step is 2-fold: (I) Phosphate buffer column
retains alkaloids (presumably emetamine and psychotrine) which
absorb in UV region between 380 and 300 nm, in which region
emetine and cephaeline do not absorb. (Retained alkaloids can
be recovered in part by elution with CHC1 3 , and remainder
with ether soln of di(ethylhexyl)phosphoric acid, which is very
effective counter-ion for extn of alkaloids.) (2) Eluate from
phosphate column carries thru pH 4.0 column material which
absorbs in UV, This material, if not removed, would accom-
pany emetine and thus give spuriously high assay values. This
is especially significant in the case of ipecac sirup, which con-
tains large amt of substance, provisionally identified as 5-hy-
droxymethyl-2-furaldehyde, spectrum of which closely resem-
bles that of emetine.
Emetine and cephaeline are sepd in final partition step.
Combined alkaloids are eluted from pH 4.0 column after rais-
ing pH to >8 in situ with soln of Et 3 N in mixt. of ether and
isooctane. This eluate continues thru 0.5N NaOH column which
retains phenolic alkaloid cephaeline. Finally, cephaeline is eluted
directly with CHC1 3 from NaOH column, with no adjustment
ofpH.
Respective eluates are extd with 0.57V H 2 S0 4 and A of acid
solns are measured at 283 nm. Since only emetine std is avail-
able, and inasmuch as cephaeline and its Me ether emetine
have essentially same molar A, this std is used for both eme-
tine and cephaeline.
B. Reagents
(a) Triethylamine . — Must pass following test: Transfer 3.0
rnL to 50 mL graduate or vol. flask contg 15 mL AN H 2 S0 4 ,
dil. to vol. with 0.57V H 2 S0 4 , and mix. Scan spectrum from
350 to 240 nm against 0.57V H 2 S0 4 as blank. If A at ca 250
nm is >0.040, purify as in 961.19B(a).
(b) Dimethyl sulfoxide (DMSO). — Spectral grade (Fisher
Scientific Co., or equiv.).
(c) Ethyl ether. — Peroxide-free. See Definitions of Terms
and Explanatory Notes, ether.
(d) Phosphate buffer.— Mix 3 vols 0.5M KH 2 P0 4 with 1
vol. 0.5M K 2 HP0 4 , and adjust to pH 6.0 ± 0.05. Dissolve
7.46 g KCl/100 mL mixt.
(e) Citrate buffer. — Mix equal vols 0.5M citric acid and 0.5M
Na citrate, and adjust to pH 4.0 ± 0.05.
(f) Emetine std soln.— Accurately weigh ca 3 mg eme-
tine. 2HC1.3H 2 and dissolve in 50 mL 0.57V H 2 S0 4 (1 mg
emetine. 2HC1.3H 2 is equiv. to 0.79 mg emetine base). If
alkaloid content of std is not known, det. as in 961. 18B.
C. Preparation of Sample
(a) Sirup. — Pipet 10 mL H 2 into 25 mL vol. flask. Using
20 mL pipet, add sirup to vol., avoiding wetting neck of flask
above graduation mark, and mix. Use 4.0 mL for prepn of
Column L
(b) Fluidextract. — Pipet 5 mL fluidext into 50 mL vol. flask,
dil. to vol. with H 2 0, and mix. Pipet 2 mL into 150 mL beaker.
Evap. almost to dryness on steam bath, using gentle stream of
air to remove alcohol. Add 3 mL H 2 and ca 1 g NaHC0 3 ,
and mix. Proceed as in 971.40D(a), beginning "... add 6 g
diat. earth, and mix."
(c) Powdered ipecac. — Accurately weigh ca 200 mg powd
ipecac (60 mesh) in 150 mL beaker, add 2 mL DMSO, and
mix thoroly with flattened stirring rod to assure complete wet-
ting of powder. Let stand ca 30 min . Add 2 mL H 2 and ca
1 g NaHC0 3 , and mix. Proceed as in 971.40D(a), beginning
"... add 6 g diat. earth, and mix."
D. Preparation of Columns
Transfer specified soln to 150 mL beaker, add specified wt
acid- washed diat. earth, and mix until uniform fluffy mixt. is
obtained.
(a) Column I. — To 4.0 mL sample soln, add 1 g NaHC0 3 ,
mix, add 6 g diat. earth, and mix. Dry-wash beaker with 1 g
diat. earth and add to column. (Since emetine is unstable in
alk. soln and in CHC1 3 , proceed with detn without delay.)
(b) Column II. — Add 3 g diat. earth to 2 mL phosphate
buffer and mix.
(c) Column III. — Add 3 g diat. earth to 2 mL citrate buffer
and mix.
(d) Column IV.— Add 3 g diat. earth to 2 mL 0.5N NaOH
and mix.
Quant, transfer mixts to sep. chromatgc tubes, 967. 31 A.
Tamp each to uniform mass and top with glass wool pad.
E. Determination
(Use H 2 0-satd solvs thruout. Rinse tips of columns with ether
before discarding columns and when changing solvs. Remove
metal leashes from separators and vol. flasks.)
Mount Column II under Column /. Pass three 50 mL por-
tions ether thru columns. Discard eluate and Column /. Mount
Column /// below Column 7/. Pass three 50 mL portions CHC1 3 -
ether (3 + 1) thru columns. Let each portion pass thru com-
pletely before next addn. Continue elution without delay. Dis-
card eluate and Column //. Pass 25 mL CHCl 3 -ether (3 + 1)
thru Column III. Discard eluate. Pass 25 mL ether-isooctane
(1 + 1) thru Column ///. Discard eluate. Prewash Column IV
with 20 mL 2% Et 3 N in ether- isooctane (I + 1) and discard
eluate. Mount Column IV below Column ///. Collect emetine
eluate in 125 mL separator contg 15 mL 4N H 2 S0 4 by passing
10 mL 20% Et 3 N in ether-isooctane (1 + 1), followed by three
10 mL portions 2% Et 3 N in ether-isooctane (1 + 1) thru Col-
umns /// and IV. Discard Column ///. Pass two 10 mL portions
586
Drugs: Part III
AOAC Official Methods of Analysis (1990)
of 2% Et 3 N in ether-isooctane (1 + 1) thru Column IV, col-
lecting eluate in same 125 mL separator. Shake separator, and
transfer acid layer to 50 mL vol. flask. Ext solv. with two "10
mL portions 0.5N H 2 S0 4 and combine in vol. flask. Dil. to
vol. with 0.5JV H 2 S0 4 .
Elute cephaeline from Column IV with 75 mL CHC1 3 . Col-
lect eluate in 250 mL separator contg 20 mL 0.5N H 2 S0 4 and
150 mL ether. Shake and transfer acid layer to 50 mL vol.
flask. Ext solv. with 2 addnl 10 mL portions Q.5N H 2 S0 4 and
combine in vol. flask. Dil. to vol. with 0.5N H 2 S0 4 .
Scan spectra of emetine std and sample fractions from 350
to 240 nm against 0.5N H 2 S0 4 . Correct A at 283 nm by sub-
tracting A at 350 nm for samples (A A) and for stds (AA').
F. Calculations
(a) Simp.— Calc. mg drug/100 mL = 0.1 x (A A/4 A') x
C X D X F, where C = concn std in |xg/mL, D - diln factor
= (25/15) x (50/4) - 20.8, and F for emetine - 0.79 if
emetine. 2HC1. 3 H 2 is used as std; if H 2 content differs from
3H 2 0, recalc. factor from MW of anhyd. salt = 554. F for
cephaeline = 0.79 X 0.971 - 0.767, where 0.971 is ratio of
MW of emetine and cephaeline.
(b) Fluidextract. — Calc. mg drug/ 100 mL as in (a), using
D = (50/2) x (50/5) - 250.
(c) Powdered ipecac. — Calc. % drug = (AA/AA') x (F
x 5C/W), where C and F are defined in (a), and W - mg
sample.
Refs.: JAOAC 54, 609, 614(1971).
CAS-483-17-0 (cephaeline)
CAS-483-18-1 (emetine)
EPHEDRA ALKALOIDS
929.13* Alkaloids in Ephedra
Final Action
Surplus 1965
See 32.047, 10th ed.
931.13 Ephedrine in Drug Inhalants
Final Action
A. Method I*
Surplus 1970
See 36.067, 11th ed.
0. Method Ifr
—Surplus 1983
See 38.050, 14th ed.
950.93* Ephedrine in Water-
Soluble Jellies, Sirups, and Solutions
of Ephedrine Salts
Gravimetric Method
Final Action
Surplus 1972
See 38.039, 12th ed.
947.14 Ephedrine in Drug Tablets
and Capsules
Final Action
A. Method I
See 961. 18B.
B. Method Ifr
—Final Action 1972
—Surplus 1975
See 38.041, 12th ed.
980.35 Ephedrine
in Solid Dosage Drugs
Spectrophotometric Method
First Action 1980
Final Action 1982
A. Principle
Ephedrine is eluted with CH 2 Cl 2 from weakly basic diat.
earth column, retained on weakly acidic column, and con-
verted to benzaldehyde by on-coJumn periodate reaction. Ben-
zaldehyde is detd by UV spectrophotometry.
B. Reagents
(a) Phosphate-chloride soln. — Dissolve 5 g KH 2 P0 4 and
7.5 g KC1 in 100 mL H 2 0. (Omit KC1 for pseudoephedrine
assay.)
(b) Sodium metaperiodate soln.—D is solve 2 g Nal0 4 in 20
mL H 2 0. Store in dark.
(c) Water-saturated methylene chloride. — Sat. ca 400 mL
CH 2 C1 2 by shaking with equal vol. H 2 I min. Use thruout
method.
(d) Ephedrine std soln. — 0.4 mg/mL. Accurately weigh ca
100 mg std ephedrine salt and transfer to 250 mL vol. flask.
Dissolve and dil. to vol. with H 2 0.
C. Apparatus
(a) Recording spectrophotometer. — For UV, with matched
1 cm cells.
(b) Chromatographic tubes and tamping rod. — See
967.31 A(a) and (b).
D. Preparation of Sample and of Chromatographic Columns
Weigh amt well mixed and ground sample contg ca 100 mg
ephedrine salt into 250 mL vol. flask. Add ca 100 mL H 2 0,
let stand ^10 min with occasional shaking, dil. to vol. with
H 2 0, mix well, and let solids settle. Prep, columns as follows:
(a) Column I. — Add 2.0 mL sample soln to 300 mg K 2 HP0 4
in 150 mL beaker. Swirl to dissolve. Add 3 g diat. earth,
960.53B, mix, transfer quant, to column, and tamp. Dry wash
beaker with 1 g diat. earth, add to column, and tamp. Cover
with small pad of glass wool. (If acidic compds such as acet-
aminophen or theophylline are present, underlay column mixt.
with 3 g diat. earth mixed with 2.0 mL 10% NaOH soln.)
(b) Column II. — Mix 3 g diat. earth and 2 mL phosphate-
chloride soln and transfer to column. Tamp and cover with
small pad of glass wool .
(c) Column III.— Mix 0.5 mL H 2 and 1 g diat. earth,
transfer to column, and tamp. Mix 3 g diat. earth and 2.0 mL
NaI0 4 soln, transfer to column, tamp, and cover with pad of
glass wool.
E. Determination
Arrange columns so that effluent from 1 flows into 11. Elute
combined columns with four 25 mL portions of CH 2 C1 2 , letting
each portion sink entirely into surface of both columns. Rinse
tip of column I into column 11 with CH 2 C1 2 , and discard col-
umn I. Elute column II with addnl 25 mL CH 2 CI 2 . Discard all
eluates.
Mount column 11 over column III and place 100 mL vol.
flask under column III. With pipet, evenly distribute 1.0 mL
cone. NH 4 OH directly onto surface of column II. Elute com-
bined columns with four 25 mL portions CH 2 C1 2 , letting each
portion sink entirely into surface of each column. Rinse tip of
AOAC Official Methods of Analysis (1990)
Ephedra Alkaloids
587
column II into column III with ca 1 mL CH 2 C1 2 - Discard col-
umn II and continue elution of column III to vol.
Prep. sep. column III, omitting glass wool at top. Mix 2.0
mL std soln and 3 g diat. earth, transfer quant, to same col-
umn, and tamp. Dry wash beaker with 1 g diat. earth, transfer
to column, tamp, and cover with pad of glass wool. Place 100
mL vol. flask under column. Wet column with 10 mL CH 2 C1 2 .
With pipet, evenly distribute 1.0 mL coned NH 4 OH directly
onto surface of column. Elute column with four 25 mL por-
tions CH 2 C1 2 , letting each portion sink entirely into surface of
column. Rinse tip of column into flask, and dil. to vol. with
CH 2 C1 2 . Elute std column with addnl 25 mL CH 2 C1 2 and col-
lect eluate for use as blank.
Scan spectra of sample and std eluates from 350 to 230 nm
against column blank eluate. If cloudy, let soln clear (ca 1
min) before detg A. Det. A and A' of sample and std solns,
resp., at min., ca 267 nm, and at max., ca 246 nm and det.
difference, A A and AA'.
mg Ephedrine sulfate/dosage unit
= (AA/AA') x C x 250 x D/(2 x W)
where C — concn std, mg/mL; D = av. wt/dosage unit; and
W - mg sample.
Ret.: JAOAC 63, 692(1980).
CAS- 134-72-5 (ephedrine sulfate)
981.26 Pseudoephedrine HCI
and Triprolidine HCI or Chlorpheniramine Maleate
in Drug Combinations
Liquid Chromatographic Method
First Action 1981
Final Action 1983
(Caution: See safety notes on acetonitrile.)
A Principle
Pseudoephedrine HCI and triprolidine HCI or chlorphen-
iramine maleate in syrups or tablets are detd by reverse phase
LC using ion-pairing. Column chromatgc cleanup of syrup re-
moves preservatives.
B. Apparatus
(Equiv. app. may be substituted.)
(a) Liquid chromato graph. — Equipped with Model U6K
injector. Model 6000 A solv. delivery system, and Model 440
UV A detector (Waters Associates, Inc.). Operating condi-
tions: flow rate 1.5 mL/min; 254 nm detector, 0.05 AUFS;
temp, ambient; 10 (xL injection.
(b) LC column. — uTJondapak Phenyl, 10 [xm particle size,
3.9 mm (id) x 30 cm long (Waters Associates, Inc.). Wash
column daily after use with H 2 followed by MeOH.
(c) Recorder. — 10 mV with 0.5 cm/min chart speed (Omni-
scribe B-5000, Houston Instrument, 8500 Cameron Rd, Aus-
tin, XX 78753).
(d) Integrator. — Chromatopac Data Processor Model El A
(Shimadzu Scientific Instruments, Inc., 7102 Riverwood Rd,
Columbia, MD 21046).
(e) Filters. — Millipore types HA and FH (pore size 0.45
|xm) and type AP prefilter (Millipore Corp.).
(f) Chromatographic tubes. — 25 X 2.5 cm od, glass col-
umn with one restricted end.
C. Reagents
(a) Solvents. — Distd-in-glass (Burdick & Jackson Labora-
tories, Inc.), or equiv.
(b) Sodium hydroxide-sodium chloride soln. — 1.0N NaOH-
0.5N NaCl. Dil. 2.0 g NaOH and 1.46 g NaCl to 50 mL with
H 2 0.
(c) Diatomaceous earth. — See 960. 53B.
(d) Internal std soln. — 0.4 mg pheniramine maleate /mL
H 2 0.
(e) Antihistamine soln. — Transfer 31.25 mg accurately
weighed NF Ref. Std Triprolidine HCI or 50 mg USP Ref . Std
Chlorpheniramine Maleate to 100 mL vol. flask and dil. to
vol. with H 2 0.
(f) LC std solns. — (I) Stock soln. — Transfer 30 mg accu-
rately weighed NF Ref. Std Pseudoephedrine HCI to 10 mL
vol. flask. Pipet 4.0 mL antihistamine soln and 5.0 mL inter-
nal std soln into flask and dilute to volume with H 2 0. (2)
Working soln. — Transfer 2.0 mL aliquot stock soln and 2.0
mL H 2 to small g-s flask and mix.
(g) LC mobile phase. — Transfer 1 bottle PIC Reagent B-5
(Waters Associates, Inc.) to 1 L vol. flask. Add 350 mL CH 3 CN,
and dil. to vol. with H 2 0. Mag. stir 5 min. Filter through type
FH filter prewetted with CH 3 CN. Place in ultrasonic bath 10
min to deaerate. (PIC Reagent B-5 contains enough pentane-
sulfonic acid and HOAc so that when 1 bottle is dild to 1 L,
conens are 0.005M and 1%, resp.)
D. Preparation of Samples
(a) Tablets. — Det. av. wt of tablets and grind to pass No.
60 sieve. Transfer accurately weighed portion of powder contg
60 mg pseudoephedrine HCI to 50 mL g-s flask. Pipet 10 mL
internal std soln and 30 mL H 2 into flask and place in ultra-
sonic bath 1 min. Stopper and shake flask vigorously. Filter
portion of soln thru type HA filter into small g-s flask. Use
type AP prefilter if necessary.
(b) Syrups. — Pipet 5.0 mL internal std soln into 10 mL vol.
flask. Carefully add sample syrup to vol. (do not wet flask
above mark) and mix. Place small glass wool plug in base of
chromatgc tube as support. Mix 0.7 mL NaOH-NaCl soln and
1.0 g diat. earth. Transfer mixt. to tube and tamp to uniform
firm mass. Piept 2.0 mL aliquot dild sample soln into 100 mL
beaker. Add 0.3 mL NaOH-NaCl soln and swirl to mix. Add
3.5 g diat. earth and mix thoroly. Transfer mixt. to tube in 3
portions, tamping after addn of each portion. Dry-wash beaker
with 1.0 g diat. earth, add wash to column, and tamp. Cover
with small glass wool plug. Elute column with four 50 mL
portions of H 2 0-satd CHC1 3 into 250 mL g-s flask contg 5 mL
alcohol and 5 drops of HCI. Evap. to dryness on steam bath
with stream of air. Add 5 mL alcohol and evap. to dryness
again. Pipet 4.0 mL H 2 into flask and swirl to dissolve res-
idue. Filter soln through type HA filter into small g-s flask.
E. Determination
Let LC system equilibrate with column in instrument and
set mobile phase at flow rate of 1.5 mL/min. Inject 10 jxL
LC working std soln. The 4 peaks (maleic acid, pseudoephed-
rine, pheniramine, and triprolidine or chlorpheniramine) should
be completely resolved and symmetrical. Make replicate in-
jections of LC working std soln and compare area response
ratios relative to internal std area response to ascertain repro-
ducibility of system. In a suitable system, the coefficient of
variation for 3 replicate injections is not >2.0%. Proceed with
sample analysis, using three 10 |llL injections each of sample
and LC std working solns. If interfering peak (methylparaben)
is observed in chromatogram of syrup sample in region of
pheniramine internal std peak, repeat column chromatgc cleanup,
using another 2.0 mL aliquot of dild sample soln. Pack column
firmly. If baseline resolution is obtained between any residual
methylparaben and pheniramine peaks, repeated cleanup is un-
necessary.
588
Drugs: Part III
AOAC Official Methods of Analysis (1990)
F, Calculations
Calc. results, using area response ratios (R and R') relative
to internal std:
mg Amine salt/tablet = (R/R f ) xCx (T/S) x 2
mg Amine salt/5 mL syrup - (R/R r ) x C
where/? and R f = area response ratio for sample and std solns,
respectively; C = mg amine salt in initial LC std stock soln;
T = av. tablet wt; S = sample wt.
Ref.: J AOAC 64, 564(1981).
CAS-1 13-92-8 (chlorpheniramine maleate)
CAS-345-78-8 (pseudoephedrine hydrochloride)
CAS-550-70-9 (triprolidine hydrochloride)
ERGOT ALKALOIDS
985.49 Colchicine in Drugs
Liquid Chromatographic Method
First Action 1985
Final Action 1987
{Caution: Colchicine is extremely poisonous.)
A. Principle
Colchicine is dild or extd with MeOH-H 2 (1 + 1) and detd
directly by LC with UV detection at 254 nm.
B. Apparatus
(a) Liquid chromato graph.— Model 100 A pump, Model 153
UV detector (replacement Model 116 Programmable Solvent
Module and Model 160, resp.) (Beckman Instruments, Inc.,
Altex Div., 2300 Camino Ramon, PO Box 5101, San Ramon,
CA 94583-0701) equipped with injection valve with 20.0 jxL
sample loop, reporting integrator (Hewlett-Packard Co.,
Avondale Div) or equiv. system equipped with strip chart re-
corder, Operating conditions: mobile phase flow rate 1.0 mL/
min, detector set at 254 nm, sensitivity 0.005 AUFS, temp,
ambient, chart speed 0.5 cm/min.
(b) LC column. — Ultrasphere Octyl (C 8 ), 5 jxm particle size,
4.6 mm id x 25 cm stainless steel (Beckman Instruments, inc.,
Altex Div.) or equiv. column packed with 5-6 (Jim octyl silane
bonded spherical particles which meets chromatgc system suit-
ability requirements. Wash column after use with MeOH-H 2
(1 + 1), and then with MeOH.
(c) Filters. —Cellulose triacetate membrane filter, pore size
0.45 |xm.
(d) Glassware. — Use low-actinic vol. flasks for all sample
and std solns.
C. Reagents
(a) Methanol. — UV or LC grade (Burdick & Jackson Lab-
oratories, Inc.) or equiv.
(b) Extraction and dilution solvent. — MeOH-H 2 (1 + 1).
Mix equal vols of MeOH and H 2 0, cool to room temp., and
pass soln thru 0.45 jim membrane filter.
(c) Potassium phosphate (monobasic) buffer. — 0.5M. Dis-
solve 68.0 g KH 2 P0 4 (LC grade, Fisher Scientific Co., or
equiv.) in H 2 and dil. to 1 L.
(d) Phosphoric acid. — 0.5M. Dil. 3.4 mL H^P0 4 (AR grade,
85%) to 100 mL with H 2 0.
(e) LC mobile phase.— MeOH-0.05M KH 2 P0 4 (55 + 45),
pH 5.5. Dil. 45 rnL 0.5M KH 2 P0 4 buffer (c) to 450 mL with
H 2 0, then further dil. to ca 1 L (e.g., 980 mL) with MeOH.
Cool to room temp, and adjust vol. to 1 L with MeOH. Adjust
pH to 5.5 (±0.05) with 4-10 drops 0.5M H 3 P0 4 (d). Filter
thru 0.45 |xm membrane filter.
(f) Colchicine std soln. — 0.006 mg/mL. Prep, all dilns with
MeOH-H 2 (1 .4- 1). Transfer 30 mg accurately weighed (3
mg if using microbalance) USP Colchicine Ref. Std, previ-
ously dried 3 h at 105°, to 50 mL vol. flask; dissolve and dil.
to vol. Dil. this soln quant, to 0.006 mg/mL. Soln is stable
>4 months when stored tightly stoppered and in dark.
D. Chromatographic System Suitability Test
(a) Repeatability. — Let chromatgc system equilibrate with
mobile phase ^45 min. Inject 20 jxL colchicine std soln and
adjust sensitivity to provide 50-70% FDS. Inject 5 replicate
20 |xL vols of same soln and compare peak response values
(peak ht or area). Coeff. of var. should be <2%.
(b) Retention. — Retention time for colchicine should be be-
tween 5.5 and 9.5 min (depending on brand and age of col-
umn).
(c) Column efficiency. — No. of theoretical plates (N) detd
from colchicine response is >4500 when calcd by following
expression:
N = 5.54(; R /W . 5 ) 2
where t R and W 0m5 are retention time and peak width at 50%
ht measured in mm, resp. Use chart speed of 2-4 cm/min to
facilitate accurate peak width measurement. Note: If small im-
purity peak elutes just before colchicine response, it should be
completely resolved from major peak.
E. Preparation of Sample
Carry out all extns and dilns with MeOH-H 2 (1 + 1) in
low- actinic glassware.
(a) Tablets. — Weigh and finely powder, to pass No. 60 sieve,
>20 tablets. Transfer accurately weighed portion of composite
equiv. to 0.6 mg colchicine into 100 mL vol. flask, add ca 50
mL MeOH-H 2 (1 + 1) and mech. shake for total of 15 min,
rinsing down walls of flask at ca 8 min. Dil. to vol. and filter
portion thru 0.45 jxm membrane filter.
(b) Injections. — Transfer accurately measured vol. (V of
liq. equiv. to 1 mg colchicine into 50 mL vol. flask and dil.
to vol. Transfer 30.0 mL of this soln into 100 mL vol. flask
and dil. to vol.
(c) Bulk drug. — Accurately weigh ca 60 mg well mixed
sample into 500 mL vol. flask, dissolve, and dil. to vol. Trans-
fer 5.0 mL of this soln to 100 mL vol. flask and dil. to vol.
F. Determination
Immediately after extn and diln of sample, inject duplicate
20 fxL vols of colchicine std soln and sample soln in alter-
nating sequence. Calc. results by using av. peak response val-
ues as follows:
Tablets: mg colchicine/tablet - (R/R f ) x C x 100 x (T/W)
Injections: mg colchicine /mL = (R/R') x C x (166. 7/V)
Bulk drug: % colchicine (as-is basis)
- (R/R f ) x C x 10 000 x (100/W0
where R and R' — peak response values of sample and std
solns, resp.; C = concn of colchicine std soln, mg/mL; J =
av. tablet wt, mg; W = sample wt, mg; V — vol. injection
taken for analysis, mL.
Ref.: JAOAC 68, 1051(1985).
CAS-64-86-8 (colchicine)
AOAC Official Methods of Analysis (1990)
Ergot Alkaloids 589
960.60 Ergotamine in Drugs
Chromatographic Method
First Action 1960
Final Action 1962
(Applicable in presence of caffeine, phenacetin, pheno-
barbital, and belladonna alkaloids)
A. Reagents
(a) Tartaric acid soln. — 1%. Dissolve 10 g tartaric acid in
H 2 and dil. to 1 L.
(b) Alcoholic tartaric acid soln. — Mix equal vols tartaric
acid soln, (a), with alcohol. Prep, fresh daily.
(c) Sodium bicarbonate soln. — 10%. Dissolve 100 g
NaHC0 3 in H 2 and dil. to 1 L.
(d) Citric acid soln. — (1 + 1). Mix equal wts of citric acid
andH 2 0.
(e) Alum soln.—0.25M. Dissolve 12 g KA1(S0 4 ) 2 .12H 2
in H 2 and dil. to 1 L. pH should be 3.5 ± 0.2.
(f) Color reagent. — Dissolve 1.25 g p-dimethylaminoben-
zaldehyde in cooled mixt. of 650 mL H 2 S0 4 and 350 mL H 2 0.
Add 0.5 mL 9% FeCl 3 soln.
(g) Diatomaceous earth. — See 960. 53B.
(h) Ergotamine tartrate std soln. — 50 (xg/mL. Dissolve 25
mg ergotamine tartrate, USP, in enough tartaric acid soln, (a),
to make 500 mL.
B. Preparation of Chromatographic Coiumn
Chromatographic tube. — Prep, chromatgc tube as in
967.314(a). Fit with packing rod, 967.31A(b). Place small
wad of glass wool in bottom of tube.
Ergotamine -retaining layer. —Add ca 4 g diat. earth to 3
mL citric acid soln in beaker. Mix thoroly with scoop- shaped
spatula until mixt. appears fluffy and uniform, and transfer to
chromatgc tube. Tap side of tube gently to settle mixt. Press
down firmly with packing rod.
Ergonovine -retaining layer. — Add ca 2 g diat. earth to 2
mL alum soln, mix, and transfer to tube on top of citric acid
layer. Press down firmly and evenly.
Water layer. — Add ca 2 g diat. earth to 2 mL H 2 0, mix,
and transfer to tube on top of alum layer. Press down firmly
and evenly and top with pad of glass wool.
C. Preparation of Sampie
(a) Tablets. — Det. av. wt and reduce to fine powder. Ac-
curately weigh portion contg ca 2.5 mg ergotamine tartrate
into beaker. Mix thoroly with 5 mL 1% tartaric acid soln and
let stand 30 min. Add 5 mL CHC1 3 and 1 mL 10% NaHC0 3
soln, and mix. (Aq. phase must be alk.) Add ca 7 g diat. earth
and stir thoroly until mass appears uniform and does not stick
to beaker. (It may be necessary to wash down sides of beaker
with small amts of CHC1 3 .) Add and mix more diat. earth as
may be necessary to make mixt. workable. Quant, transfer mixt.
to another chromatgc tube fitted with glass wool plug, in sev-
eral portions, pressing down firmly with packing rod. Wash
packing rod, spatula, and sides of beaker with small amt (ca
5 mL) of CHC1 3 . Add enough diat. earth to make mixt. work-
able. Scrub sides of beaker and add mixt. to tube. Again rinse
rod, spatula, and beaker with CHC1 3 and pour wash onto col-
umn.
(b) Suppositories. — Place suppositories contg 3-5 mg er-
gotamine tartrate in 125 mL separator. Add 10 mL 0.2N H 2 S0 4
and 75 mL ether. Shake until sample dissolves and then 1 min
more. Drain acid layer into second separator. Complete extn
with three 10 mL portions acid. Discard ether layer. Combine
exts and make alk. with NH 4 OH. Promptly ext alkaloids with
four 10 mL portions CHCI3. Pass each CHC1 3 ext directly onto
prepd column, 960.60B. Let column drain completely between
addn of successive exts. Proceed as in 960. 60D, second par.
D. Separation of Ergotamine
Place tube contg sample so that effluent will flow directly
onto water layer of second column. Add 50 mL H 2 0-satd ether
to top column and receive eluate from bottom column in 250
mL erlenmeyer. Follow with 50 mL H 2 0-satd CHC1 3 . (Since
effluent may flow faster thru sample column than thru second
column, do not add too much CHC1 3 at a time.) Rinse tip of
sample column with CHC1 3 from wash bottle and discard sam-
ple column.
Let column drain completely and then rinse down sides with
small amt of CHC1 3 . Pass thru addnl 25 mL H 2 0-satd CHC1 3
into same flask and rinse tip of column with alcohol. Discard
effluent if ergotamine was properly retained. {See Note.)
Inspect column for proper retention of ergotamine and for
presence of H 2 0-sol. alkaloids by holding column under UV
light very briefly. (Caution: See safety notes on hazardous ra-
diations.) Blue fluorescent band must not be at bottom of col-
umn. (See Note.) Extrude column into 400 mL beaker. Rinse
tube with H 2 0. Add 8 g NaHC0 3 and ca 25 mL H 2 to form
aq. liq. layer. Break up column with spatula and mix. Wash
mixt. with H 2 from wash bottle into 250 mL separator. Add
10 mL CHC1 3 and shake. Check aq. layer to assure that it is
alk. Be sure that layers are well sepd. It may be necessary to
break CHC1 3 bubbles with wire. Drain CHC1 3 layer thru glass
wool filter into 100 mL vol. flask. Ext aq. layer with four 10
mL portions CHC1 3 and filter solv. layers into the 100 mL vol.
flask. DiL to vol. with CHC1 3 , and mix.
E Determination
Evap. 10 mL aliquot CHC1 3 soln in 50 mL erlenmeyer to
dryness with air current. Do not heat. (Ergotamine is easily
decomposed. If assay cannot be completed in 1 day, dried res-
idue after evapn of CHCI3 may be stored in refrigerator over-
night.) Dissolve residue, equiv. to 0.25 mg ergotamine tar-
trate, in 5.0 mL ale. tartrate soln. Pipet 5.0 mL std soln into
50 mL erlenmeyer. Add, to each, 10 mL color reagent drop-
wise while swirling continuously in ice-H 2 bath. After 30,
but <60 min, det. A of sample and of std, A', at 550 nm
against blank prepd by mixing 5 mL H 2 and 10 mL reagent.
mg Ergotamine tartrate in sample weighed = (A /A') X 2.5
Note: Ergot alkaloids fluoresce bright blue when exposed to
UV light at ca 360 nm. If fluorescent band has reached bottom
of trap layer, sample must be discarded. If desired, sample
can be salvaged by combining column and effluent, shaking
with CHC1 3 , and passing thru another acid trap. Use of eluant
which is not H 2 0-satd will cause loss of ergotamine from col-
umn. Blue fluorescent ring at top of alum layer indicates pres-
ence of H 2 0-sol. ergot alkaloids. If detn of H 2 0-sol. alkaloid
content is desired, repeat detn on new portion of sample,
changing citric acid trap to one prepd by mixing 3 g diat. earth
with 3 mL alum soln. Cover alum layer with mixt. of 2 g diat.
earth and 2 mL H 2 0.
Refs.: JAOAC 43, 224(1960); 46, 634(1963).
CAS-379-79-3 (ergotamine tartrate)
961.20 Ergotamine in Drugs
Paper Chromatographic Identification
First Action 1961
Final Action 1962
A. Reagents
(a) Mobile solvent. — Dissolve 7.1 g Na citrate in H 2 and
dil. to 100 mL. Adjust pH to ca 4.7 with 2N HC1 and transfer
590
Drugs: Part
AOAC Official Methods of Analysis (1990)
to separator. Add 70 mL formamide and 9 mL di methyl -
phthalate. Shake vigorously, Jet sep., and drain and discard
lower layer. Adjust to pH 5.2 with 2N HC1 or NaOH.
(b) Immobile solvent. — Dimethylphthalate-CHC1 3 (1 + 9).
Prep, immediately before use.
(c) Ergotamine std solns. — Accurately weigh 10 mg ergo-
tamine tartrate, USP, into small separator contg 5 mL 1% tar-
taric acid soln and mix gently. Make alk. with few drops 10%
NaHC0 3 soln, add 2.0 mL CHC1 3 , and shake vigoroLisly. Draw
off CHC1 3 layer (std soln J). Dil. 1.0 mL std soln 1 to 25 mL
with CHCI3 {std soln 2).
B. Identification
For details of app. and technic see 970.52G and 970.52BB*.
Blotter paper liners must be used in tank and tank must be
sealed.
Equilibrate mobile solv, in sealed tank ^3 hr with liners
dipping into solv. Just before use, quickly dip marked 8 x 8"
paper once in freshly prepd immobile solv. and let dry 15 min.
Prep, soln of ergotamine in CHC1 3 , as in 960. 60D, contg
2.5 mg/0.5 mL (remainder of CHC1 3 soln from assay may be
evapd to this concn). Spot 10 jxL each of sample and std solns
on paper and let solv. evap. Place paper in tank, seal tank,
and let chromatogram develop until solv. front is ca 2.5 cm
from top (ca 3 hr). Let paper dry overnight in hood and ex-
amine under UV light. There should be one yellow primary
spot (and there may be a "tail," probably as result of ergota-
mine changing to ergotaminine during developing) corre-
sponding to 50 |mg ergotamine spot (std 1) in position and in-
tensity. If any other spot is more intense than that of std 2,
>2 |xg other ergot alkaloids, expressed as ergotamine tartrate,
are present.
Note: Ergot alkaloids produce blue fluorescence which on
overnight contact with formamide and air changes to yellow
fluorescence. Paper must be completely dry and std 2 spot clearly
visible. In humid weather it may be necessary to dry developed
paper 2-3 days in well ventilated hood.
Ref.: JAOAC44, 288(1961).
CAS-379-79-3 (ergotamine tartrate)
PHYSOSTIGMINE ALKALOIDS
982.37 Physostigmine Salicylate
and Physostigmine Sulfate
in Drug Solutions and Ointments
Chromatographic Method
First Action 1982
Final Action 1987
A. Principle
Physostigmine salicylate or sulfate is dild with CH 3 CN and
detd by LC with UV (254 nm) detector and with flurazepam
as internal std.
B. Apparatus
(a) Liquid chromatography — Model 204 equipped with 2
Model 6000 pumps, Model 660 solv. programmer, 254 nm
UV detector, Model U6K injector (Waters Associates, Inc.)
and Model 3380A integrator (Hewlett-Packard). Equiv. LC
system with strip chart recorder may be used.
(b) LC column. — (xBondapak C 18 , 3.9 mm id x 30 cm
(Waters Associates, Inc.) or equiv. reverse phase column pro-
viding appropriate retention times and sepn for physostigmine
and internal std.
C. Reagents
(a) Ammonium acetate. — 0.05M. Dissolve 3.85 g NH 4 OAc
in H 2 and di 1 . to 1 L. Filter thru 4.7 cm Whatman GF/F
glass microfiber filter, or equiv., in Millipore-type filter holder.
Adjust filtrate to pH 6.0 ± 0.1 with HOAc or NH4OH.
(b) Solvents. — UV grade hexane and CH 3 CN (Burdick &
Jackson Laboratories, Inc., or equiv.); filtered thru same filter
as in (a).
(c) Mobile phase.— CH 3 CN~0.05M NH 4 OAc (1 + 1) at flow
rate of ca 2.0 mL/min. Mobile phase ratio and flow rate
may be varied to give retention time of ca 3-4 min for
physostigmine peak (first) and sepn of flurazepam internal std
peak (second).
(d) Internal std soln. — Dissolve 50 mg flurazepam HC1 in
MeOH and dil. to 100 mL with MeOH.
(e) Physostigmine std solns. — 3.0 mg/100 mL. Transfer 60
mg accurately weighed USP Physostigmine, Physostigmine
Salicylate, or Physostigmine Sulfate to 100 mL vol. flask and
diL to vol. with CH 3 CN. Transfer 5,0 mL aliquot to 100 mL
vol. flask contg 5.0 mL internal std soln and dil. to vol. with
CH 3 CN. Use physostigmine and salicylate stds without drying.
Dry sulfate std 2 h at 105°.
D. Sample Preparation
(a) Solutions. — Transfer aliquot of sample (V) contg ca 3
mg physostigmine or its salts to 100 mL vol. flask contg 5.0
mL internal std soln and diL to vol. with CH 3 CN.
(b) Ointments. — Transfer accurately weighed sample (W)
contg ca 3 mg physostigmine or its salts to 60 mL separator.
Add 20 mL n-hexane and ext with four 20 mL portions of
CH 3 CN. Collect exts in 100 mL vol. flask contg 5.0 mL in-
ternal std soln and dil. to vol. with CH 3 CN.
E System Suitability (Chromatographic System) Check
(a) Repeatability. — Let system equilibrate with flow rate of
ca 2 mL/min. Then make four 10.0 (jlL injections of any std.
soln. Measure coeff. of variation of peak response for 4 in-
jections by following formula:
cv, % = 100
2(jc - x) 2
n - 1
where x = ratio of area of physostigmine peak divided by area
of internal std peak, x = mean of these ratios, and n = number
of injections.
Coeff. of variation should be <2%. If reproducibility is un-
satisfactory, let system equilibrate longer and repeat test.
(b) Resolution. — Retention time for physostigmine peak
should be 2.5-4.5 min. Resolution f actor , R, for physostig-
mine peak and internal std peak should be >3.0, using fol-
lowing formula:
R = 2(t' - t)/(PW + PW)
where t and t' = mm retention of physostigmine and internal
std peaks, respectively; and PW and PW = mm peak widths
measured at baseline of physostigmine and internal std, re-
spectively.
F. Determination
Make duplicate 10 |mL injections each of sample soln and
appropriate std soln, alternating sample and std solns. Calc.
results by using response ratios (RR) reL to internal std, based
on peak areas:
For solns:
Physostigmine (or salt), mg/mL
100 X (RR/RR f ) X (C/V)
AOAC Official Methods of Analysis (1990)
Physostigmine Alkaloids
591
For ointments:
Physostigmine (or salt), mg/g
- 100 X (RR/RR F ) X (C/W)
where RR and RR' = response ratio of sample and std, resp.;
C = concn of std (mg/100 mL); V = mL soln; W = g oint-
ment. Identification is based on same retention times for sam-
ples and stds.
Refs.: JAOAC 65, 132(1982); 66, 339(1983).
(b) Individual tablets. — Transfer tablet to 50 mL c&ntrf. tube,
powder if coated, and add 6.0 mL pH 5.8 phosphate buffer
by pipet. Stopper, shake mech. 30 min, and centrf. 5 min at
high speed. Pipet 2.0 mL clear supernate into 150 mL beaker
and proceed as in 974. 42D.
(c) Ophthalmic soln. — Dil. accurately measured vol. sam-
ple soln to 2.5 mg neostigmine bromide/mL with pH 5.8
phosphate buffer soln. Pipet 2.0 mL dild sample soln into 150
mL beaker and proceed as in 974. 42D.
D. Determination
948.31 *
Physostigmine
in Drug Ointments
Final Action
Surplus 1965
See 32.089, 10th ed.
See 961. 18B.
942.31*
Physostigmine Salicylate
in Drug Tablets
Final Action
Neostigmine in Drugs
Distillation Method
Final Action
Surplus 1974
See 38.159, 12th ed.
974.42 Neostigmine in Drugs
Chromatographic Method
First Action 1974
Final Action 1976
A. Apparatus
(a) Spectrophotometer. — Suitable for measurement in range
265-400 nm.
(b) Chromatographic tube and tamping rod. — See 967. 31 A.
B. Reagents
(a) Phosphate buffer. -^-^H 5.8. Mix 1 vol. 1A/K ? HP0 4 (17.4
g/100 mL) with 4 vols. \M KH 2 P0 4 (13.6 g/100 mL). Adjust
pH, using pH meter, to 5.80 ± 0.05 with either component.
(b) Washed chloroform. — Shake equal vols CHC1 3 and H 2
in separator. Let layers sep. 5 min and discard upper layer.
(c) Washed ether. — Shake equal vols ether and H 2 in sep-
arator. Let layers sep. 5 min and discard lower layer.
(d) Bis(2-ethylhexyl) hydrogen phosphate (DEHP) soln. —
2.5% Mix 2.5 mL DEHP with 97.5 mL H 2 0- washed CHC1 3 .
(e) Diatomaceous earth. — See 960. 53B.
(f) Neostigmine std soln. — Dry neostigmine bromide 3 hr
in 105° oven. Accurately weigh ca 5 mg dry std, using mi-
crobalance, and transfer to 150 mL beaker. Add 2.0 mL pH
5.8 phosphate buffer, mix by swirling gently, and proceed as
in 974.42D.
C. Preparation of Sample
(a) Tablets. — Accurately weigh portion powd tablets contg
5 mg neostigmine bromide into 150 mL beaker, add 2.0 mL
pH 5.8 phosphate buffer, mix by swirling gently, and proceed
as in 974.42D.
(Caution: See safety notes on distillations, diethyl ether, chlo-
roform, and isooctane. Use H 2 0- washed sotvs thruout.)
Treat std and sample solns similarly. Add 3.0 g diat. earth,
mix with metal spatula until fluffy, and transfer quant, in 3
portions to chromatgc tube contg 1 g diat. earth mixed with
0.5 mL pH 5.8 phosphate buffer. Pack uniformly. Dry- wash
beaker with 0.2 g diat. earth, transfer wash to tube, and pack
uniformly. Wipe beaker and all app. used in column prepn
with glass wool and add to column. Proceed without delay.
Wash column with 75 mL ether and then with 75 mL CHCl 3 .
Discard washings. Elute neostigmine bromide with 75 mL 2.5%
DEHP soln into 500 mL separator contg 20 mL 0. IN H 2 S0 4 .
Complete elution with 25 mL CHC1 3 . Add 175 mL isooctane
to eluate and shake vigorously 2 min. Let stand >5 min to
completely sep. layers. Transfer lower aq. layer to 250 mL
beaker. Repeat extn with two 20 mL portions 0.17V H 2 S0 4 ,
and combine aq. layers in the 250 mL beaker.
Add 10 mL 10% NaOH soln to beaker, mix by swirling
gently, cover with watch glass, and heat 45 min on vigorous
steam bath. Cool, transfer quant, to 100 mL vol. flask, dil. to
vol. with H 2 0, and mix. Centrf. portions of std and sample
solns. Record spectra of clear sample and std solns between
400 and 255 nm against 1% NaOH soln in 1 cm cells. Det.
A A of each soln by subtracting A at 340 nm from A at max.,
ca 293.5 nm.
mg Neostigmine bromide in final soln
- (AA/ZU') x C x 100
where C = mg neostigmine bromide std/mL, and A A and A A'
refer to sample and std, resp.
Ref.: JAOAC 57, 725(1974).
CAS- 11 4-80-7 (neostigmine bromide)
971.41 Neostigmine Methylsulfate
in Drugs
Spectrophotometric Method
First Action 1971
Final Action 1975
(Applicable only to injections)
A. Apparatus and Reagents
(a) Recording spectrophotometer. — Suitable for measure-
ment in range 230-350 nm.
(b) Neostigmine methylsulfate std soln. — 0.5 mg/mL. Ac-
curately weigh 50 mg neostigmine methylsulfate of known pu-
rity, transfer to 100 mL vol. flask, add 1 mL \N H 2 S0 4 , and
dil. to vol. with H 2 0.
B. Preparation of Sample
(a) Interfering UV -absorbing preservatives absent. — Transfer
aliquot contg 5.0 mg neostigmine methylsulfate to 150 mL
beaker and hydrolyze as in 971. 41D.
592
Drugs: Part
AOAC Official Methods of Analysis (1 990)
(b) Injection solns containing phenol or parabens. — Pro-
ceed as in 971.41C.
C. Extraction of Interferences
Transfer sample aliquot (or sample diln if necessary) contg
5.0 mg neostigmine methylsulfate to 125 mL separator, add 1
mL IN H 2 S0 4 , and add H 2 to total vol. of 21 mL. Add 35
mL CHC1 3 and shake vigorously 2 min. Transfer CHC1 3 layer
into second 125 mL separator contg wash soln of 10 mL H 2
and 1 mL IN H 2 S0 4 ; shake, let layers sep. completely, and
discard CHC1 3 phase. Repeat extn with 5 addnl 35 mL portions
CHCI3. Rinse stem of each separator with CHC1 3 after last extn
and discard CHC1 3 . Combine aq. layers in 150 mL beaker.
Rinse each separator in succession with two 5 mL portions
H 2 0, rinse stem of each separator with H 2 0, transfer rinsings
to beaker, and proceed as in 971. 41D.
D. Hydrolysis
Add 25 mL 10% NaOH soln and H 2 to ca 80 mL. Cover
with watch glass and heat 30 min on vigorous steam bath.
Cool, quant, transfer soln thru loose glass wool plug, pre-
washed with 1% NaOH soln, to 250 mL vol. flask, and dil.
to vol. with H 2 0. In sep. beaker, similarly treat 10 mL aliquot
std neostigmine methylsulfate soln. Perform blank detn, omit-
ting neostigmine methylsulfate. Proceed as in 971.41E.
E. Determination
Record spectra of sample and std solns, relative to blank,
in 1 cm cells, from 350 to 230 nm. Det. A A of each soln by
subtracting A at 350 nm from A at max., ca 239 nm.
mg/mL Neostigmine methylsulfate in sample
= (AA/AA') x 0.5 X (10/mL sample aliquot)
where A A and A A' refer to sample and std, resp.
Ref.: J AOAC 54, 21(1971).
CAS-5 1-60-5 (neostigmine methylsulfate)
CH1NCHONA ALKALOIDS
Quinine in Drugs
Final Action
(a) Microchemical tests.— See Table 930.40.
(b) Optical cry stallo graphic properties. — See 955.52 and
955.58.
(c) With acetanilid and caffeine. — See 916.05*.
(d) With acetanilid, caffeine, and morphine. — See 916.06*.
(e) With diacetylmorphine . — See 955.56*.
942.32* Quinine in Drugs
Spectrophotometric Method
Final Action
Surplus 1974
See 38.066-38.067, 12th ed.
947.15* Quinine Ethylcarbonate
in Drugs
Titrimetric Method
Final Action
Surplus 1974
See 38.068, 12th ed.
962.22 Elixirs of Iron, Quinine,
and Strychnine in Drugs
First Action 1962
Final Action 1965
A. Method I— Surplus 1974*
See 38.069-38.074, 12th ed.
Method II
B. Apparatus
See 967.31 A.
C. Reagents
(a) Strychnine sulfate std solns.- — (I) Stock soln. — 250 (xg/
mL. Dissolve 25.0 mg strychnine sulfate in MeOH and dil. to
100 mL with MeOH. (2) Working soln.— 50 |xg/mL. To 10
mL stock soln add 5 drops HC1 and dil. to 50 mL with CHC1 3 .
(b) Triethylamine. See 961.196(a).
D. Preparation of Sample and Columns
Sample. — Pipet 10 mL sample into 100 mL beaker, add 0.2
g /?-toluenesulfonic acid, and heat on steam bath under gentle
air current to remove alcohol.
Column /.—Add 2 mL 2/V NaOH to 3 g diat. earth, 960.53B.
Mix thoroly by kneading with flexible spatula, transfer to col-
umn, and tamp, using gentle pressure, to uniform mass. Add
8 g diat. earth to alcohol-free sample. (If sample is too sirupy
from excess evapn of H 2 0, add small amt of H 2 0.) Mix tho-
roly, transfer to column above NaOH layer, and tamp. Dry-
wash with ca 1 g diat. earth for quant, transfer.
Column If. — Mix 3 g diat. earth and 2 mL 2N NaOH, and
tamp as above. Mix 8 g diat. earth and 7 mL IN HC1, transfer
to column above NaOH layer, and tamp.
Column III .-—Mix 3 g diat. earth and 2 mL 17V tartaric acid,
and tamp as above.
Place small pad of glass wool above each column.
E. Determination
(Use H 2 0-satd solvs thruout.)
Pass 100 mL ether thru Column /, discarding eluate contg
aromatic flavoring components and bulk of transformation
product of quinine which forms in aged prepns.
Mount columns so that eluate from / passes thru // onto ///.
Pass 100 mL CHCI3 thru columns and discard Column /. Pass
50 mL CHCI3 thru Column // onto /// and finally pass 50 mL
CHCI3 thru Column ///. Discard eluate. Column // may be
used for quinine detn as in 38.074*, 12th ed.
Place 50 mL vol. flask contg 10 mL MeOH and 1 mL HC1
under Column /// which contains strychnine. Pass thru column
5 mL CHCI3 contg 1 mL triethylamine, followed by 32 mL
1% triethylamine in CHC1 3 . Dil. to vol. with CHC1 3 . Det. A
at 350, 320, and 288 nm against CHC1 3 or, preferably, record
spectrum over this region. (Film of Et 3 N.HCl may adhere to
walls of cells. Rinse cells carefully with H 2 and alcohol; then
wipe clean before use.) Background A at 310-360 nm should
be <0.02. Deduct av. reading at 320 and 350 nm from reading
at inflection at 288 nm. (Max. A of strychnine is at ca 255 nm
but nature of solvs makes it undesirable to use this wave-
length.)
Deduct A of blank of 10 mL MeOH, 1 mL triethylamine,
and 1 mL HC1 dild to 50 mL with CHC1 3 . Compare net A. with
that of dild strychnine sulfate std soln, A', and calc. strychnine
content.
AOAC Official Methods of Analysis (1990)
Rauwolfia Alkaloids
593
Ref.: JAOAC 45, 595(1962).
CAS- 130-95-0 (quinine)
CAS-57-24-9 (strychnine)
947.16*
Pamaquine in Drugs
Titrimetric Method
Final Action
Surplus 1974
See 38.169-38.170, 12th ed.
944.15* Quinacrine Hydrochloride
in Drugs
Volumetric Method
Final Action
Surplus 1972
See 38.216-38.217, 12th ed.
970.83 Quinacrine Hydrochloride
in Drugs
Fluorometric Method
First Action 1970
Final Action 1974
{Caution: See safety notes on photofluorometers.)
A. Apparatus
Spectrophotofluorometer . — Scanning, with 1 cm cell path,
Xe lamp, excitation wavelength 420 nm, and sensitivity to
produce 80% fluorescence intensity (F) for std soln.
B. Reagent
Quinacrine hydrochloride std soln.— 0.00050 mg/mL. Weigh
5.0 mg USP Quinacrine. HC1 Ref. Std in 1 L vol. flask and
dil. to vol. with H 2 0. Mix well and dil. 10.0 mL to 100 mL
with 0.17V HCl, Alternatively, weigh 50.0 mg quinacrine. HCJ
into 1 L vol. flask and dil. to vol. with H 2 0. Mix well and
dil. 10.0 mL to 1 L with 0.1N HCl. Prep, fresh daily.
C. Preparation of Sample
(a) Tablets and powders,— Weigh amt of well mixed or well
ground sample contg 100 mg quinacrine. HCl into 200 mL vol.
flask. Dil. to vol. with 0.\N HCl, mix 2 min, and filter if
necessary. Dil. 10.0 mL clear sample soln to 1 L with H 2
and mix. Finally dil. 10 mL to 100 mL with 0.17V HCl to
obtain sample soln.
(b) Liquids. — Pipet accurate sample contg ca 100 mg quin-
acrine. HCl into 200 mL vol. flask. Proceed as in (a), begin-
ning "Dil. to vol. with 0.1 JV HCl, . . ."
D. Determination
Adjust spectrophotofluorometer to ca 80% fluorescence in-
tensity (F) at 500 nm with std soln. Transfer ca 3 mL 0AN
HCl to clean 10 x 10 mm cell and record the blank scan be-
tween 350 and 650 nm. Repeat with std and sample solns. In
each case, draw baseline from 350 to 650 nm. Det. %F at peak
max. (ca 500 nm) of sample and std solns relative to 0.1 N
HCl blank.
Calc. as follows:
Liqs: mg quinacrine. HCl /mL
- 200,000 x C x (F/F') x (1/V)
Solids: % by wt quinacrine. HCl
= 200,000 x C x (F/F r ) x (100/W)
where C = mg/mL std soln; F and F f , resp., = fluorescence
of sample and std solns at 500 nm, each corrected for blank;
W - mg sample; and V = mL sample.
E. Identification
Set emission wavelength monochromator at wavelength of
max. fluorescence, i.e., 500 nm. Scan std and sample solns
used for quantitation with excitation wavelength monochro-
mator from 200 to 750 nm.
Use same instrument parameters as for quantitation except
set sensitivity at ca 40. Sample and std spectra exhibit identical
max. and min.
Refs.: JAOAC 53, 117(1970); 65, 484(1982).
CAS-69-05-6 (quinacrine hydrochloride)
RAUWOLFIA ALKALOIDS
958.17 Reserpine in Drugs
Spectrophotometric Method I
Final Action 1965
A. Reagents
(a) Sulfamic acid soln. — 2.5%. Prep, fresh every 2-3 days.
(b) Alcoholic sodium nitrite soln. — Dissolve 10 g NaN0 2
in 100 mL H 2 0. Store in refrigerator. Mix 1 mL of this aq.
soln with 50 mL alcohol.
(c) Reserpine std soln.- — 50 (xg/mL. Dissolve 25 mg USP
Reserpine Ref. Std, previously dried 3 hr at 60°, in ca 40 mL
boiling alcohol, cool, and dil. to 100 mL with alcohol. Dil.
10 mL of this stock soln to 50 mL with alcohol. When stored
in tightly stoppered brown bottle in dark, solns are stable for
weeks.
B. Determination
(a) Crystalline reserpine. — Accurately weigh ca 25 mg res-
erpine, dissolve in ca 40 mL boiling alcohol, cool, and dil. to
100 mL with alcohol. Transfer 10.0 mL to separator contg 50
mL 1% NaHC0 3 soln. Ext with 20, 10, and 10 mL CHC1 3 ,
washing each CHC1 3 ext in second separator with 50 mL 2%
citric acid soln. Filter CHC1 3 exts thru cotton into 50 mL vol.
flask contg 5 mL alcohol, dil. to 50 mL with CHC1 3 , and mix.
Transfer duplicate 5.0 mL aliquots to 25 mL vol. flasks contg
15 mL alcohol. Transfer duplicate 5.0 mL aliquots dil. reser-
pine std soln to 25 mL vol. flasks contg 10 mL alcohol and
4.5 mL CHCl 3 . Add 1.0 mL ale. NaN0 2 soln to 1 std and 1
sample soln. Add 10 drops HCl to all flasks, swirl, and let
stand 30 min. Add 1.0 mL sulfamic acid soln, dil. with al-
cohol to 25 mL, and mix. Let stand 15 min and det. A in
matched 1 cm cells at 390 nm against alcohol.
mg Reserpine in sample weighed
= 25
x (A - A )/(A' - AS)
where A and A refer to nitrite-treated and untreated sample,
resp., and A' and A' refer to corresponding std aliquots.
(b) Tablets. — Transfer accurately weighed portion powd
tablets contg ca 5 mg reserpine to 100 mL beaker. Add 20 mL
alcohol, cover with watch glass, and heat to simmering. Boil
gently 20 min, stirring occasionally, adding small portions al-
cohol to maintain vol. Cool to <50°, add 10 mL CHC1 3 , and
mix. Filter thru pledget of cotton, and collect filtrate in 50 mL
vol. flask. Wash filter and solids with several portions CHC1 3 .
Cool, dil. to 50 mL, and mix. Transfer 25 mL aliquot to sep-
594
Drugs: Part
AOAC Official Methods of Analysis (1990)
arator contg 50 mL 1% NaHC0 3 . Add 5 mL CHC1 3 and shake
vigorously. Transfer CHC1 3 layer to separator contg 50 mL 2%
citric acid soln, and shake. Repeat extns with two 10 mL por-
tions CHC1 3 . Filter exts thru cotton and collect in 50 mL vol.
flask contg 5 mL alcohol. Proceed as in (a), second par,, after
dilg to vol. with CHC1 3 .
mg Reserpine in portion powd tablets weighed
= 5 X (A - A )/(A'
ReL: JAOAC 41, 488(1958).
CAS-50-55-5 (reserpine)
M)
969.51
A. Reagents
Reserpine in Drugs
Spectrophotometry Method II
First Action 1969
Final Action 1976
(Prep, std, sample, and blank solns from same lots of CHC1 3
and MeOH.)
(a) Treated fiberglass. — Soak Pyrex fiberglass, Corning
Glass Works No. 3950, in CHC1 3 , rinse several times with
CHCI3, and air dry on filter paper or dry in forced-draft oven.
(b) Dimethylsulfoxide {DMSO). —See 972.50A(f).
(c) Sodium nitrite in dilute methanol soln. — 0.3% in MeOH
(1 + 1). Stable 5:1 month when stored in refrigerator. Bring
to room temp, before use.
(d) Methanolic hydrochloric acid soln. — Dil. 6.0 mL HC1
to 100 mL with MeOH.
(e) Reserpine std soln. — 20 (xg/mL. Dissolve 25.0 mg ac-
curately weighed USP Reserpine Ref. Std , previously dried 3
hr at 60°, in 0.25 mL CHC1 3 . Mix with ca 30 mL MeOH,
previously warmed to 50°; transfer mixt. to 250 mL vol. flask
with warm MeOH. Cool soln to room temp., dil. to vol. with
MeOH, and mix. Protect soln from light. Just prior to use,
pipet 10 mL into 50 mL vol. flask, add 36 mL CHC1 3 , and
dil. to vol. with MeOH.
(f) Diatomaceous earth. — See 960.53B.
B. Preparation of Column
Place small pledget of treated fiberglass in base of 200 x
22 mm id tube. Lower layer: Mix 1 g diat. earth, 960. 53B,
with 0.5 mL freshly prepd 2% NaHC0 3 , transfer to column,
and tamp to uniform mass. Acid layer: Mix 1 g diat. earth
with 0.5 mL freshly prepd 0.5% citric acid soln, transfer to
column, and tamp. Water layer: Mix 1 g diat. earth with 0.5
mL H 2 0; transfer to column, and tamp.
Proceed with entire assay quickly, without interruption,
avoiding exposure of sample to direct light. Read UV spec-
trum immediately after column elution is completed.
C. Chromatography
Powder tablets and pass thru No. 60 sieve. Transfer accu-
rately weighed amt, contg ca 1 mg reserpine, but <1 g of the
powder, to 150 mL beaker. Dry-mix powder with ca 500 mg
diat. earth. Add 1 mL DMSO and wet sample thoroly by mix-
ing with spatula. Let mixt. stand, with spatula remaining in
beaker, 5 min. Add addnl 500 mg diat. earth and mix thoroly.
Add addnl diat. earth to total wt of 2 g and mix thoroly. Quant,
transfer to column thru wide-mouth funnel. Dry- wash beaker
with ca 1 g diat. earth and transfer to column. Wipe beaker,
spatula, and funnel with small pledget of treated glass wool.
Tamp sample, dry wash, and glass wool firmly. Pass ca 45 mL
CHCI3 thru sample column. Collect eluate in 50 mL vol. flask
contg 14 mL MeOH, rinsing tip of column with CHCI3, and
dil. sample to vol. with CHC1 3 .
Prep, and elute blank column exactly as above, replacing
sample layer with 1 mL DMSO + 2 g diat. earth.
D. UV Assay
Scan UV absorption spectrum of sample eluate from 250 to
350 nm, against column blank eluate. Likewise, scan spectrum
of std soln in same range against ref. blank of 3.6 parts CHC1 3
and 1.4 parts MeOH.
mg Reserpine in sample portion = (A /A') x (C x 50)
where A and A r refer to sample and std, resp., at 268 nm, and
C — mg reserpine /mL in std soln (0.02).
E. Colorimetric Assay
Pipet duplicate 5.0 mL aliquots of sample eluate and std soln
into sep. 10 mL vol. flasks. Add 2.0 mL methanolic HC1 soln
to each flask and swirl. To one std and one sample flask, add
1 .0 mL MeOH (1 + 1) (blanks). To remaining std and sample
flasks, add 1.0 mL 0.3% NaN0 2 soln and mix. Let stand ex-
actly 30 min. Add 0.5 mL freshly prepd 5% NH 4 sulfamate
soln to each flask, dil. with MeOH, and let stand 5=10 min.
Read A from 450 to 350 nm for each soln against blank of 3.6
parts CHCI3, 5.4 parts MeOH, and 1 part H 2 0.
mg Reserpine in sample portion
= [(A ~A )XCX 50]/(A' - K)
where A, A , A' , and A$ refer to sample, sample blank, std,
and std blank, resp., at 390 nm and C — mg reserpine /mL in
std soln (0.02).
Refs.: JAOAC 52, 113(1969); 53, 1106(1970).
CAS-50-55-5 (reserpine)
972.54 Reserpine in Drugs
Single Tablet Assay
First Action 1972
Final Action 1973
A. Reagents
(a) Vanadium pentoxide-phosphoric acid {VP-PA) solns. —
(7) Stock soln. — Sat. 85% H 3 P0 4 with V 2 O s by shaking mech.
2 hr. Filter thru medium porosity fritted glass funnel. (Satd
soln contains ca 0.8 mg V 2 5 /mL.) Soln is stable ca 1 month.
(2) Working soln. — Dil. 10 mL stock soln to 100 mL with
H 2 0. Prep, fresh daily.
(b) Reserpine std solns. — (1) Stock soln.—OA mg/ mL.
Accurately weigh ca 10 mg USP Reserpine Ref. Std, previ-
ously dried 3 hr at 60°, into 100 mL vol. flask. Dissolve in
0.1 mL CHC1 3 ; then add 30 mL alcohol previously warmed to
50°. Cool to room temp, and dil. to vol. with alcohol. (2)
Working soln L — 0.002 mg/mL. Transfer 2.0 mL stock soln
to 100 mL vol. flask contg ca 50 mL alcohol. Add following
vols CHC1 3 : for 1 mg tablets, 1.0 mL; 0.5 mg, 2 mL; 0.25
mg, 2.4 mL. Dil. to vol. with alcohol. (3) Working soln II. —
0.001 mg/mL. Transfer 1.0 mL stock soln to 100 mL vol.
flask contg ca 50 mL alcohol, add 2.0 mL CHC1 3 , and dil. to
vol. with alcohol. Protect all std solns from light as in
961.20B(c).
B. Apparatus
Spectrophotofluorometer .— Adjusted so that reserpine
working std solns // and / give ca 40 and 80% F, resp. Wave-
lengths of max. excitation and fluorescence of reserpine treated
with VP-PA are 400 and 500 nm, resp. {Caution: See safety
notes on photofluorometers.)
AOAC Official Methods of Analysis (1990)
Rauwolfia Alkaloids 595
C. Preparation of Sample
Drop single tablet into 100 mL vol. flask. Add 2 mL H 2 0,
crush tablet with fire-polished glass rod, and, leaving rod in
flask, heat on steam bath ca 15 min or until tablet is dispersed.
Frequently crush particles with rod to aid soln. Cool, Rinse
rod into flask with following vols CHC1 3 : 2 mL for 0.1 mg
tablets; 3 mL for 0.25; and 5 mL for 0.5 and 1. Remove rod.
Protect CHC1 3 solns of reserpine from light. Vigorously shake
flask ca 2 min. Dil. to vol. with alcohol, shake vigorously,
and filter thru rapid paper, discarding first 25 mL filtrate. Col-
lect remaining filtrate in g-s erlenmeyer. Further dil. filtrate
as follows: For 0.1 mg tablets, use directly; 0.25 mg, dil. 20
mL to 25 mL; 0.5, 20 to 50; and 1, 10 to 50.
D. Determination
Transfer 5.0 mL std soln // (for 0. 1 mg tablets) or std soln
/ (for all others) and 5.0 mL final sample diln to sep. 50 mL
g-s erlenmeyers. Add 5.0 mL VP-PA working soln to each
flask, shake vigorously, and let stand 15-60 min. Det. fluo-
rescence of sample and std solns. (Blank is unnecessary, since
its reading is negligible compared to sample and std solns.)
Relative fluorescence = %F x meter multiplier reading
mg Reserpine/tablet = (R/R') x C xf
where R and R' = relative fluorescence of sample and std solns,
resp., C = concn reserpine stock soln, and factor/ = 1, 2.5,
5, and 10 for 0.1, 0.25, 0.5, and 1 mg tablets, resp.
Ref.: JAOAC 55, 149(1972).
CAS-50-55-5 (reserpine)
976.34 Reserpine in Drugs
Semiautomated Fluorometric Method
First Action 1976
Final Action 1978
A. Principle
Reserpine is dissolved in 0.25M H3PO4 soln contg 20% MeOH
and mixed with V 2 5 , and fluorescence of oxidized reserpine
is detd.
B. Apparatus
(Other equiv. instruments may be used.)
(a) Automatic analyzer. — AutoAnalyzer with following
modules (Technicon Instruments Corp.): Sampler II with 30/
hr (2:1) cam; proportioning pump I; manifold (see Fig. 976.34).
(b) Ratio fluorometer . — Equipped with flowcell and Kopp
Glass No. C5\ 13 filter for 395 nm excitation and Wratten No.
8 filter for 495 nm emission.
(c) Recorder. — Texas Instruments Servo /Riter II (Texas
Instruments, Inc., 24500 Hwy 290, PO Box 1444, Sypress,
TX 77429), or equiv.
C. Reagents
(a) Phosphoric acid-methanol soln. — Add 20 mL H 3 P0 4 to
200 mL MeOH and dil. to 1 L with H 2 0.
(b) Vanadium pentoxide-phosphoric acid (VP-PA) solns. —
(7) Stock soln. — Sat. 85% H3PO4 with V 2 5 by stirring mag.
3 hr. Let settle overnight. (Satd soln contains ca 0.8 mg V 2 5 /
mL.) Soln is stable ca 1 month. (2) Working soln. — Dil. 200
mL stock soln to 1 L with H 2 0. Prep, fresh daily.
(c) Reserpine std solns. — (J) Stock soln. — 0.125 mg/mL.
Accurately weigh ca 25 mg USP Reserpine Ref. Std, previ-
ously dried 3 hr at 60°, into 200 mL vol. flask, dissolve in
H 3 P0 4 -Me0H soln, and dil. to vol. (2) Working soln L— 0.0025
mg/mL. Dil. 5.0 mL stock soln to 250 mL with H 3 P0 4 -Me0H
soln. (3) Working soln II. — 0.002 mg/mL. Dil. 4.0 mL stock
soln to 250 mL with H 3 P0 4 -MeOH soln.
D. Preparation of Sample
Place tablet in suitable vol. flask to give reserpine concn of
0.002-0.0025 mg/mL. Add H 3 P0 4 -MeOH soln to ca V2 vol.
of flask and place in ultrasonic generator to disintegrate tablet.
After complete disintegration, agitate 15 min on mech. shaker.
Dil. to vol. and mix. Let soln settle 2 hr.
E. Analytical System
Sample is withdrawn, segmented with air, and dild with
H 3 P0 4 -MeOH soln. Soln is resampled into stream of solv. that
has been segmented with air and then mixed with VA-PA
working soln. After flowing thru full delay coil (ca 10 min
delay), soln is debubbled and passed thru flowcell, and fluo-
rescence is measured at excitation and emission wavelengths
of 395 and 495 nm, resp.
F. Start-Up and Shut-Down Operations
Place all tubes in resp. solns and pump until steady baseline
is obtained. To shut down system, place all lines in H 2 and
pump 15 min. Remove lines from H 2 reservoir and pump
system dry.
G. Determination
Fill 3 mL sample cups in following order: 5 cups std soln,
5 cups sample solns, 1 cup std soln, 5 cups sample solns, etc.,
ending with 2 cups std soln. Start Sampler II. After last cup
has been sampled, let system operate until steady baseline is
obtained. Draw tangent to initial and final baselines. Subtract
baseline to det. net fluorescence for each sample, F, and std,
F', peak, resp. Using av. of 2 stds which bracket sample peak,
calc. reserpine as follows:
mg Reserpine in sample taken = (F/F r ) x C x D
where C — concn of std in mg/mL and D = diln factor.
Ref.: JAOAC 59, 289(1976).
CAS-50-55-5 (reserpine)
977.31 Reserpine-Rescinnamine Group
Alkaloids in Rauwolfia serpentina Drugs
Spectrophotofluorometric Method
First Action 1977
Final Action 1979
A. Principle
Reserpine-rescinnamine is extd with DMSO-MeOH. After
addn of H 2 S0 4 , drug is extd into CHC1 3 , then sepd from in-
terfering materials by chromatgy on QAN NaOH-diat. earth
and silica gel columns. Reserpine-rescinnamine is eluted from
latter column with CHCl 3 -MeOH and detd by spectrophoto-
fluorometry against std treated similarly.
B. Apparatus
(a) Chromatographic tube and tamping rod. — 200 x 22 (id)
mm. See 967.31A.
(b) Shaker. — Wrist action (Model BT, Burrell Corp., or
equiv.).
(c) Spectrophotofluorometer . — Excitation and emission
wavelengths 400 and 502 nm (uncorrected), resp. Use exci-
tation and emission slit widths consistent with good quanti-
tation according to manufacturer's recommendations. Sensi-
tivity setting depends upon slit widths. (Caution: See safety
notes on photofluorometers.)
596
Drugs: Part
AOAC Official Methods of Analysis (1990)
ALL TRANSMISSION TUBING STANDARD WALL 4 mm O.D. GLASS EXCEPT
FROM DELAY COIL TO CELL -.065" TYGON
luPPER y^, 0\
'level O O O
Sp n o°
2.4 mm I.D,
24 TURN
MIXING COIL
2,4 mm KD.
14 TURN
MIXING COIL
-@-
TO WEIR BOX
2.4 mm ©
24TURN O
MIXING §
COIL «
-@-
.60 T
ATE (per hour)
30 2:1
ps 2.00 T ? H^POA-MeO H
K4
rH9 — © 1 - 60 T
HqP04-MeO H
©
(
©
■ 60 T RESAMPLE
/
/C3
®
5)
1.60 T
2.00 T V205-H.TPO d
^@-
3.90 T
H3PO4-
PRI, FILT. 395 nmPEAK
CORNING 5113
SEC. FILT. 495 nm PEAK
WRATTEN 8
PS = PULSE SUPPRESSOR 116-0480
T = TYGON
FLUOROMETER RECORDER
FIG. 976.34— Flow diagram for semiautomated fluorometric analysis for reserpine
C. Reagents
(a) Acidic alcohol soln. — Add 10 mL 85% H3PO4 to 40 mL
H 2 and 50 mL alcohol . Mix well and cool before use.
(b) Chloroform-methanol mixture. — (1 + 1). Mix equal vols
CHC1 3 and MeOH; 105 mL/detn is required.
(c) Diatomaceous earth. — See 960. 53B.
(d) Dimethyl sulfoxide-methanol mixture. — (1 + 1). Mix
equal vols DM SO and MeOH. 10 mL/detn is required. {Cau-
tion: DMSO can be harmful. Avoid skin contact by wearing
heavy rubber gloves. Use effective fume removal device.)
(e) Silica gel for column chromatography. — 0.063-0.2 mm.
See 968.22B(a).
(f) Sulfuric aeid.—0.5N. Add 7 mL H 2 S0 4 to 500 mL H 2 0,
and mix well.
(g) Vanadium pentoxide-phosphoric acid (VP-PA) solns. —
(/) Stock soln.— 0.8 mg V 2 5 /mL. Sat. 85% H 3 P0 4 with V 2 5
by shaking mech. 2 hr. Filter thru medium porosity fritted glass
funnel. Soln is stable ca 1 month. (2) Working soln. — 0.08
mg V 2 5 /mL. Dil. 10 mL stock soln and 40 mL H 2 to 100
mL with alcohol. Prep, fresh daily.
(h) Reserpine std solns. — (I) Stock soln. — 40 jxg/mL. Ac-
curately weigh 20 mg USP Reserpine Ref. Std, previously dried
3 hr at 60°, into 500 mL vol. flask. Dissolve with 50 mL hot
alcohol, cool, and dil. to vol. with alcohol. Protect soln from
direct sunlight. Soln, when stored in g-s brown glass bottle,
is stable for weeks. (2) Working soln. — 2 fxg/raL. Pipet 5 mL
stock soln into 100 mL vol. flask, and dil. to vol. with alcohol.
Prep, fresh daily.
D. Preparation of Sample
Grind tablets to pass No. 60 sieve. Mix well. Use powd root
samples as received.
£ Preparation of Columns
(a) Column /. — Mix 2 mL 0. IN NaOH with 3 g diat. earth.
Transfer to chromatgc tube plugged with glass wool, and tamp.
Cover with pad of glass wool.
(b) Column II. — Pour silica gel into chromatgc tube (plugged
with glass wool) to ht of ca 4 cm. Tap side of tube lightly
with tamping rod. Cover silica gel with pad of glass wool.
Arrange columns so that eluate from Column I flows directly
into Column II. Wet columns with 25 mL freshly prepd H 2 0-
satd CHC1 3 .
F. Cleanup
(Perform in subdued light.)
Accurately weigh prepd sample contg ca 200 mg Rauwolfia
serpentina and transfer to 50 mL g-s centrf. tube. Add 10.0
mL DMSO-MeOH (1 + 1), stopper, and shake vigorously by
hand until entire sample is thoroly wetted. Shake mech. 30
min at most vigorous setting. (Wrap tube with Al foil if shaker
is not in dark place.) Centrf. 5 min at 1200 rpm. Pipet 5 mL
aliquot of supernate into 125 mL separator contg 50 mL 0.5N
H 2 S0 4 . (Caution: DMSO can be harmful. Do not use mouth
suction to fill pipet.) (Do not pipet any undissolved residue.)
Ext with four 25 mL portions CHC1 3 .
In second separator contg 25 mL 0.57V H 2 S0 4 , shake each
25 mL CHCI3 ext individually, and drain into Column I. Let
each ext sink entirely into both columns before adding next 25
mL ext. After draining fourth ext into second separator, rinse
tip of first separator into second with 1-2 mL CHC1 3 . Then
rinse tip of second separator into Column I, and tip of Column
I into Column II. Discard all CHC1 3 eluted from Column II.
Sep. columns and discard Column I.
Place 100 mL vol. flask under Column II, and elute with
ca 90 mL CHCl 3 -MeOH (1 + 1). Rinse tip of Column II into
vol. flask with ca 5 mL CHCl^-MeOH (1 + 1), and dil. eluate
to vol. with CHCL-MeOH (1 + 1).
G. Determination
Transfer duplicate 5.0 mL aliquots CHCl 3 -MeOH eluate to
sep. 25 mL vol. flasks. Transfer duplicate 5.0 mL aliquots
working std soln to sep. 25 mL vol. flasks, each contg 5 mL
CHCl 3 -MeOH (1 + 1). Add 5.0 mL VP-PA working soln,
(g)(2), to 1 flask of each set, and mix well. Add 5.0 mL acidic
alcohol soln, (a), to other (blank) flasks, dil. to vol. with al-
AOAC Official Methods of Analysis (1990)
Rauwolfia Alkaloids 597
cohol, and mix well. Det. fluorescence intensity of each soln
within 15-60 min.
H. Calculations
(a) Tablets. — Calc. % reserpine-rescinnamine group alka-
loids in labeled amt Rauwolfia serpentina /tablet:
[(F - F )/(F' - Fi)] x (C/W)x (TjL) x 100 x D
where F and F' = fluorescence of sample and std, resp.; F
and Fq — fluorescence of sample and std blanks, resp.; C =
mg reserpine/mL in working std soln (0.002); W - g sample;
T — av. tablet wt in g; L — labeled mg Rauwolfia serpentina/
tablet; and D — sample diln factor (200 mL).
(b) Powdered root. — Calc. % reserpine-rescinnamine group
alkaloids in Rauwolfia serpentina powd root:
[(F - F )/(F' - FJ)] x (C/W) x 100 x D
where symbols are defined in (a).
Ref.: J AOAC 59, 811(1976).
CAS-248 15-24-5 (rescinnamine)
CAS-50-55-5 (reserpine)
tube just stops (avoid prolonged heating in absence of solv.).
Wipe outsides of warm tubes, place in vac. desiccator, and
evap. to dryness under vac. Dissolve residues by agitating with
5.0 mL alcohol.
Take duplicate 5 mL aliquots reserpine std soln, and add
2.0 mL 0.5N H 2 S0 4 to one sample tube and to one std tube
(blanks). To other tubes add 1.0 mL 0.57V H 2 S0 4 and 1.0 mL
0.3% NaN0 2 soln. Mix contents of each tube, and warm in
H 2 bath 20 min at 50-60°. Cool, add 0.5 mL sulfamic acid
soln to each tube, and mix. Let stand 15 min and det. A in
matched 1 cm cells at 390 nm against alcohol-H 2 (2 + 1).
mg Reserpine-rescinnamine alkaloids in sample weighed
= 5 X (A - A Q )/(A' - A' Q )
where A and A refer to nitrite-treated and untreated samples,
resp., and A' and Aq refer to std soln aliquots.
Refs.: J. Am. Pharm. Assoc. Sci. Ed. 45, 708(1956). JAOAC
40, 64(1957).
CAS-248 15-24-5 (rescinnamine)
CAS-50-55-5 (reserpine)
956.09 Reserpine-Rescinnamine
Group Alkaloids in Rauwolfia serpentina Drugs
Spectrophotometric Method
Final Action 1965
A. Reagents
(a) 1,1 J -Trichloroethane. — (Caution: Trichloroethane is
toxic.) Redistil in all-glass app., collecting fraction boiling at
73-76°.
(b) Reserpine std soln.— -20 ng/mL. Dissolve 20.0 mg USP
Reserpine Ref. Std, previously dried 3 hr at 60°, in 25 mL hot
alcohol, cool, and dil. to 50 mL with alcohol. Dil. 5 mL of
this soln to 100 mL with alcohol.
(c) Dilute sulfuric acid. — 0.5N. Dissolve ca 30 mL H 2 S0 4
in 2 L H 2 0.
(d) Sulfamic acid soln.— 5% aq. soln. Prep, fresh every 2—
3 days.
8. Apparatus
Soxhlet extraction apparatus. — Medium size extractor with
250 mL flask and 35 x 80 mm thimble is most convenient,
although smaller app. may be used.
C. Determination
Ext 2-3 g finely powd Rauwolfia serpentina root, or equiv.
in powd tablets, in Soxhlet extn app. 4 hr, using ca 100 mL
vigorously boiling alcohol. Protect flask and thimble, and all
solns of rauwolfia alkaloids, from strong or direct light.
Wash ext into 100 mL vol. flask with alcohol, cool, dil. to
vol., and mix. Transfer 20 mL aliquot to separator contg 200
mL 0.57V H 2 S0 4 , mix, and ext with three 25 mL portions tri-
chloroethane. Drain lower solv. phase as completely as pos-
sible. Wash each trichloroethane ext in second separator contg
50 mL 0.57V H 2 S0 4 , and discard.
Ext main aq. soln with 25, 15, 15, 10, 10, and 10 mL CHC1 3 .
Wash each CHC1 3 ext with the acid in second separator, and
then with two 10 mL portions 2% NaHC0 3 soln in third and
fourth separators. Filter CHC1 3 exts thru cotton into 100 mL
vol. flask contg 10 mL alcohol. Dil. to 100 mL with CHC1 3
and mix.
Transfer duplicate 10.0 mL aliquots to 18 X 150 mm test
tubes and mix each with 4 mL alcohol. Add two or three "20-
mesh" SiC boiling chips, and heat to boiling in H 2 bath at
ca 70°. Gradually raise bath temp, to 100°, or until boiling in
961.21 Rescinnamine in Drugs
Spectrophotometric Method
First Action 1961
Final Action 1970
A. Reagents
(a) Ammonium sulfamate soln. — 2.5%. Prep, fresh every
2-3 days.
(b) Alcoholic sodium nitrite soln. — See 958.17A(b).
(c) Rescinnamine std soln. — 40 (xg/mL. Dissolve 20.0 mg
USP Rescinnamine Ref, Std in 0.5 mL CHC1 3 , transfer to 50
mL vol. flask, and dil. to vol. with alcohol. Protect all res-
cinnamine solns from direct or strong light. Ale. soln is stable
several weeks in dark. Dil. 5.0 mL std soln to 50 mL with
CHC1 3 .
B. Determination
(a) Crystalline rescinnamine . — Accurately weigh ca 20 mg
sample, dissolve in 0.5 mL CHC1 3 , transfer to 50 mL vol.
flask, and dil. to vol. with alcohol. Pi pet 5 mL aliquot into
separator contg 50 mL 0.5N H 2 S0 4 , add 22 mL CHC1 3 and 3
mL alcohol, and shake vigorously 2 min. Transfer CHC1 3 layer
to second separator contg 50 mL 1% NaHC0 3 soln, and shake
again. Filter CHC1 3 layer thru cotton previously washed with
CHC1 3 into 50 mL vol. flask contg 5.0 mL alcohol. Ext acid
and alk. solns with 2 addnl 10 mL portions CHC1 3 , filter into
vol. flask, and dil. to vol. with CHCI 3 .
Transfer duplicate 10 mL aliquots prepd sample soln and
std soln, (c), to 25 mL vol. flasks, each contg 10 mL alcohol.
Add 1 mL ale. NaN0 2 soln, 958.17A(b), to 1 flask of each
set; to remaining flasks add 1 mL alcohol. Add 10 drops HO
to all flasks, swirl, and let stand 30 min. Add 1 mL 2.5% NH 4
sulfamate soln, dil. to vol. with alcohol, mix, and let stand
10 min.
Det. A in matched 1 cm cells at 390 nm against mixt. of
CHC1 3 , alcohol, and H 2 (9 + 15 + 1) as ref.
mg Rescinnamine in sample weighed
= 20 x (A - A )/(A' - AJ)
where A and A refer to nitrite-treated and untreated sample,
resp., and A f and Aq refer to corresponding std aliquots.
(b) Tablets. — Transfer accurately weighed portion powd
tablets contg ca 2.5 mg rescinnamine to 50 mL beaker. Insert
small glass rod and cover with watch glass. Add 10 mL al-
598
Drugs: Part
AOAC Official Methods of Analysis (1990)
cohol, mark vol., and boil gently 20 min with occasional stir-
ring, maintaining original vol. by adding alcohol when nec-
essary. Cool to <50°, add 5 mL CHCI3, and filter thru pledget
of cotton previously washed with CHC1 3 into 25 mL voL flask.
Wash filter and solids with CHC1 3 , cool, and dil. to vol. Mix,
and let settle ca 10 min. (If soln is not clear, transfer to g-s
graduate and let settle 10 min more.)
Pipet 20 mL aliquot into separator contg 50 mL 0.57V H 2 S0 4 ,
add 10 mL CHC1 3 , and shake vigorously 2 min. Transfer CHC1 3
to second separator contg 50 mL 1% NaHC0 3 soln, and shake
again. Filter CHC1 3 layer thru cotton previously washed with
CHC1 3 into 50 mL vol. flask contg 5.0 mL alcohol. Ext acid
and alk. solns with two 10 mL portions CHC1 3 , filter into vol.
flask, and dil. to vol. with CHC1 3 .
Proceed as in (a), second par.
mg Rescinnamine in sample weighed
- 2.5 X (A - A )/G4' - AQ
C. Determination of Total Alkaloids
Transfer 10 mL aliquot prepd sample soln and 10 mL std
soln, (c), to sep. 25 mL vol. flasks, and dil. to vol. with al-
cohol. Det. spectrum of each soln in region 250-360 nm against
blank of 9 mL CHC1 3 dild to 25 mL with alcohol.
mg Total alkaloids in sample weighed = 2.5 x T/S
where T and S are A of sample and std solns at max. near 304
nm, resp.
Presence of other alkaloids is indicated by difference be-
tween the 2 spectra; presence of reserpine in particular is in-
dicated by difference between colorimetric and UV detns.
Ref.: JAOAC 44, 303(1961).
CAS-248 15-24-5 (rescinnamine)
OTHER ALKALOIDS
932.24* Aconitine in Aconite Root
Qualitative Test
Procedure
Surplus 1965
See 32.028, 10th ed.
Arecoline Mydrobromide in Drugs
See 960.53B.
Cocaine in Drugs
See 960.53B.
Pilocarpine Hydrochloride in Drugs
See 960.53B.
B. Apparatus
(a) Liquid chromato graph. — Equipped with 7000 psi injec-
tion valve with 10 |xL injection loop and printer-plotter; UV-
visible detector set at 220 nm, 0.04 AUFS, time const set at
4 s; data integration system with peak width set to peak thresh-
old ratio of 1:60 (Model 4200 or 4270, Spectra-Physics, Inc.,
3333 N. First St, San Jose, CA 95134, or appropriate settings
for equiv. chromatgc data system).
(b) Liquid chromatographic column. — 10 jxm reverse phase
phenyl bonded column, 30 cm x 3.9 mm id (Waters Asso-
ciates, Inc.).
C. Reagents
(a) Mobile phase. — UV quality LC grade H 2 and CH 3 CN
(97 + 3). Add KH2PO4 to make 5% soln. Adjust pH to 2.5
± 0.1, using 85% H3PO4. Filter soln thru 5 |Jim mixed cel-
lulose acetate and nitrate filter.
(b) Std soln. — Dry USP Ref. Std Pilocarpine Nitrate Salt
2 h at 105°. Accurately weigh 10 mg into 100 mL vol. flask.
Dissolve and dil. to vol. with mobile phase. Filter thru 5 pjn
mixed cellulose acetate and nitrate filter. Check std soln daily
by LC injection; prep, new std soln if pilocarpine concn, as
detd by peak area, has changed >2%.
(c) System suitability std soln.—DW. 10 mg pilocarpine and
1 mg isopilocarpine to 100 mL with mobile phase.
D. Preparation of Sample
Prep, soln contg 10 mg pilocarpine nitrate salt/ 100 mL (based
on label concn) using mobile phase as diluent. Filter soln thru
5 fim mixed cellulose acetate and nitrate filters.
E. Liquid Chromatography Test
Equilibrate overnight at 26 ± 1° with mobile phase at flow
rate of 03 mL/min. Circulate sol v. continuously thruout du-
ration of analyses, without interruption.
System suitability test. — Inject 10 u.L aliquots of system
suitability std soln into chromatgc column. Retention time for
pilocarpine should be 50-54 min, and isopilocarpine, 45-49
min. Resolution factor, R^, for pilocarpine/isopilocarpine should
be>1.13.
F. Determination
Inject 1.0 |xL aliquots of pilocarpine std soln and sample prepn
in triplicate. Calc. quantity, in g/100 mL of pilocarpine, in
each sample by formula:
g pilocarpine (as nitrate)/ 100 mL = PA /PA' X C x D
where PA and PA' = area of pilocarpine peak for sample and
std solns, resp.; C = g std/ 100 mL; D - diln factor of sample
prepn. Altho not necessarily quant., isopilocarpine (retention
time 45-49 min) and pilocarpic acid (retention time 33-38
min) may be estd by use of stds and similar calcns. When
using different pilocarpine salts, use correction factor for dif-
ferent mol. wts: F = MW pilocarpine salt/MW pilocarpine
std (corrects for differences in extinction coefficients between
pilocarpine and pilocarpic acid).
Ref.: JAOAC 67, 924(1984).
984.39 Pilocarpine, Isopilocarpine,
and Pilocarpic Acid in Drugs
Liquid Chromatographic Method
First Action 1984
Final Action 1987
A. Principle
Pilocarpine is detd by LC, using acidified phosphate buffer-
CH 3 CN (97 + 3) mobile phase, reverse phase phenyl bonded
column, and detection at 220 nm.
920.211
Strychnine in Liquid
Drug Preparations
Final Action
(Other alkaloids absent. See 38.069-38.074*, 12th ed.)
Into evapg dish measure 50 mL sample, or enough to yield
>0.065 g strychnine, and remove alcohol by evapn. Transfer
to separator, add 1 mL NH 4 OH, or enough to render soln alk.,
AOAC Official Methods of Analysis (1990)
Digitalis
599
and proceed as in 961. 18B, beginning "... ext with four 25
mL portions CHC1 3 use ..."
Refs.: JAOAC 3, 379(1920); 4, 572(1921).
CAS-57-24-9 (strychnine)
Strychnine in Drug Tablets
(Other alkaloids absent)
See 968. 18B.
938.17* Cinchophen in Drugs
Final Action
Surplus 1975
A In Presence of Salicylates
See 38.151-38.152, 12th ed.
B. In Presence of Sodium Bicarbonate
See 38.153-38.154, 12th ed.
976.35 Nikethamide in Drugs
Gas Chromatographic Method
First Action 1976
Final Action 1977
A Principle
Nikethamide prepn is dild with acetone contg anthracene as
internal std and detd by GC with flame ionization detector.
Method is applicable to levels of nikethamide normally en-
countered in injectable prepns (25% w/v). Store all solns contg
anthracene in low-actinic glassware and complete detn within
1 day.
B. Apparatus and Reagents
(Caution: See safety notes on acetone.)
(a) Gas chromatography — Hewlett-Packard Model 838, or
equiv., with flame ionization detector and 1.8 m (6') X 4 mm
id glass tube packed with 4% XE-60 on 80-100 mesh Gas-
Chrom Q (Applied Science, precoated, or equiv., or prep, as
in 976. 35C). Operating conditions: temps (°) — column 180,
injection port 210, detector 210; gas flows (mL/min)— N car-
rier gas 56, air 200, H 25; sensitivity 10" 9 amp full scale,
attenuation lx. Before use, condition column 24 hr at 240-
250° with 100 ± 20 mL N/min. If necessary, vary column
temp, or gas flow to attain retention times of ca 6 and 4-5
min for anthracene and nikethamide, resp. Also vary detector
sensitivity or injection vol. (4-7 pL) to attain peak hts of 50-
90% full scale.
(b) Internal std soln. — 0.8 mg/mL. Accurately weigh ca
0.8 g anthracene, heat ca 15 min on steam bath with acetone
to dissolve, and dil. to 100 mL with acetone.
(c) Nikethamide std solns. — (1) Stock soln. — Approx. 1.0
mg nikethamide (Sigma Chemical Co., No. D4378)/mL ace-
tone, accurately prepd. (2) Working soln. — Approx. 0.5 mg
nikethamide /mL acetone. Accurately measure equal vols (^5.0
mL each) of (b) and (c)(7) and mix thoroly.
C. Preparation of Column
Wash tube and small amt fine glass wool with 5% (v/v)
dichlorodimethylsilane in toluene; rinse with acetone and dry
thoroly at room temp. (Caution: Dichlorodimethylsilane is toxic.
Avoid contact with skin and eyes. Use effective fume removal
device.) Dissolve 3.0 g XE-60 in 100 mL CHC1 3 . Transfer
soln to 250 mL beaker and slowly add 20.0 g 80-100 mesh
Gas-Chrom Q with const but gentle stirring. Continue stirring
ca 30 sec after adding all support. Place beaker under bell jar
and apply vac. Carefully increase vac. and hold at max. ca 1
min to degas.
Swirl slurry rapidly and transfer in small portions to buchner
fitted with 9 cm Whatman No. 4 paper. Maintain vac. 5 min
after last portion is added. Air dry coated support 1 hr by
spreading on smooth surface, and oven-dry addnl hr at 105°.
Plug column exit with small wad of silanized fine glass wool
and thru-hole septum. Apply vac. to exit and slowly add coated
support thru injection end, tapping very gently to aid com-
paction. Pack to within 1 cm of area heated by flash heater.
Plug inlet with <3 mm wad of silanized fine glass wool and
condition as in 976.35B(a).
D. Determination
Pipet 5 mL sample (25% w/v com. prepn) into 250 mL vol.
flask, dil. to vol. with acetone, and mix. Dil. 20.0 mL to 100
mL with acetone (dil. sample soln). Prep, assay soln by mix-
ing equal vols of dil. sample soln and internal std soln. Inject
5 juL working std soln into gas chromatograph and record
chromatogram; then inject 5 jxL assay soln and record.
% Nikethamide (w/v)
- (P x /' X C X 100)/(/" X / X C)
where P and P' = peak hts (or areas) of nikethamide in assay
soln and working std soln, resp.; / and /' = peak hts (or areas)
of anthracene (internal std) in assay soln and working std soln,
resp.; C" — g niketharnide/mL working std soln; and C = mL
sample/mL assay soln.
Ref.: JAOAC 59, 93(1976).
CAS-59-26-7 (nikethamide)
DIGITALIS
954.16 Digitoxin in Drugs
Spectrophotometric Method
Final Action 1965
A. Reagents
(a) Formamide. — Shake 1 L HCONH 2 (99% grade) with ca
30 g anhyd. K 2 C0 3 15 min and filter. Distil under vac. in
all-glass app. Reject first portion of distillate contg H 2 0, and
collect fraction boiling at ca 101°/12 mm Hg (1.6 kPa) (115°/
25 mm Hg; 3.3 kPa). Store over H 2 S0 4 until odor of NH 3 is
no longer detected.
(b) Alkaline picrate reagent. — Mix 20 mL 1% aq. picric
acid soln with 10 mL 5% NaOH soln, dil. to 100 mL with
H 2 0, and mix. Reagent is stable 2-3 days.
(c) Digitoxin std soln. — 0.04 mg/mL. Dissolve 20.0 mg
USP Digitoxin Ref. Std in alcohol, and dil. to 50 mL with
alcohol. Dil. 10.0 mL of this stock soln to 100 mL with al-
cohol.
(d) Diatomaceous earth. — See 960. 53B.
B. Preparation of Chromatographic Column
Chromatographic tube. — See 967.31A.
Wash layer. — Add ca 2 g diat. earth to 1 mL H 2 in 100
mL beaker. Mix thoroly with stirring rod or scoop until the
mixt. appears fluffy and uniform, and transfer to chromatgc
tube. Press down lightly with packing rod. (Wash layer should
be 15-20 mm thick.)
Trap layer. — Add 3 g diat. earth to 3 mL formamide-H 2
600
Drugs: Part
AOAC Official Methods of Analysis (1990)
soln (2 -f 1) in 150 mL beaker, mix thoroly, and transfer to
tube containing wash layer. Press trap layer down lightly and
evenly.
C. Preparation of Sample
(a) Crystalline digitoxin. — Dissolve 20 mg digitoxin, ac-
curately weighed, in 20 mL CHC1 3 . Transfer to 100 mL vol.
flask with several portions of benzene, dil. to vol. with ben-
zene, and mix. Transfer 10.0 mL to chromatgc column,
954. 16B. When liq. has passed into column, proceed as in
954. 16D.
(b) Tablets. — Thoroly mix accurately weighed powd sam-
ple contg 2 mg digitoxin with 2 mL H 2 in 250 mL beaker.
Add 4 mL formamide, stir thoroly, and cover beaker with watch
glass. Heat mixt. 20 min on steam bath, with frequent stirring.
Cool; add 2 mL H 2 and ca 8 g diat. earth. Stir thoroly until
mass appears uniform and does not stick to beaker. Quant,
transfer mixt. to prepd chromatgc tube, 954. 16B, thru powder
funnel in several portions, pressing it down with stirring rod.
Use rubber policeman to sweep adhering particles from beaker
and funnel into tube. Scrub beaker and stirring rod with ca 1
g diat. earth, and add dry washings to tube thru funnel. Repeat
washing with 2 addn.1 portions diat. earth. Place cotton pad in
tube and press it down on column with packing rod, sweeping
diat. earth on sides of tube before it. (Over-all ht of column
should be 120-150 mm.)
D. Separation of Digitoxin
Elute digitoxin with ca 240 mL benzene-CHCl 3 (3 + 1),
collecting eluate in 250 mL vol. flask at rate <4 mL/min.
Wash stem with stream of CHC1 3 , dil. to 250 mL with CHC1 3 ,
and mix.
Continue elution as in 954. 16F.
E. Colorimetric Determination
Transfer 25 mL aliquot eluate to small erlenmeyer and evap.
to dryness on steam bath with aid of air current. Moisten res-
idue with ca 0.5 mL alcohol, and again evap. to dryness. Add
5.0 mL alcohol to cooled flask, stopper, and let stand 15 min
with occasional shaking.
Transfer 5.0 mL aliquot dild std digitoxin soln to small flask
and 5 mL alcohol to another flask as blank. Add 3.0 mL alk.
picrate reagent to each flask, and mix by swirling. Protect soln
from intense light. After 10 min, det. A of std and sample
solns relative to blank at 495 nm, repeating measurements at
2 min intervals until max. values are attained. Calc. digitoxin
content of sample.
F. Tests for Other Digitoxosides
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
After digitoxin seps, elute other digitoxosides with 200 mL
CHC1 3 , collecting eluate in separator. Shake with 100 mL H 2 0.
Transfer lower layer to beaker, ext H 2 with 30 mL CHC1 3 ,
and add CHC1 3 washings to beaker. Evap. to dryness. Pipet 5
mL dild digitoxin std soln into second beaker and evap. to
dryness. Add 4 mL Keller- Kiliani reagent, 959.17A(b), to each
of the cooled residues and mix thoroly. After 15 min, filter
thru glass wool if necessary, and det. A of clear sample and
std relative to reagent blank, at 590 nm; repeat measurements
at 5 min intervals until max. values are attained. Calc. content
of other digitoxosides in sample as digitoxin.
Refs.: J. Am. Pharm. Assoc. Sci. Ed. 43, 580(1954). JAOAC
41, 487(1958).
CAS-7 1-63-6 (digitoxin)
975.58 Digoxin in Drugs
Automated Method
First Action 1975
Final Action 1976
A. Principle
KIO4 oxidizes czs-2-deoxy sugars to malonyldialdehydes
which are condensed with 2-thiobarbituric acid to yield stable,
intensely colored methine dyes. Glycoside moiety of digoxin
consists of 3 molecules of digitoxose, 2,6-dideoxy-D-ribohex-
ose, which yields colored compd with max. A at 530 nm.
B. Apparatus
(a) Automatic analyzer. — AutoAnalyzer with following
modules (Technicon Instruments Corp.): Sampler II with 40/
fir (2:1) cam; proportioning pump I; manifold; const temp, bath
(75°) with two 40' X 1.6 mm id coils; Model 1 colorimeter,
with 50 mm tubular flow cell, matched 530 nm filters; Bristol
recorder linear in T, and paper printed in A units. (See Fig.
975.58.)
(b) Shaker. — Model BT, wrist- action (Burrell Corp.).
(c) Ultrasonic generator. — 150 watt.
C. Reagents
(a) Arsenic trioxide soln. — Add 20.0 g As 2 3 and 7.0 g
NaOH pellets to 100 mL H 2 0, and heat to bp to dissolve.
(Caution: See safety notes on arsenic trioxide.) Add 800 mL
H 2 and 60 mL HC1, and dil. to 1 L.
(b) Potassium metaperiodate soln. — Add 3.6 g KI0 4 to 900
mL H 2 0. Heat and stir to dissolve. Cool. Add 3.0 mL H 2 S0 4
and dil. to 1 L.
(c) Thiobarbituric acid (TBA) soln.— Add 15.0 g TBA and
4.5 g NaOH pellets to 900 mL H 2 0, and stir to dissolve. Add
HC1 slowly to pH 3.5-4.0. Filter and dil. to 1 L.
(d) Digoxin std solns. — (J) Stock soln. — 0.05 mg/mL.
Accurately weigh ca 25 mg USP Digoxin Ref. Std into 500
mL vol. flask and dil. to vol. with 50% alcohol. (2) Working
soln. — 5 jjug/mL. Pipet 10 mL stock soln into 100 mL vol..
flask and dil. to vol. with 50% alcohol. Prep, fresh daily.
D. Preparation of Sample
Disintegrate individual tablet or disperse weighed composite
in accurately measured vol. 50% alcohol to give digoxin concn
of 5 (xg/mL. Use ultrasonic generator >5 min to assure tablet
disintegration. Shake mech. 1 hr. Let soln settle ^2 hr.
E. Analytical System
Sample is withdrawn, segmented with air, and oxidized with
KIO4 in mixing coil. As 2 3 is added to remove excess KI0 4 ;
then TBA is added. Color is developed in 75° heating bath,
and A of soln at 530 nm is measured in 50 mm flowed!.
F. Start-Up
Place all lines in their resp. solns, and let system equilibrate
30 min.
G. Shut-Down
Place KIO4, As 2 3 , and TBA lines in H 2 0. Remove all other
lines from their solns. After 10 min, remove remaining lines
from H 2 and pump system dry.
H. Determination
Fill sample cups in following order: 4 cups std soln, 5 cups
sample soln, 1 cup std soln, 5 cups sample soln, etc. Place 2
cups std soln at end of each run. (Extra cups of std solns at
start and end of sampling pattern will eliminate carryover ef-
fect in transitions from wash soln to std soln and vice versa.
Three extra cups at start and 1 extra cup at end should suffice,
but det. number needed for equilibrium by experiment. System
AOAC Official Methods of Analysis (1990)
Digitalis
601
To Weir Box -«a—
1
O O O SAMPLER
. Q oJ RATE (per hour) 40 2:1
Lower . Tube Size^- 2 -^
50% Ethyl Alcohol
Air
Sample
KI0 4
H2O
As 2 3
Proportioning
Pump
Colorimeter Recorder
FIG. 975.56 — Flow diagram for automated analysis for digoxin
>T - Tygon, S = Solvaflex.A ~ Acidflex
should give uniform response for at least final pair of extra std
cups before sample pattern is started.) Start Sampler II. After
last cup has been sampled, let system operate until steady
baseline is obtained. Draw tangent to initial and final base-
lines. Subtract baseline to det. A A and A A' for each sample
and std peak, resp. Discard values for first 3 and last std peaks
and calc. av. A A'.
mg Digoxin in portion taken = (AA/AV) X C X D
where C — concn of std in mg/mL and D = diln factor.
Ref.: J AOAC 58, 70(1975).
CAS-20830-75-5 (digoxin)
959.17 Digoxin and Total Digitoxosides
in Drugs
Spectrophotometric Method
Final Action 1976
A. Reagents
(a) Alkaline dinitrobenzene soln. — (1) Prep. 5% soln
m-dinitrobenzene in benzene, and store in g-s brown glass bot-
tle. (2) Mix 1 mL 10% tetramethyl ammonium hydroxide soln
with 140 mL absolute alcohol, titr. with 0.01 N HO, using Me
red, and adjust to 0.OOSW with absolute alcohol. Just before
use, mix 60 mL (J) with 40 mL (2).
(b) Keller -Kiliani reagent. — Mix 60 mL HOAc with 1 mL
9% FeCl 3 .6H 2 soln and 5 mL H 2 S0 4 , and cool.
(c) Digoxin std soln. — 25.0 p,g/mL. Dissolve 25.0 mg USP
Digoxin Ref. Std, C 4I H 64 0| 4 , in hot alcohol, cool, dil. to 100
mL, and mix. Dil. 10.0 mL of this soln to 100 mL with al-
cohol and mix.
B. Preparation of Sample
(a) Crystalline digoxin. — Prep. ale. soln contg 125 [xg dig-
oxin/mL. Transfer 10.0 mL to separator, add 50 mL H 2 and
1 mL 2N H 2 S0 4 , and ext with three 30 mL portions CHC1 3 .
Wash each CHC1 3 ext in second separator by shaking with 10
mL H 2 and 1 g powd anion-cation exchange resin (Amberlite
MB-1, anal, grade, indicator-free, has been found satisfactory;
available as Mallinckrodt Cat. No. 5890), and filter thru pledget
of cotton moistened with CHC1 3 into 100 mL vol. flask. Dil.
to vol. with CHC1 3 and mix well. This soln is Assay Soln.
(b) Elixirs and injections. — Transfer aliquot contg 1.25 mg
digoxin to separator, and proceed as in (a), beginning: "...
add 50 mL H 2 and 1 mL IN H 2 S0 4 , ..."
(c) Tablets. — Accurately weigh, into 100 mL beaker, por-'
tion of powd tablets contg L25 mg digoxin. Add 10 mL al-
cohol, cover with watch glass, and heat to bp on steam bath.
Let simmer 20 min with frequent stirring. Cool, wash quant,
into separator with 30 mL CHC1 3 and 50 mL H 2 0, add 1 mL
IN H 2 S0 4 , and proceed as in (a), beginning: "... ext with
three 30 mL portions CHC1 3 ."
C. Determination
(a) Digoxin. — Pipet 5.0 mL digoxin std sotn and 10.0 mL
assay soln into similar erlenmeyers, and evap. to dryness on
steam bath with aid of air current. Cool, and to each flask add
5.0 mL freshly prepd alk. dinitrobenzene reagent. Let stand 5
min at <30°, with frequent mixing. Det. A of developing blue
colors relative to reagent blank at 620 nm at 1 min intervals,
using matched 1 cm cells and spectrophtr. Record max. A of
aliquot of assay soln and that of digoxin std soln, A'. Digoxin
(mg in assay soln) = 1.25 A /A'.
(b) Other digitoxosides. — Pipet 20 mL assay soln and 10
mL digoxin std soln into sep. beakers and evap. to dryness on
steam bath with aid of air current. Cool, add 4.0 mL Keller-
Kiliani reagent at <30° to each beaker, and mix thoroly. After
15 min, det. A of sample and std at 590 nm relative to reagent
blank at 5 min intervals. Record max. A of sample and that
of std, A'. Total digitoxosides calcd as digoxin (mg in sample
soln) = 1.25 A/A'. Difference between this value and that
obtained in (a) is amt of other digitoxosides in sample soln.
Ref.: JAOAC 42, 453(1959).
CAS-20830-75-5 (digoxin)
602
Drugs: Part
AOAC Official Methods of Analysis (1990)
OTHER NATURAL PRODUCTS
926.18* Camphor in Drugs
Polarimetric Determination
Final Action
Surplus 1972
(Not applicable to synthetic camphor)
See 39.061, 12th ed.
945.100* Camphor in Spirits
Gravimetric Determination
Final Action
Surplus 1972
See 39.062-39.063, I2th ed.
972.55 Camphor in Drugs
Gas Chromatographic Method
First Action 1972
Final Action 1974
A. Apparatus
(a) Gas chromato graph. — With H flame ionization detec-
tor and strip chart recorder. Operate instrument in accordance
with manufacturer's instructions. Operating conditions: temps
(°) — column 180, detector 220, flash heater 250; N flow rate
ca 60 mL/min adjusted to elute phenol in ca 9 min. Approx.
retention time of camphor is 2.5 min. Adjust electrometer sen-
sitivity so that 2.5 |xg phenol gives ca 50% deflection.
(b) GC column. — (Material available from Applied Science
has been found satisfactory.) Dissolve 1.2 g Carbowax 20M
in ca 50 mL CH 2 C1 2 on steam bath. (Caution: Use effective
fume removal device when heating or evapg CH 2 C1 2 .) Add 1.0
g 100-140 mesh Gas-Chrom P and stir as sol v. evaps. Dry 1
hr at 105°. Pour dry, coated packing material into 1.8 m (6')
x 4 mm glass tube, vibrating with hand vibrator. Place glass
wool plugs at each end; then insert septums. Condition prepd
column overnight at 200° with N flow.
B. Reagents
(a) Camphor std soln. — 0.5 mg/mL. Dissolve and dil. 100
mg camphor to 200 mL with CHC1 3 .
(b) Phenol internal std soln. — 1 mg/mL. Dissolve and dil.
100 mg phenol to 100 mL with CHC1 3 .
(c) Menthol internal std soln. — 0.5 mg/mL. Dissolve and
dil. 100 mg menthol to 200 mL with CHC1 3 .
(d) Methyl salicylate internal std soln. — 1 mg/mL. Dis-
solve and dil, 100 mg Me salicylate to 100 mL with CHC1 3 .
(e) Working std soln.- — Pipet equal vols of appropriate in-
ternal std soln, (b), (c), or (d), and camphor std soln, (a), into
g-s flask. Phenol is preferred internal std but sample must not
contain substance selected as internal std.
C. Preparation of Sample
(a) Oily solns. — Dil. aliquot of sample with CHC1 3 to ca
0.5 mg camphor/mL.
(b) Ointments. — Dissolve weighed sample in CHC1 3 ,
warming very slightly if necessary. Dil. with CHC1 3 to ca 0.5
mg camphor /mL.
D. Determination
Pipet equal vols sample soln and internal std into g-s flask.
Inject 5 |xL into gas chromatograph and record chromatogram.
Inject 5 |xL working std soln and record chromatogram. Mea-
sure ht of each peak above baseline.
Concn (mg/mL or mg/g) in sample
= (P x /' x C')/(P' x / x C)
where P' and P = hts std and sample peaks, resp.; /' and/ =
hts internal std peak in std and sample, resp.; C = mg std/
mL std soln used to prep, working std; and C ~ mL or g
sample/mL sample soln.
Ref.: JAOAC 55, 610(1972).
CAS-76-22-2 (camphor)
922.14* Camphor (Monobromated)
in Drug Tablets
Gravimetric Method
Final Action
Surplus 1975
See 39.068-39.069, 12th ed.
929.14*
Menthol in Drugs
Saponification Method
Final Action
Surplus 1975
See 39.070, 12th ed.
939.18* Cod Liver Oil in Emulsions
Final Action
Surplus 1965
See 32.299, 10th ed.
932.25*
Aloin in Drugs
Gravimetric Method
Final Action
Surplus 1975
See 39.071, 12th ed.
933.11* Podophyllum in Drugs
Gravimetric Method
Final Action
Surplus 1975
See 39.072, 12th ed.
931.14* Chenopodium Oil in Drugs
Titrimetric Method
Final Action
Surplus 1975
See 39.073-39.074, 12th ed.
AOAC Official Methods of Analysis (1990)
Natural Products
603
952.28 Rutin in Drugs
Spectrophotometry Method
Final Action
A. Reagents
(a) Acid-alcohol reagent. — Mix 550 mL alcohol with 50
mL HO Ac and dil. to 1 L with H 2 0.
(b) Rutin std soln. — 0.02 mg/mL. Accurately weigh 100
mg rutin (obtainable from ICN-K&K Laboratories, Inc.) and
dissolve in 50 mL acid-alcohol. Transfer to 250 mL vol. flask
with small portions acid-alcohol. Dil. to vol. with reagent and
mix well. Pipet 5 mL aliquot into 100 mL vol. flask and dil.
to vol. with H 2 0.
(c) Quercetin std soln. — 0.01 mg/mL. Prep, as in (b), us-
ing 50 mg quercetin. Pure quercetin may be prepd as in J. Am.
Pharm. Assoc. Sci. Ed. 42, 66(1953).
B. Apparatus
(a) Spectrophotometer. — Capable of isolating 338.5, 352.5,
and 366.5 nm, with isolated spectrum <5 nm.
(b) Absorption cells. — Matched 1 cm.
(c) Glass stirring rods. — Of small enough diam. to dis-
lodge material from tips of 50 mL conical centrf. tubes.
C. Preparation of Sample Solution
Weigh directly into 50 mL centrf. tube number of tablets
required to give 0.05-0.50 g rutin (^5 tablets). Record num-
ber and wt (If tablets are coated, dissolve coating with distd
H 2 after weighing, discard aq. washings, and transfer rutin-
contg core to centrf. tube.) Add 20 mL acid-alcohol reagent
and break up tablets with stirring rod. After tablets are thoroly
disintegrated, heat mixt. 10 min in H 2 bath held at 70-80°,
resuspending material occasionally by stirring. Remove stir-
ring rod, rinse with acid-alcohol reagent, and centrf. 15 min.
at ca 2000 rpm.
Decant supernate into 250 mL vol. flask, using funnel and
decanting with one smooth motion, and let tube drain ca 10
sec. While still inverted, rinse mouth of tube with acid-alcohol
reagent. Ext twice more, starting with "Add 20 mL acid-al-
cohol reagent ..." After third extn, dil. combined supernates
to 250 mL with acid-alcohol reagent. Any insol. material may
be removed by filtration after diln if first 15-20 mL filtrate is
discarded. Depending on original wt rutin taken, make diln
with H 2 to give final concn of 0.01-0.03 g rutin/L. Ppts
forming during aq. diln may be removed by filtration if first
portion of filtrate is discarded to guard against concn changes
due to adsorption.
D. Determination
Det. A of sample soln against H 2 blank at 338.5, 352.5,
and 366.5 nm. Also det. A of std rutin soln, Ar, and std quer-
cetin soln, Aq, against H 2 blank at 352.5 and 366.5 nm. (In
absence of std quercetin, values Aq 352 .5 - 0.553 and A Q)365 _ 5
= 0.631 may be used. Any error introduced by use of these
predetd values should be of second order.) Calc. as follows:
R} ~ ^338.5/^3
and
R?
5 /A 3
If 7?i = 0.914 ± 0.009 and R 2 = 0.842 ± 0.013, extd material
can be considered pure rutin and wt rutin /tablet can be calcd:
mg Rutin /tablet = A 352 . 5 X d
X W X 0.02/A' R>352 . 5 x w
where d = sample diln factor; W = av. wt/tablet; and w =
wt sample.
(Value of/?( beyond its upper limit while R 2 remains within
its range indicates interfering absorption which diminishes rap-
idly enough to be ineffective at 352.5 nm. Under this condi-
tion, A observed at 352.5 nm is accepted as correct, and rutin
content is calcd as for pure rutin. Increase in R 2 while R { re-
mains within or below its limits usually indicates presence of
quercetin. Simultaneous increase or decrease of both ratios be-
yond their respective limits indicates invalidating condition.)
Amts of rutin and quercetin may be calcd by solution of fol-
lowing simultaneous equations:
= (K,
r/0.02) + (Aq, 352 . 5 X <y/0.01)
^366.5 = 6^,366.5 X r/0.02) + (A^ 366 . 5 X 0/0.01)
where r = mg rutin/mL in sample soln, and q - mg quer-
cetin /mL in sample soln.
Refs.: JAOAC 35, 566(1952); 36, 85, 699(1953).
CAS- 11 7-39-5 (quercetin)
CAS- 153- 18-4 (rutin)
932.26 Santonin in Drug Mixtures
Final Action
A. Langer Method (Modified)*
—Surplus 1970
See 36.515, 11th ed.
Dinitrophenylhydrazine Method
B. Reagent
Dinitrophenylhydrazine sulfate soln. — Dissolve 1 g 2,4-di-
nitrophenylhydrazine in mixt. of 90 mL H 2 and 10 mL H 2 S0 4
by warming; cool, and filter.
C. Determination
(Caution: See safety notes on distillation, flammable solvents,
toxic solvents, and benzene.)
Weigh 2.5 g ground sample into gooch and wash with ca
100 mL pet ether satd with santonin. Discard washings. Ext
with ca 100 mL benzene, collecting filtrate in beaker. Evap.
to dryness, warm residue with alcohol until dissolved, transfer
to 100 mL vol. flask, cool, dil. to vol. at 20° with alcohol,
and filter if necessary. To 25 mL of this soln add 50 mL dini-
trophenylhydrazine sulfate soln and let stand 48 hr in dark.
Collect ppt in gooch and wash with ca 150 mL alcohol (1 +
2). Dry residue 1 hr at 100°, cool, and weigh. Wt ppt x 0.5775
— wt santonin.
Refs.: J. Pharm. Chim. 8th ser. 16, 49(1932). JAOAC 18,
526(1935).
962.23 Santonin in Drug Mixtures
Ultraviolet Absorption Method
First Action 1962
(Applicable in presence of starch and calomel)
A. Reagent
Santonin std soln. — 10 (xg/mL. Weigh 50 mg santonin NF
XI, transfer to 50 mL vol. flask, dissolve in alcohol, and dil.
to vol. with alcohol. Pipet 2 mL aliquot into 200 mL vol. flask
and dil. to vol. with alcohol.
604
Drugs: Part III
AOAC Official Methods of Analysis (1990)
B. Determination
Accurately weigh portion powd sample contg ca 35 mg san-
tonin, transfer to 100 mL vol. flask, dil. to vol. with alcohol,
and shake frequently during 15 min. Let settle ca 15 min, transfer
5 mL aliquot supernate to 200 mL vol. flask, dil. to vol. with
alcohol, and mix. Det. A of this soln and of std soln, A\ against
alcohol at 240 nm.
Grains santonin/tablet = (wt std, mg) x A x 4000
X tablet wt (mg)/A r X mg sample x 64.8
C. Identification
Ext portion of powd tablets with alcohol or use ale. soln
from detn and evap. to dryness. Santonin gives white tabular
crystals, mp 170-173°.
Ref.; JAOAC 45, 593(1962).
965.45 Santonin in Drug Mixtures
Infrared Method
First Action 1965
Final Action 1966
(Applicable to tablets in presence of calomel)
A. Apparatus
Infrared spectrophotometer. — For operation in 2-15 \xm re-
gion; equipped with 2 matched NaCl cells 1 .0 mm thick, suit-
able for CS 2 solns. (Cells of shorter path length are not suitable
because of low solubility of santonin.)
B. Determination
(Caution: See safety notes on distillation, pipets, flammable
solvents, toxic solvents, carbon disulfide, and chloroform.)
Transfer 25 mg Santonin NF XI, accurately weighed, to 125
mL separator contg ca 15 mL H 2 0. Ext as for sample.
Transfer accurately weighed portion powd tablets, contg ca
25 mg santonin, to 125 mL separator contg ca 15 mL H 2 0.
Make just ammoniacal with NH 4 OH (1 + 9) (ca 1 drop) and
ext with four 25 mL portions CHC1 3 . Filter each ext thru cotton
plug, moistened with CHC1 3 , in long-stem glass funnel into
250 mL beaker. Evap. combined CHC1 3 exts to ca 5 mL on
steam bath with aid of air current. Transfer quant, to 25 mL
g-s erlenmeyer with ca 10 mL CHC1 3 in 2 mL portions, and
evap. to dryness. Wash down sides of flask with few mL anhyd.
ether, repeating if necessary to form dry residue. Use caution
to avoid loss of sample by spattering. Add 10 mL CS 2 from
pipet, stopper flask, and mix by swirling. Filter any insol. ma-
terial thru cotton, and immediately det. baseline A of sample
and std (A') solns relative to CS 2 at max. of 9.75 jjliti, drawing
baseline between minima of 9.6 and 9.95 |xm.
% Santonin = A X mg std X 100/4' X mg sample
Record spectra of sample and std solns from 2 to 15 |xm
and compare for sample identity.
Ref.: JAOAC 48, 592(1965).
CAS-48 1-06-1 (santonin)
932.27* Santonin in Santonica
(Levant Worm Seed)
Dinitrodiphenylhydrazine Method
Final Action
Surplus 1975
937.15* Gums in Drugs
Spot Test Identification
Final Action
Surplus 1972
{See also 920.126.)
See 39.088-39.090, 12th ed.
(pomea in Drugs
Final Action
Proceed as in 932.28*.
932.28
Jalap in Drugs
Gravimetric Method
Final Action
See 39.083, 12th ed.
Place 10 g sample, as "60-mesh" powder, in 250 mL er-
lenmeyer and add 50 mL alcohol. Fit flask with stopper thru
which is inserted glass tube ca 1 m long to act as condenser,
and heat gently on simmering steam bath 30 min, shaking oc-
casionally. Transfer contents to small percolator and percolate
slowly with warm alcohol until ca 95 mL collects.
To test for complete extn, collect 10 mL more percolate and
pour few drops into cold H 2 0; if more than faint cloudiness
appears, continue percolation with warm alcohol until test for
resin fails. Cone, the addnl percolate by evapn and add to flask
before dilg to vol. Cool percolate to room temp, and dil. to
100 mL with alcohol. Mix well.
Evap. 25 mL of the prepd tincture (representing 2.5 g drug)
on H 2 bath in beaker or flask and dry residue until alcohol-
free. Add 15 mL H 2 0, bring mixt. to bp, let cool ca 3 min,
and stir well with flat-end rod 2 min to ensure thoro washing
of resin. Cool mixt. by placing container in jar of ice-cold H 2
and decant wash H 2 onto 9 cm filter. Repeat washing of resin
with another 15 mL portion H 2 0, boiling and cooling mixt.,
kneading resin as before, and decanting washings into filter as
before. Repeat washing and kneading process with hot H 2
third time.
Dissolve residue in container in 10 mL warm alcohol and
pour soln onto filter, collecting filtrate in weighed beaker or
flask. Use enough hot alcohol in small portions to completely
transfer soln of resin to filter and ensure thoro washing of fil-
ter. Evap. combined filtrate and washings to apparent dryness,
add 1 mL absolute alcohol, and evap. sol v., taking care to
rotate container in inclined position as last portions of solv.
are dissipated. Dry residue at 80° to const wt.
Refs.: JAOAC 15, 448(1932); 16, 375(1933).
912.03* Acidity (Volatile)
of Tragacanth Drugs
Titrimetric Method
Final Action
Surplus 1972
See 39.093, 12th ed.
982.38 Allergenic Extracts in Drugs
Protein Nitrogen Unit Precipitation Method
First Action 1982
Final Action 1985
A. Principle
Protein is pptd from allergenic ext by phosphotungstic acid,
and N in ppt is detd by appropriate Kjeldahl procedure. Protein
nitrogen unit (PNU) is equiv. to 1 x 10" s mg N.
AOAC Official Methods of Analysis (1990)
Natural Products
605
B. Reagents
(a) Phosphotungstic acid (PT A) precipitating solution . — 15%
PTA in 10% HO. Dissolve 15.0 g PTA in ca 70 mL H 2 0.
Add 22.2 mL HC1 (sp. gr. 1.19 g/m'L, 37.8% HC1) and dil.
to 100 mL with H 2 0.
(b) Sulfuric acid. — Sp. gr. 1.84, N-free.
(c) Copper sulfate. — CuS0 4 .5H 2 0, N-free. Prep, satd aq.
soln.
(d) Acid soln. — Add ca 40 mL satd aq. CuS0 4 to 9 lb bottle
of H 2 S0 4 in 10 mL portions with thoro mixing. After several
days, excess anhyd. CuS0 4 crystallizes and supernatant acid
is ready for use (CuS0 4 -H 2 S0 4 soln).
(e) Potassium sulfate. — N-free.
(f) Sodium hydroxide soln.— 50%.
(g) Boric acid soln. — 2%.
(h) Indicator soln. — Me red-bromocresol green soln. Mix
1 part 0.1% ale. Me red soln with 5 parts 0.1% ale. bromo-
cresol green soln.
(i) Hydrochloric acid. — 0.01N. Prep, as in 936.15, or use
0.01000N HC1 (purchased as std).
C. Apparatus
(a) Digestion rack. — With either gas or elec. heaters which
will supply enough heat to 30 mL flask to cause 15 mL H 2
at 25° to come to rolling boil in >2 but <3 min.
(b) Distillation apparatus. — One-piece or Parnas-Wagner
distn app. recommended by Committee on Microchemical Ap-
paratus, ACS.
(c) Digestion flasks. — Use 30 mL regular Kjeldahl or
Soltys-type flasks (Ref.: Anal. Chem. 23, 523(1951)). For small
samples, 10 mL Kjeldahl flasks may be used.
D. Preparation of Sample
Combine vol. of allergenic ext indicated below with 0.25
mL HC1 in 12 mL conical centrf. tube. Use 2 mL sample when
approx. PNU value of ext is not known. When approx. PNU
value of ext is known, analyze following vols:
Allergenic ext, PNU/mL
Vol., mL
>35 500
15 500-35 500
<15 500
Add 1 mL PTA pptg soln. Mix thoroly. Let stand 1 h at
room temp. (22 ± 3°).
Centrf. mixt. at room temp, at 2700 rpm (rotor radius =
10.80 cm) for 10-15 min (rel. centrifugal force measured to
tip of sample tube = g = 880).
Test for completeness of pptn by adding 5 drops PTA soln.
Check visually for turbidity in supernate. If turbidity develops,
add addnl 0.5 mL PTA soln. Let mixt. stand 1 h at room temp.
Recentrf. at 2700 rpm for 10-15 min (room temp.).
Pour off supernate. Invert centrf. tube to drain ppt. Do not
wash ppt.
Dissolve ppt in 10 mL 2% NaOH by first adding 3 mL 2%
NaOH with vol. pipet. Use vortex mixer to loosen ppt. Add
7 mL 2% NaOH (vol. pipet). Mix thoroly.
E. Determination
Pipet 9 mL prepd sample into 30 mL digestion flask. Add
ca 500 mg K 2 S0 4 > 3 boiling stones, and 2 mL CuS0 4 -H 2 S0 4
soln. Place flask in digestion rack. Heat carefully and digest
sample until soln turns colorless. Continue digestion for addnl
V2 h. Cool and place thin film of pet. jelly on rim of flask.
Transfer digest and boiling chips to distn app. and rinse flask
5 or 6 times with 1-2 mL portions of H 2 0. Place 125 mL
erlenmeyer contg 5 mL 2% H3BO3 soln and 5 drops of indi-
cator under condenser with tip extending below surface of soln.
Add ca 6 mL NaOH (50% w/w) to still. If distn app. uses
steam distn. distil at rate of 5 mL/min and collect ca 50 mL.
If app. does not introduce steam into distg flask, collect 10-
15 mL distillate and dil. to ca 50 mL with H 2 0. Titr. distillate
with 0.0 IN HO to end point (pinkish purple). Perform blank
detn in same manner, using H 2 in place of sample.
F. Calculation
mg N/mL = [(mL HC1 - mL HC1 blank)
X normality x 14.007 X 10/9]/mL sample
Calc. PNU/mL as follows:
PNU/mL = 10 5 x mg N/mL
Ref.: JAOAC64, 1435(1981). 69, 231,292(1986).
606
Drugs: Part
AOAC Official Methods of Analysis (1990)
Common and Chemical Names of Drugs in this Chapter
Common Name
Chemical Name
Acetanilide
Aconitine
Aloin
Apomorphine (hydrochloride)
Arecoline (hydrobromide)
Atropine
Benztropine mesylate
Caffeine
Camphor
Cephaeline
Chlorpheniramine maleate
Cinchophen
Cocaine (hydrochloride)
Codeine (phosphate, sulfate,
monohydrate)
Colchicine
Digitoxin
Digoxin
Emetine (hydrochloride)
Ephedrine (hydrochloride, sulfate)
Ergotamine (tartrate)
Ethylmorphine (hydrochloride)
Homatropine (hydrobromide,
hydrochloride)
Hydrocodone (bitartrate, hydrochloride)
Menthol
Morphine (hydrochloride, sulfate,
diacetate)
Neostigmine (bromide, methylsulfate)
Nikethamide
Pamaquine
Phenacetin
Physostigmine (salicylate, sulfate)
Pilocarpine (hydrochloride, nitrate)
Procaine hydrochloride
Quercetin
Quinacrine hydrochloride
Quinine (ethylcarbonate)
Rescinnamine
Reserpine
Rutin
Santonin
Strychnine (sulfate, nitrate)
Terpin hydrate
Theobromine
Theophylline
Triprolidine (hydrochloride)
A/-Phenylacetamide
1 6-Ethyl- 1,16,1 9-trimethoxy-4-(methoxymethyl)aconitane-3,8,1 0, 1 1 , 1 8-pentol 8-acetate 1 0-benzoate
10-Glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone
5,6,6a,7-Tetrahydro-6-methyl-4f/-dibenzo{de,g)quinoline-1 0, 1 1 -diol
1,2,5,6-Tetrahydro-1-methyl-3-pyridinecarboxylic acid methyl ester
a-(Hydroxymethyl) 8-methyl-8-azabicyclo[3.2.1]oct-3-yi ester benzeneacetic acid
3-(Diphenylmethoxy)-8-methyl-8-azabicyclo[3.2. 1 ]octane methanesulfonate
3,7-Dihydro-1 ,3,7-trimethyl-1 H-purine-2,6-dione
1 ,7,7-Trimethylbicyclo[2.2. 1 ]heptan-2-one
7',10,11-Trimethoxyemetan-6'-ol
7-(4-Chlorophenyl)-A/,A/-dimethyl-pyridinepropanamine-2-butenedioate
2-Phenyl-4-quinolinecarboxylic acid
3-(Benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester
(5a,6«)-7,8-Didehydro-4,5-epoxy-3-methoxy-17-methymorphinan-6-ol
A/-(5,6,7,9-Tetrahydro-1 ,2,3, 1 0-tetramethoxy-9-oxobenzo[a]heptalen-7-yl)acetamide
(3p,5p)-3[(C^2,6-Dideoxy-(i-D-r/Do-hexopyranosyl-(1^4)-0-2,6-dideoxy-[3-D-/'/ J (?o-hexopyranosy!-(1^4)-2,6-di-
deoxy-p-D-r/bo-hexopyranosyl)oxy]-14-hydroxycard-20(22)-enolide
(3fi,5f},12p)-3-[(0-2,6M3ideoxy-p-D-r/bo-hexopyra^
dideoxy-p-D-/vfc>o-hexopyranosyl)oxy]-1 2, 1 4-dihydroxycard-20(22)-enolide
6' ,7',1 0, 11 -Tetramethoxyemetan
a-[1-(Methylamino)ethyl]benzenemethanol
1 2'-Hydroxy-2'-methyl-5'a-(phenylmethyl)ergotaman-3' ,6' ,1 8-trione
(5a, 6a)-7,8-Didehydro-4,5-epoxy-3-ethoxy-17-methylmorphinan-6-ol
a-Hydroxy-8-methyl-8-azabicyclo[3.2.1]oct-3-yl ester benzeneacetic acid
5a-4,5-Epoxy-3-methoxy-17-methylmorphinan-6-one
(1a,2(i,5a)-5-Methyl-2-(1-methylethyl)cyclohexanoi
(5a,6a)-7,8-Didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol
3[[(Dimethylamino)carbonyl]oxy]-A/ J A/ J A/-trimethylbenzeneaminium
A/,A/-Diethyl-3-pyridinecarboxamide
A/ 7 ,A/ 1 -Diethyl-A/ 4 -(6-methoxy-8-quinolinyl)-1,4-pentanediamine
A/-(4-Ethoxyphenyl)acetamide
1 ( 2,3,3a,8,8a-Hexahydro-1,3a,8-trimethylpyrrolo(2,3-b)indol-5-ol methylcarbamate (ester)
3-Ethyldihydro-4-[(1-methyl-1H-imidazole-5-yl)methyl]-2(3H)-furanone
2-(Diethylamino)ethyl ester 4-aminobenzoic acid
2-(3,4-Dihydroxyphenyl)3,5,7-trihydroxy-4H-1-benzopyran-4-one
A/ 4 -(6-Chloro-2-methoxy-9-acridinyl)-A/ 1 , A/ 1 -diethyl-1 ,4-pentanediamine dihydrochloride
6'-Methoxycinchonan-9-ol
Hjy-Dimethoxy-IS-tfl-oxo-S-fS^^-trimethoxyphenyO^-propenylloxyl-S^O-yohimban-ie-carboxylicacid methyl
ester
11,17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester
3-[[6-0-(6-Deoxy-a-L-mannopyranosyl)-p-D-glucopyranosyl]oxy]-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-
benzopyran-4-one
3a,5,5a,9b-Tetrahydro-3,5a,9-trimethylnaphtho(1 I 2-b)furan-2,8(3H,4H)-dione
Strychnidin-10-one
4-Hydroxy-a,a,4-trimethyl-cyclohexanemethanol monohydrate
3,7-Dihydro-3,7-dimethyl-1H-purine-2,6-dione
3,7-Dihydro-1 ,3-dimethyl-1 H-purine-2,6-dione
2-[1-(4-Methylphenyl)-3-(1 -pyrrolidinyf)- 1 ~propenyl]pyridine
Source: USAN and the USP Dictionary of Drug Names (1983; 1989) U.S. Pharmacopeial Convention, Rockville, MD.
21. Drugs: Part IV
Thomas G. Alexander, Associate Chapter Editor
Food and Drug Administration
NATURAL ESTROGENS
961.22* Conjugated Estrogens
in Drugs
Spectrophotometric Method
First Action 1961
Final Action 1977
Surplus 1988
See 39.001-39.005, 14th ed.
955.55* Ketosteroids in Drugs
Colorimetric Methods
First Action
Surplus 1974
See 39.006-39.012, 12th ed.
946.05* Beta-Estradiol in Drugs
Colorimetric Method
First Action
Surplus 1970
fe 36.249-36.254, 11th ed.
973.76 Estradiol Valerate in Drugs
Spectrofluorometric Method
First Action 1973
Final Action 1975
A. Principle
Oils are eluted with heptane from CH 3 N0 2 -diat. earth col-
umn. Estradiol valerate is eluted with addnl heptane, and detd
by fluorometry at max. intensity, ca 328 nm.
B. Apparatus
(a) Recording spectrophotofluorometer . — With 1 cm cell
path, excitation wavelength 285 nm, and sensitivity to produce
70% fluorescence for std soln at 328 nm.
(b) Glass chromatographic tubes. — 250 X 25 mm id.
C. Reagents
(a) Heptane. — Redistd.
(b) Nitromethane. — Spectral grade, or equiv.
(c) Diatomaceous earth. — See 960. 53B.
(d) Estradiol valerate std solns. — (1) Stock soln. — 0.4 mg/
mL, Accurately weigh ca 40 mg USP Estradiol Valerate Ref.
Std in 100 mL vol. flask and dil. to vol. with absolute alcohol.
(2) Working soln. — 16 (xg/mL. Dil. 2 mL stock soln to 50
mL with absolute alcohol.
D. Preparation of Sample
Using "to contain" pi pet (or hypodermic syringe fitted with
1 V2", 18 gage needle), transfer accurately measured vol. sam-
ple contg ca 40 mg estradiol valerate to 100 mL vol. flask.
Wash pipet with heptane and add wash to vol. flask. Dil. to
vol. with heptane and mix.
E. Preparation of Column
Place glass wool plug in base of chromatgc tube. To 10 g
diat. earth in 250 mL beaker, add 1 1 mL CH 3 N0 2 . {Caution:
CH3NO2 is toxic and flammable. Wear resistant rubber gloves
when using it. Use effective fume removal device.) Mix until
fluffy and add to tube in portions, packing moderately after
each addn. Top column with glass wool pad and prewash col-
umn with 50 mL heptane.
F. Determination
Transfer 2 mL sample soln to column. Wash with 5,5, 10,
10, and 40 mL heptane (70 mL total), allowing each portion
to pass thru column before adding next. Discard eluate. (Cau-
tion: See safety notes on distillation and flammable solvents.)
Change receiver to 250 mL beaker and continue eluting with
heptane, collecting ca 150 mL. Evap. eluate to dryness and
quant, transfer residue to 50 mL vol. flask, using absolute al-
cohol. Dil. to vol. with absolute alcohol.
Adjust spectrophotofluorometer to ca 70% fluorescence in-
tensity at 328 nm with working std soln. Scan sample and std
solns from ca 280 to 450 nm, reading % fluorescence at max.,
ca 328 nm. Use absolute alcohol as blank.
mg Estradiol valerate/mL = 100 X C x (F/F') X (\/V)
where C = concn of std soln (mg/mL); F and F' = fluores-
cence of sample and std solns, resp., at 328 nm, each cor-
rected for blank; and V = vol. of sample taken.
Refs.: JAOAC54, 1192(1971); 56, 86(1973).
CAS-979-32-8 (estradiol valerate)
974.43 Ethinyl Estradiol
in Drugs
Spectrophotometric Method
First Action 1974
Final Action 1976
A. Reagents
(a) Methanol-sulfuric acid. — In ice bath, cautiously add
chilled H 2 S0 4 in small increments, with mixing, to 60 mL
chilled anhyd. MeOH in 200 mL vol. flask. Cool to room temp.,
dil. to vol. with H 2 S0 4 , and mix. Reagent is stable at room
temp, ca 1 month. (Caution: Wear face shield and heavy rub-
ber gloves to protect against splashes.)
(b) Washed chloroform. — Vigorously shake ca 500 mL
CHCI3 with 30 mL H 2 S0 4 in 1 L separator ca 2 min. Discard
H 2 S0 4 (bottom) layer. Wash CHC1 3 with 400 mL H 2 by
shaking vigorously 1 min; discard H 2 0. Repeat H 2 washing
3 times as above. Filter clear CHC1 3 layer thru funnel contg
pad of glass wool covered with ca 50 g granular anhyd. Na 2 S0 4 .
Prep, fresh daily. Use same batch of washed CHC1 3 for all
samples and stds thruout series.
(c) Ethinyl estradiol std solns. — (/) Stock soln. — 0.8 mg/
607
608
Drugs: Part IV
AOAC Official Methods of Analysis (1990)
inL. Accurately weigh ca 40 mg USP Ref. Std Ethinyl Estra-
diol, dissolve in anhyd. MeOH in 50 mL vol. flask, dil. to
vol. with MeOH, and mix. (2) Intermediate soln. — 20 jxg/
mL. Pipet 5.0 mL stock soln into 200 mL vol. flask, dil. to
vol. with isooctane, and mix. (3) Working soln. — 4 jig/mL.
Pipet 20 mL intermediate soln into 100 mL vol. flask, dil. to
vol. with isooctane, and mix. (This soln is stable at room temp.
ca 3 weeks.)
(d) Diatomaceous earth. — See 960. 53B.
B. Preparation of Column
Trap layer. — Transfer ca 5 g granular anhyd. Na 2 S0 4 to 25
x 250 mm chromatgc tube contg pad of glass wool in base.
Thoroly mix 3 mL 10% NaOH soln with 3 g diat. earth in 100
mL beaker. Transfer mixt. to tube in 1 portion and tamp mod-
erately.
Sample layer. — Accurately weigh portion of ground tablet
composite contg ca 40 |JLg ethinyl estradiol into 100 mL beaker.
Add 3 mL CHC1 3 and 2 mL H 2 0, and stir frequently 2 min
to dissolve max. amt of sample. Mix with 4 g diat. earth 1
min, transfer quant, to tube in 1 portion, and tamp moderately.
Dry- wash beaker with ca 0.5 g diat. earth and transfer wash
to column. Wipe tamper, spatula, and beaker with glass wool
and place glass wool on column.
C. Chromatography
Rinse tamper, spatula, and beaker with 25 mL isooctane and
add rinse to column. Discard eluate. Using total of 55 mL
CHCl 3 -isooctane (1 + 9), repeat rinsing as above and discard
eluate. Wash column with 15 mL isooctane and discard eluate.
Finally, elute ethinyl estradiol with 50 mL washed CHC1 3 , fol-
lowed by 25 mL isooctane, collecting eluate in 250 mL sep-
arator.
D. Determination
Pipet 10 mL each of std soln and isooctane (reagent blank)
into sep. dry 250 mL separators. To each add 50 mL washed
CHC1 3 and 15 mL isooctane, and mix gently. Pipet 10 mL
MeOH-H 2 S0 4 into sample, blank, and std separators, letting
pipet drain completely, (Caution: See safety notes on pipets
and sulfuric acid.) Shake vigorously 4 min, and let layers sep.
ca 15 min; protect from strong light. Within 30 min, scan spectra
between 700 and 500 nm of pink (lower) phases of std and
sample in 1 cm cells against reagent blank as ref., set at at
700 nm.
|xg Ethinyl estradiol in final soln = (A /A') x C X 10 (mL)
where A and A' refer to sample and std solns, resp., at max.,
ca 537 nm; and C = (Jig/mL std soln.
Ref.: J AOAC 57, 747(1974).
CAS-57-63-6 (ethinyl estradiol)
988.25 Cortisone Acetate
in Bulk Drug and Dosage Forms
Liquid Chromatographic Method
First Action 1988
A. Principle
Bulk drug or dosage form is dissolved in CH 3 CN-0.025M
acetate pH 4 buffer (1 + 1) and analyzed by exernal std method.
Cortisone acetate is resolved from extaneous components by
reverse phase liq. chromatgy and detected at 254 nm.
B. Apparatus
(a) Liquid chromatography. — Equipped with isocratic pump
system with UV detector (254 nm) and suitable recorder. Op-
erate at ambient temp.
(b) LC column. — Reverse phase octadecylsilane, 10 u-m.
(c) Ultrasonic bath.
(d) Table top centrifuge.
C. Reagents
Use LC grade reagents.
(a) Sodium acetate pH 4 buffer.— Mix 20 mL IN HC1, 150
mL 0.5N KC1, and 50 mL 0.5M NaOAc in 1 L vol. flask.
Dil. to vol. with H 2 (0.025M and 0.1 jul soln).
(b) Diluent.— Mix CH 3 CN and pH 4 buffer (1 + 1). Let
mixt. equilibrate to room temp.
(c) Mobile phase. — Degas mixt. of 450 mL CH 3 CN and
550 mL H 2 0. Adjust vol. of CH 3 CN as needed to obtain suit-
able retention time.
D. Preparation of Standard Solutions
Accurately weigh ca 30 mg USP Ref. Std and transfer to
100 mL vol. flask. Add diluent, sonicate until std is dissolved,
and dil. to vol. with diluent. Further dil. soln 10 mL to 50
mL, 10 mL to 25 mL, and 5 mL to 10 mL to prep. 3 std solns.
E. Preparation of Samples
Bulk drug. — Accurately weigh ca 25 mg bulk drug and
transfer to 250 mL vol. flask. Dissolve in and dil. to vol. with
diluent. Use an aliquot, concn ca 0. 1 mg/mL, for LC analysis.
Tablets. — Weigh and finely powder 10 tablets. Accurately
weigh equiv. of ca 2 tablets or amt necessary to prep, soln not
to exceed 1.0 mg/mL concn. Transfer sample to vol. flask,
dissolve in diluent, and sonicate ca 5 min. Dil. as needed to
concn of ca 0.1 mg/mL. Centrf. aliquot of prepd soln, and
use portion of supernate for LC analysis. Filter supernate thru
0.45 |xm filter before analysis if necessary.
Suspension. — Measure sample vol. of 1 or more vials as
follows: Shake vial vigorously until product is homogeneous
(but >15 s). Remove sample immediately by successive use
of clean, dry hypodermic syringes of appropriate size. Deliver
samples into same stdzd cylinder graduated to contain. Read
vol. Transfer contents of cylinder to vol. flask. Rinse all glass-
ware twice with diluent, and add rinses to vol. flask. Concn
of soln should not exceed 1.0 mg/mL. Dil. soln as needed to
concn of ca 0.1 mg/mL for LC analysis. Filter soln thru 0.45
fxm filter before analysis if necessary.
F. System Suitability Tests
Condition column with mobile phase until baseline is ac-
ceptable. Cortisone acetate peak should fulfill following per-
formance specifications: column efficiency, >rl500 theoretical
plates; asymmetry or tailing factor (at 5% peak ht), <2; ca-
pacity factor, k\ >2; relative std deviation <].% for 5 repli-
cate 20 jjlL injections.
G. Determination
Inject each of 3 std solns before and after all samples. Use
peak area to calc. amt of each sample, fig/mL, with respect
to stds. Curve-fit samples and calc. results mathematically or
by calculator.
H. Calculations
(Y) Calc. concn, jxg/mL, of cortisone acetate in each std
soln (C std ) as follows:
C std - (W std /V sld ) x (v d V df ) x (F/100) x 1000
where W sld ~ wt of std, mg; V std — vol. of std, mL; V d = vol.
of aliquot transferred for diln, mL; V df - vol. of flask used
for diln, mL; P = purity of std as %.
(2) Use linear regression procedure of PA std vs C std to prep,
std curve mathematically or by computer. Correlation coeffi-
cient (r) should be >0.999 and intercept <±3.0.
AOAC Official Methods of Analysis (1990)
Synthetic Estrogens
609
Substitute calcd values for consts m and c, and variable X
in following equation to calc. individual Y values:
Y = mX to c
where Y = std concn, (xg/rnL; and X = av. of peak areas for
stds injected before and after samples.
Enter /M sam (av. peak areas for samples = X) on std curve
and obtain value for C sam (concn of sample, jxg/mL = K).
(3) Calc. cortisone acetate in bulk drug and dosage forms
as follows:
Bulk drug:
Cortisone acetate, mg = (C sam x D)/1000
Suspension:
Cortisone acetate, mg/mL = [C sam x (D/N)]/ 1000
Tablets:
Cortisone acetate, mg/tablet
- C sam x D X [^/(^ x 1000)]
where D = diln factor; N = vol. for vials sampled, mL; W, ab
= av. tablet wt, mg; W sam = sample wt, mg.
Ref.: JAOAC 71, 534(1988).
CAS-50-04-4 (cortisone acetate)
SYNTHETIC ESTROGENS
960.61 Diethylstilbestrol in Drugs
Spectrophotometric Method
First Action 1960
Final Action 1965
A. Reagent and Apparatus
(a) Diethylstilbestrol std soln. — Accurately weigh suitable
amt of USP Diethylstilbestrol Ref. Std, dissolve in alcohol,
and prep, soln contg 20.0 (xg/mL by accurate stepwise diln
with alcohol. Prep, working std soln by mixing 25 mL of this
soln with 25 mL 1.8% K 2 HP0 4 soln.
(b) Irradiation containers . — Quartz cells ^4 mL capacity
with clear sides, or 18 x 150 mm Vycor test tubes, held in
rack that does not obstruct effective light beam of cylindrical
15 watt germicidal lamp, may be used conveniently.
B. Preparation of Assay Solution
(a) Oil solns containing 2 mg or less diethylstilbestrol /mL. —
Using accurately calibrated hypodermic syringe, transfer vol.
sample contg 2 mg diethylstilbestrol to separator contg 50 mL
isooctane. Shake mixt. with 10 mL IN NaOH and transfer well
defined aq. layer as completely as possible to second separator
contg 50 mL isooctane. Shake vigorously and transfer clear
aq. layer to third separator. Repeat extn of the 2 isooctane
layers successively with two 10 mL portions IN NaOH, collect
aq. layers in third separator, and discard extd isooctane layers.
Acidify combined aq. exts with 3 mL H 2 S0 4 (1 + 1), cool,
and ext diethylstilbestrol with three 30 mL portions CHC1 3 .
Wash CHC1 3 exts successively in 2 separators, first contg 20
mL 1% NaHC0 3 soln and second, 20 mL H 2 0.
Filter washed CHC1 3 exts thru cotton pledget moistened with
CHC1 3 into 100 mL vol. flask, dil. to vol. with CHC1 3 , and
mix.
Transfer 10.0 mL CHCl 3 soln, contg 200 fxg diethylstilbes-
trol, to small erlenmeyer and evap. just to dryness on steam
bath with aid of air current. Cool in vac. desiccator 10 min.
Add 10.0 mL alcohol, stopper, and dissolve residue by swirl-
ing. After 15 min, mix with 10.0 mL 1.8% K 2 HPO A to prep.
assay soln.
(b) Oil solns containing more than 2 mg diethylstilbestrol /
mL. — Dil. convenient accurately measured vol. oil soln with
CHC1 3 to obtain soln contg 0.5 mg diethylstilbestrol /mL.
Transfer 4 mL aliquot to separator contg 50 mL isooctane and
proceed as in (a), beginning "Shake mixt. with 10 mL IN
NaOH ..."
(c) Tablets. — Transfer accurately weighed portion powd
material contg 2 mg diethylstilbestrol to separator contg 30 mL
CHCI3. Add 10 mL H 2 and 1 mL H 2 S0 4 (1 + 1) and shake
vigorously. Drain CHC1 3 layer into second separator, wash with
5 mL H 2 6, and filter thru cotton pledget moistened with CHC1 3
into 100 mL vol. flask. Repeat extn with three 20 mL portions
CHC1 3 , dil. combined exts to 100 mL, and mix.
Proceed as in (a), fourth par.
C. Irradiation
(Caution: Protect eyes from direct rays of UV light.)
Test transparency of several irradiation containers as fol-
lows: Transfer convenient vols of working std soln to tubes,
place them ca 7 cm from 15 watt germicidal lamp, and irra-
diate soln transversely ca 10 min. Measure A of yellow solns
at 418 nm in suitable spectrophtr in matched 1 cm cells, against
H 2 0. Re-irradiate for 1-3 min intervals, and note irradiation
time required for max. A. Repeat irradiation process, varying
distance of tubes from lamp, and det. most convenient con-
ditions for developing stable, repeatable colors of max. A (ca
0.7 at 418 nm).
Transfer portions of working std soln and assay soln to clean,
dry irradiation containers, and irradiate under optimum con-
ditions previously detd. Calc. wt diethylstilbestrol in sample.
D. Total Phenols
Transfer 20 mL CHC1 3 ext, 960.61B, contg 400 *xg dieth-
ylstilbestrol to beaker. Transfer ale. soln contg 400.0 \xg USP
Diethylstilbestrol Ref. Std to similar beaker, and treat both
solns as follows: Evap. to dryness on steam bath with aid of
air current. Dissolve residues in 2.0 mL HO Ac with gentle
warming. Cool to room temp., add 10 drops H 2 S0 4 (1 + 1),
and mix. Cool, add 5 drops 10% NoN0 2 soln, and let stand
45 min with occasional mixing. Wash quant, into 25 mL vol.
flask with ca 20 mL ale. NH 4 0H soln, prepd by mixing equal
vols alcohol and dil. NH 4 OH (4 + 6). Cool in ice bath, and
let stand at room temp. 1 hr. Dil. to vol. with the ale. NH 4 OH
soln, and mix. If ppt forms, filter thru dry paper, rejecting first
few mL filtrate. Dot. A of clear, yellow alk. solns at 420 nm
in tightly stoppered 1 cm cells, in suitable spectrophtr, against
alcohol (1 + 2). Calc. % total phenols, as diethylstilbestrol,
in sample.
Ref.: JAOAC 43, 248(1960).
CAS-56-53-1 (diethylstilbestrol)
973.77 Dienestrol in Drugs
Spectrophotometric Method
First Action 1973
Final Action 1977
(Caution: See safety notes on distillation, pi pets, benzene,
diethyl ether, isooctane, and methanol.)
A. Reagents
(a) Dienestrol std soln. — Approx. 15 |xg/mL. Accurately
weigh USP Dienestrol Ref. Std, dissolve in MeOH, and se-
rially dil. to concn. Store in low-actinic vol. flask.
610
Drugs: Part IV
AOAC Official Methods of Analysis (1990)
(b) Methanolic sulfuric acid. — Carefully add, with swirl-
ing, 50 mL H 2 S0 4 to 50 mL cold MeOH, while continuously
chilling mixt. in ice-H 2 0. Use reagent at room temp. Reagent
is stable 3-4 days in g-s flask.
(c) Ethyl ether. — Test as follows on day of use: Evap. , with
gentle heat and air stream, mixt. of 10.0 mL dienestrol std
soln in ca 200 mL H 2 0- washed ether. Dissolve residue in 10.0
mL MeOH. Proceed as in 973. 77D, using this soln and 5.0
mL dienestrol std soln. Resulting solns should be clear and
exhibit single max. at ca 303 nm, and corrected A, 973. 77E,
should differ <3%. If necessary, wash 750 mL ether with three
50 mL portions ION KOH in 1 L separator. Percolate upper
ether layer thru 300 x 22 mm glass chromatgc tube contg glass
wool plug and 20 g diat. earth thoroly mixed with 15 mL ION
KOH and tamped moderately tight. Discard first 30 mL eluate
and collect remainder for use. Column will only purify max.
of 1 L ether.
(d) Diatomaceous earth. — See 960. 53B,
B. Preparation of Columns
Trap column. — Mix 4 g diat. earth and 3 mL 0.25M KOH
and transfer to 200 X 22 mm glass chromatgc tube contg glass
wool plug. Tamp mixt. tightly and top with glass wool pad.
Prewash column with 25 mL H 2 0- washed ether, followed by
25 mL benzene.
Sample column. — Accurately weigh freshly ground sample
contg ca 400 (xg dienestrol into 150 mL beaker. Add 3 mL
03M K 3 P0 4 and wet sample completely. Add 5 g diat. earth
and mix thoroly with spatula. Transfer quant, in 2 equal por-
tions to 200 x 22 mm glass chromatgc tube contg glass wool
pad, tamping each portion moderately tight. Dry- rinse beaker
with .1-2 g diat. earth and add rinse to column. Wipe tamper,
spatula, and beaker with glass wool pad and add pad to top of
column.
C. Chromatography
Arrange columns so that eluate from sample column passes
into trap column. Add 25 mL benzene to trap column; then
add 175 mL benzene-isooctane (9 + 1) to sample column, us-
ing several portions to rinse sample beaker. Maintain layer of
eluant over trap column. (To maintain this reservoir in trap
column, connect the 2 columns with air-tight stopper, i.e.,
hollow No. 4 Nalgene stopper with hole drilled to accom-
modate stem of sample column.) Discard sample column when
elution is complete. Wash trap column with addnl 25 mL ben-
zene-isooctane (9 + 1) and discard eluates.
Elute dienestrol from trap column with 225 mL H 2 0- washed
ether into 250 mL g-s conical flask contg 10 mL absolute al-
cohol. Without delay, evap. to near dryness, using air stream
and gentle heat. Rinse flask walls with small amt of absolute
alcohol and evap. soln to dryness. Pipet 25 mL MeOH into
flask, stopper tightly, and let stand several min with frequent
vigorous swirling.
D. fsomerization
Into sep. 25 mL g-s conical flasks, pipet 5 mL dienestrol
std soln, 5 mL sample prepn, and 5 mL MeOH as reagent
blank. Add 5.0 mL methanolic H 2 S0 4 to each flask with swirl-
ing (solns will become warm). Stopper flasks tightly and shake
vigorously; then let cool S:25 min at room temp.
B. Determination
Det. A of sample and std solns between 400 and 240 nm in
1 cm cells against reagent blank. Correct A at ca 303 nm by
subtracting A at 360 nm.
mg Dienestrol /tablet = [(A /A') x C x V x W]/Q
where A and A' refer to sample and std solns, resp.; C = exact
concn of std in mg/mL; V = mL sample diln (25 mL); W
av. tablet wt (g); and Q = sample wt (g).
Refs.: JAOAC 55, 190(1972); 56, 674(1973).
CAS-87-17-3 (dienestrol)
965.46 Hexestrol in Drugs
Spectrophotometry Method
First Action 1965
Final Action 1967
A. Determination
Grind tablets to fine powder. Weigh amt powder contg ca
5 mg hexestrol into 125 mL separator contg 25 mL H 2 and
1 mL HC1 (1 + 9). Ext with 25, 15, 10, and 10 mL CHC1 3 .
Drain each ext thru CHCl 3 -satd cotton pledget into 100 mL
beaker. Evap. combined exts to ca 25 mL on steam bath in
air current. Check for completeness of extn by evapg addnl 10
mL ext to dryness.
Quant, transfer coned CHC1 3 exts to 125 mL separator contg
10 mL isooctane. Ext with 25, 15, 15, and 10 mL ca 0.1W
NaOH, rolling or shaking separator gently 90 sec each time;
emulsions may form. Drain lower org. layer into second 125
mL separator, each time including any small emulsion layer
present. Continue alk. extn of org. phase, draining it alter-
nately into two 125 mL separators and combining alk. exts by
pouring each time into original separator. Discard org. phase.
Make combined alk. exts acid with HC1. Ext with 25, 15,
15, and 10 mL CHC1 3 , collecting combined exts in 125 mL
separator. Wash CHC1 3 exts with two 15 mL portions H 2 0.
Discard H 2 washes.
Pass combined CHC1 3 exts thru 1 cm column of granular
anhyd. Na 2 S0 4 in coarse fritted glass funnel, ca 3.5 cm id,
into 100 mL vol. flask. Rinse column and stem tip with small
portions CHCJ 3 . Dil. to vol. with CHCI 3 . Place 50.0 mL ali-
quot in g-s flask and evap. just to dryness on steam bath, with
aid of air current. Remove last traces of CHC1 3 with air current
and without heat. Pipet 50 mL alcohol onto dry residue; shake
1 min to dissolve. This is sample soln.
Prep, std soln by dissolving pure hexestrol in enough al-
cohol to make concn ca 2.5 mg/50.0 mL. Use alcohol as ref,
blank with sample and std solns.
Det. baseline A of sample and std solns at 280 nm with
spectrophtr. If recording UV spectrophtr is used, record spec-
tra between 320 and 240 nm. Adjust instrument to begin at
320 nm with zero A, and record spectra to 240 nm.
mg Hexestrol in assay sample
= (A J A') X (mg/mL std soln) X total mL sample soln
where A refers to sample and A' refers to std soln at 280 nm.
B. Qualitative Identification
(a) Ultraviolet spectra. — Dil. ale. soln of sample and std
previously used for quant, assay to ca 20 fxg/mL with alcohol.
Compare UV spectrum from 215 to 320 nm with similar spec-
trum from authentic hexestrol.
(b) Infrared spectra. — Prep. KBr disk contg 0.3-0.6%
hexestrol from residue obtained by evapg portion of remaining
CHCI3 sample soln from assay. Compare IR spectrum from 2
to 16 (xm with similar spectrum from authentic hexestrol. (Ex-
traneous peak at 5.85 |xm appears in spectra of tablet prepns
that does not appear in std.)
Ref.: JAOAC 48, 613(1965).
CAS-5635-50-7 (hexestrol)
AOAC Official Methods of Analysis (1990)
Synthetic Estrogens
611
971.42 Mestranol in Drugs
Spectrophotometric Method
First Action 1971
Final Action 1973
(Applicable in presence of norethindrone and norethynodrel;
not applicable in presence of ethynodiol diacetate or chlor-
madinone acetate)
A. Reagents
(a) Dicitomaceous earth. — See 960. 53B.
(b) Immobile solvent. — Mix equal vols DMF and form-
amide (either redistd or stabilized formamide may be used).
(c) n-Heptane. — Redistd (may be prepd by fractionating thru
all-glass column). A against alcohol in 1 cm cells should be
<0.500 in range 250-360 nm (limit of aromatic content). Res-
idue from evapn of 25 mL distillate, dissolved in 10 mL al-
cohol, should have A <0.01 in range 230-360 nm (nonvolatile
residue limit).
(d) Spectrophotometric solvent. —Transfer 10.0 mL CHC1 3
to 100 mL vol. flask, add ca 80 mL rc-heptane, warm to room
temp., and dil. to vol. with n-heptane.
(e) Mestranol std soln. — 0.06 mg/mL. Dissolve accurately
weighed amt USP Mestranol Ref. Std in CHC1 3 and dil. quant,
to ca 0.6 mg mestranol /mL. Transfer 10.0 mL aliquot to 100
mL vol. flask, add ca 80 mL n-heptane, warm to room temp.,
and dil. to vol. with n-heptane.
B. Preparation of Assay Mixture
Finely powder tablets. Transfer accurately weighed portion
contg ca 0.6 mg mestranol to 100 mL beaker. Add 2.0 mL
immobile sol v., mix, and warm 5 min on steam bath with oc-
casional stirring with spatula to ensure that powder is thoroly
wetted. Cool, add 4 g diat. earth, and mix with spatula until
fluffy.
C. Column Chromatography
Pack pledget of fine glass wool in base of 25 x 250 mm
chromatgc tube. Transfer 3,0 mL immobile solv. to 100 mL
beaker, add 1 g anhyd. Na 2 S0 4 , and mix by swirling. Add 5
g diat. earth and mix until fluffy. Transfer to tube and tamp
gently to compress to uniform mass. Quant, transfer prepd
sample to column, scrub beaker with 0.5 g diat. earth, and
tamp as before. Wipe beaker, spatula, and funnel with pad of
glass wool Place pad on top of column and tamp lightly.
Add /i-heptane to column. Discard first 20 mL eluate and
then collect ca 99 mL eluate in 100 mL vol. flask. Wash tip
of column with heptane, dil. eluate to vol. with heptane, and
mix. Transfer 50.0 mL aliquot to 125 mL g-s conical flask
and evap. on steam bath with aid of air current to ca 1 mL.
(Caution: Use effective fume removal device when evapg hep-
tane.) Remove last traces of solv. without heat. Wash sides of
flask with ca 2 mL alcohol and evap. solv. on steam bath as
before, removing last traces of solv. without heat. Add 5.0
mL spectrophtric solv. to flask, stopper, let stand ca 5 min,
and swirl to ensure soln of residue.
D. Determination
Record A of sample and std solns in 1 cm cells against spec-
trophtric solv. Construct baseline by extending line passing be-
tween points on spectrum at 302 and 315 nm. Det. baseline-
corrected A of sample (A A) and std (A A') at max., ca 287
nm.
mg Mestranol in portion of tablets taken
= 10C x (A A/A A')
where C - exact concn, mg/mL, of mestranol std.
Ref.: JAOAC 54, 590(1971).
CAS-72-33-3 (mestranol)
975.59 Mestranol with Ethynodiol
Diacetate in Drugs
Spectrophotometric Method
First Action 1975
Final Action 1976
A. Principle
Mestranol is sepd on partition column, eluted with n-hep-
tane, and extd into MeOH-H 2 S0 4 reagent to form colored ste-
roid complex with max. A at ca 540 nm.
B. Apparatus and Reagents
(Caution: Dimethyl sulfoxide (DMSO) and formamide can be
harmful. Avoid skin contact by wearing heavy rubber gloves.
Use effective fume removal device.)
(a) Chromatographic tubes and tamping rod. — Glass, 25
(od) X 300 mm. See 967.31A.
(b) Formamide. — Reagent grade contg no stabilizing agent
or H 2 0. Use recently opened bottle.
(c) Washed n- heptane. — Vigorously shake n-hcptanc (bp
98-99°) with ca 10% of its vol of H 2 S0 4 in separator 5:5 min.
Discard H 2 S0 4 (lower) layer and wash heptane with H 2 until
washings are neut. to pH test paper. Filter thru firm plug of
absorbent cotton covered with ca 50 g anhyd. Na 2 S0 4 , dis-
carding first 5 mL. Use same batch of washed heptane for all
samples and stds thruout series.
(d) Methanol- sulfuric acid reagent. — Cautiously add in small
increments, with mixing, chilled H 2 S0 4 (min. 95%) to 60 mL
chilled anhyd. MeOH in 200 mL vol. flask in ice bath. Adjust
to room temp., dil. to vol. with H 2 S0 4 , and mix. Reagent is
stable at room temp, ca 1 month. (Caution: See safety notes
on sulfuric acid and methanol.)
(e) Mestranol std solns. — (1) Stock soln. — 1 mg/mL. Dis-
solve ca 25 mg USP Ref. Std Mestranol, accurately weighed,
with 3 mL CHC1 3 in 25 mL vol. flask, dil. to vol. with n-
heptane, and mix well. (2) Intermediate soln. — 30 (mg/mL.
Pipet 3 mL stock soln into 100 mL vol. flask, dil. to vol. with
rc-heptane, and mix. (3) Working soln. — 0.75 jjig/mL. Pipet
5 mL intermediate soln into 200 mL vol. flask, dil. to vol.
with /7-heptane, and mix. Prep, fresh daily.
C. Preparation of Column
Thoroly mix 3 g diat. earth, 960. 53B, and 1 mL H 2 in 100
mL beaker, transfer to chromatgc tube contg pledget of glass
wool at base, and tamp moderately tight.
Thoroly mix 7 g diat. earth and 3.5 mL DMSO (spectral
grade) -formamide (10 + 9) in 150 mL beaker, transfer to tube
in 2 portions, and tamp each moderately tight.
Accurately weigh portion of ground tablets contg ca 150 jxg
mestranol into 100 mL beaker. Add 2 mL formamide and stir
continuously 2 min to wet and disperse sample completely.
Mix thoroly with 4 g diat. earth, transfer quant, to column in
1 portion, and tamp moderately tight. Scrub beaker with ca
0.5 g diat. earth and transfer to column. Wipe tamper, spatula,
and beaker with glass wool, and place as pad above column
contents.
D. Determination
Rinse tamper, spatula, and beaker with 65 mL n-heptane,
and pour rinse into column. Discard eluate contg ethynodiol
diacetate. Elute mestranol with total of 135 mL w-heptane, col-
lecting eluate in 200 mL vol. flask. Dil. to vol. with ^-heptane
and mix.
612
Drugs: Part IV
AOAC Official Methods of Analysis (1990)
Pipet 50 mL each of mestrano] working std soln and sample
eluate in sep. dry 250 ml separators. Pipet 10 mL MeOH-
H 2 S0 4 (Caution: See safety notes on pipets.) reagent into each,
draining pipet completely. Shake vigorously 4 min and let stand
45 min, protected from light. Within ^25 min, scan pink (lower)
phase in each separator between 700 and 500 nm in 10 mm
cells against MeOH~H 2 S0 4 reagent as ref , , setting instrument
to A at 700 nm for each scan.
|xg Mestranol in sample taken = 200 x C x (A /A')
where 200 = mL sample diln, C = fig mestranol std/mL std
soln, and A and A' refer to sample and std, resp., at max., ca
540 nm.
Ref.: JAOAC 58, 75(1975).
CAS-72-33-3 (mestranol)
PROGESTATIONAL STEROIDS
mg Steroid/tablet - (A/A r ) x W x (T/W)
where A. and A' refer to sample and std, resp., at max., ca
380 nm; and W, W, and T = mg sample, mg std/ 100 mL,
and av. tablet wt in mg, resp.
(b) Norethynodrel. — Add 1,0 mL methanolic HC1 to sep.
flasks contg 100 mL sample ext, 100 mL norethynodrel std,
and 100 mL CHC1 3 as blank. Shake vigorously 3 min (rnixts
may be hazy) and let stand 70 min. Add 1.0 mL MeOH to
each flask and mix thoroly (mixts become clear).
Transfer 5.0 mL each soln to sep. 50 mL g-s conical flasks
and continue as in (a).
Refs.: JAOAC 53, 831(1970); 54, 617(1971).
CAS-79-64-1 (dimethisterone)
CAS-7 1-58-9 (medroxyprogesterone acetate)
CAS-68-22-4 (norethindrone)
CAS-5 1-98-9 (norethindrone acetate)
CAS-68-23-5 (norethynodrel)
971.43 Progestational Steroids in Drugs
Spectrophotometry Method
First Action 1971
Final Action 1973
A. Principle
CHC1 3 ext of norethindrone, norethindrone acetate, dimeth-
isterone, or medroxyprogesterone acetate is treated directly with
isonicotinic acid hydrazide to produce stable color measured
at 380 mm Norethynodrel in CHC1 3 ext is isomerized with HC1
prior to same reaction.
B. Reagents
(a) Isonicotinic acid hvdrazide (INH) soln. — Transfer 100
mg INH (mp 171-173°) to 200 mL vol flask. Add ca 150 mL
MeOH and 0.1 mL HCL Shake to dissolve, and diJ. to vol.
with MeOH.
(b) Washed cotton.- — Wash absorbent cotton with CHC1 3
and air dry.
(c) Washed chloroform. — Shake CHC1 3 with equal vol, H 2
in separator. After CHC1 3 layer clears, filter thru pledget of
washed cotton covered with bed of ca 50 g anhyd. Na 2 S0 4 .
Use thruout method.
(d) Methanolic HCl soln. — Oil. 3.0 mL HC1 to 50 mL with
MeOH.
(e) Std soln. — Dissolve accurately weighed amt std drug in
CHCI3 and dil,, if necessary, with CHC1 3 to ca 10 mg/1.00
mL,
C. Preparation of Sample
Finely powder tablets. Transfer accurately weighed portion
of powder contg ca 10 mg steroid to 125 mL separator contg
10 mL H 2 0, Add 25 mL CHC1 3 , shake continuously 5 min,
and filter ext thru pledget of cotton and ca 30 g anhyd. Na 2 S0 4
into 100 mL vol. flask. Repeat extn with two 25 mL portions
CHCI3, combine exts, rinse filter with CHC1 3 , and dil. filtrate
to vol. with CHCI3.
0. Determination
(a) Norethindrone, norethindrone acetate, dimethisterone,
and medroxyprogesterone acetate. — To sep. 50 mL g-s con-
ical flasks, transfer 5.0 mL sample ext, 5.0 mL std soln, and
5.0 mL CHCI3 as blank. To each flask add 25.0 mL INH soln,
stopper, mix, and let stand 30 min. Record spectra from 500
to 350 nm against reagent blank,
977.32 Progestational Steroids in Drugs
Single Tablet Assay
First Action 1977
Final Action 1979
A. Principle
Principle is same as in 97 1.43 A, except that before colori-
metric detn, sample is eluted from H 2 0»diat. earth column with
CHCI3. Elution vols and vols of isonicotinic acid hydrazide
used vary with individual tablet dosage levels ranging from
0.35 to 10 mg.
B. Apparatus and Reagents
(a) Glass chromatographic tube and tamping rod. - — 250 X
25 (od) mm. See 967.31A.
(b) Isonicotinic acid hydrazide (INH) soln. — Prep. 500 mL
as in 971.43B(a), using 500 mL vol. flask and 2.5-fold amts
of all reagents.
(c) Washed chloroform. — See 971.43B(c),
(d) Methanolic hydrochloric acid soln, — 10%. Dil. 5.0 mL
HCl to 50 mL with MeOH.
(e) Std solns, — Dissolve in individual vol. flasks accurately
weighed amt of each std material in CHCI 3 , and dil. with CHC1 3
to ca 0.035 mg norethindrone /mL, 0,025 mg norgestrel/mL,
and 0.25 mg norethynodrel/mL. Prep, fresh daily.
C. Preparation of Column
Soak 1 accurately weighed tablet with 1 mL H 2 in 100 mL
beaker. Thoroly mix 1 g diat. earth, 960.53B, with 0.5 mL
H 2 in another 100 mL beaker with small metal spatula, trans-
fer to chromatgc tube contg small pledget of glass wool at
base, and tamp tight. Carefully triturate tablet with spatula,
add 3 mL CHCl 3s and mix gently to dissolve as much as pos-
sible of tablet. Mix sample thoroly with 3 g diat. earth, trans-
fer to tube in 1 portion, and tamp tight. Scrub beaker with ca
0.5 g diat. earth and transfer to tube. Wipe tamper, spatula,
and beaker with small pledget of glass wool and add to col-
umn.
D. Elution
(a) Norethindrone (0.35 mg/ tablet).- — Pipet 15 mL INH soln
into 25 mL vol. flask and place to collect eluate from column.
Rinse tamper, spatula, and beaker with 10 mL CHC1 3 , and
pour rinse into column. When elution stops, continue elution
AOAC Official Methods of Analysis (1990)
Adrenocortico Steroids
613
by adding 1 mL portions CHC1 3 until eluate fills flask to within
ca 0.5 mL of mark. Gently swirl flask occasionally during elu-
tion without detaching it from column. Detach flask from col-
umn, add CH.C1 3 to vol., mix, and let stand 45 min before
colorimetric detn.
For >0.35 mg norethindrone, increase elution vol. and /or
dil. eluate.
(b) Norgestrel (0.5 mg/ tablet). — Pipet 30 mL INH soln into
50 mL vol. flask and place it to collect eluate from column.
Rinse tamper, spatula, and beaker with 10 mL CHC1 3 , and
pour rinse into column. When elution stops, continue elution
by adding 2 mL portions CHC1 3 until eluate fills flask to within
ca 0.5 mL of mark. Continue as in (a).
(c) Norethynodrel (2.5-5.0 mg /tablet). — Place 100 mL vol.
flask marked to indicate ca 75 mL vol. to collect eluate from
column. Rinse tamper, spatula, and beaker with 10 mL CHC1 3 ,
and pour rinse into column. When elution stops, continue elu-
tion by adding 5 mL portions CHC1 3 until eluate fills flask to
75 mL mark. Pipet 10 mL norethynodrel std soln into another
100 mL vol. flask and mix with 65 mL CHC1 3 . Add 75 mL
CHCI3 into third 100 mL vol. flask as blank. Add ca 5 small
boiling chips and 1 .0 mL methanol ic 10% HC1 into each flask.
Stopper, and shake vigorously 1-2 min. Remove stoppers and
heat soln to bp on steam bath. Continue heating 15 min with
occasional swirling. Remove flasks and cool to room temp.
Dil. each to vol. with CHC1 3 and mix. (CHC1 3 solns should
be completely clear.)
£ Determination
(a) Norethindrone. — Into 25 mL vol. flask, pipet 10 mL
norethindrone std soln and 15.0 mL INH soln. Stopper, and
shake ca 1 min. Into another 25 mL vol. flask, pipet 10 mL
CHCI3 and 15 mL INH soln as blank for both norethindrone
and norgestrel. Stopper flask and mix. Let all flasks stand 1
hr.
(b) Norgestrel. — Pipet 20 mL norgestrel std soln into 50
mL vol. flask and add 30.0 mL INH soln. Stopper, and shake
ca 1 min. Let soln and blank, (a), stand 1 hr.
(c) Norethynodrel. — Into sep. 25 mL vol. flasks, pipet 10
mL each of isomerized sample, std, and blank. Add 15.0 mL
INH soln to each. Stopper, shake ca 1 min, and let stand 1
hr.
Record spectra of samples and stds from 550 to 350 nm
within next hr, against corresponding ref. blank, setting in-
strument to A at 550 nm for each scan.
jxg Progestin/tablet = (A/ A') X C X (W/W)
where A and A' refer to sample and std, resp., at ca 380 nm;
C = |xg of corresponding std in 10.0 mL norethindrone or
norgestrel std soln, or 20.0 mL norethynodrel std soln; W =
av. tablet wt; and W = individual tablet wt.
Ref.: J AOAC 60, 922(1977).
CAS-68-22-4 (norethindrone)
CAS-68-23-5 (norethynodrel)
CAS-797-63-7 (norgestrel)
963.31* Ethisterone in Drugs
Infrared Spectroscopic Method
First Action 1963
Surplus 1974
See 39.043-39.046, 12th ed.
ADRENOCORTICO STEROIDS
984.40 Hydrocortisone in Drugs
Liquid Chromatographic, Infrared Spectroscopic,
and Thin Layer Chromatographic Methods
First Action 1984
Final Action 1988
A. Principie
Sample is dissolved in MeOH and CH 2 C1 2 , and hydrocor-
tisone is detd by liq. chromatography using acetaminophen in-
ternal std. Identity is confirmed by IR or TLC.
B. Apparatus and Reagents
(a) Liquid chromatograph . — Equipped with sampling valve
capable of introducing 10 jjlL injections, 25 cm x 4.6 mm id
stainless steel column packed with spherical 5-6 |xm diam.
porous silica particles, and 254 nm UV detector set at sensi-
tivity to produce ca 1 / 2 full scale peak ht on suitable recorder
for 10 julL injection of std soln. Mobile solv. flow rate 1.5
mL/min, ambient temp.
(b) Water-washed 1 ,2-dichloroethane . — Shake 500 mL LC
grade 1,2-dichloroethane with 250 mL H 2 for 1 min, let lay-
ers sep., and filter lower layer thru 0.5-1 |mm porosity poly-
tetrafluoroethylene (PTFE) membrane.
(c) Mobile solvent.— -Mix 55 mL H 2 0-LC grade MeOH (5
+ 95) soln with 1.0 mL glacial HOAc and dil. to 1 L with
H 2 Owashed 1,2-dichloroethane. Degas mixt. Adjust H 2 and
MeOH content to obtain suitable retention times.
(d) Internal std soln. — Dissolve 200 mg acetaminophen
(100% pure, Aldrich Chemical Co.) in 4 mL MeOH and dil.
to 200 mL with H 2 0-washed 1,2-dichloroethane.
(e) Std soln. — Accurately weigh ca 10 mg USP Ref. Std
Hydrocortisone, add 2 mL MeOH and 4.0 mL internal std soln,
and dil. to 50 mL with LC grade CH 2 C1 2 .
C. Chromatographic System Suitabiiity Test
Equilibrate LC system and inject 10 |xL portions of std soln.
Retention times for hydrocortisone and acetaminophen should
be ca 8 and 10.5 min, resp. Column efficiency, n, calcd using
hydrocortisone peak, should be >5000 theoretical plates; res-
olution, R s , between hydrocortisone and acetaminophen peaks
should be >2.5. Inject 6 replicate 10 |mL portions of std soln
and calc. response ratios, R, of hydrocortisone peak relative
to acetaminophen peak. Relative std deviation for six R values
should be <1.0%.
D. Sample Preparation
(a) Bulk material. — Accurately weigh ca 50 mg previously
dried (3 h at 105°) sample, add 10 mL MeOH and 20.0 mL
internal std soln, and dil. to 250.0 mL with CH 2 C1 2 .
(b) Tablet composite. — Weigh and finely powder >20 tab-
lets. Accurately weigh portion of powder equiv. to 1 tablet and
transfer to vol. flask of a size to yield final hydrocortisone
concn of 0.2 mg/mL. Add 2 mL MeOH for each 10 mg of
labeled hdyrocortisone content. Place flask in ultrasonic bath
2 min. Add CH 2 C1 2 until flask is ca 72-fiill and return to ul-
trasonic bath 1 min. Add 4.0 mL internal std soln for each 10
mg labeled hydrocortisone content. Dil. to vol. with CH 2 C1 2 ,
mix, and filter thru 0.5-1.0 \xm porosity Teflon membrane.
(c) Individual tablets. — Place 1 tablet in g-s or vol. flask
of a size to yield final hydrocortisone concn of ca 0.2 mg/
mL. Place 100 |xL H 2 for each 10 mg hydrocortisone directly
on tablet and let soak 30 min. Add 2 mL MeOH for each 10
mg of labeled hydrocortisone content and place flask in ultra-
sonic bath 10 min or until tablet disintegrates. Proceed as in
Tablet composite, starting with "Add CH 2 Cl 2 until . . .".
614
Drugs: Part IV
AOAC Official Methods of Analysis (1990)
E. Determination
Equilibrate LC system by passing mobile phase thru column
ca V2 h. Inject 10 \xL portions of std soln until R values for 3
consecutive std chromatograms agree within 1%, then inject
10 |ulL sample prepn.
mg Hydrocortisone in sample (R/R') x (V/V) x W
where R and R' — response ratios of hydrocortisone peak rel-
ative to internal std peak for sample and std, resp.; V and V
= mL internal std soln in std and sample solns, resp.; W =
mg hydrocortisone in std soln.
F. Infrared Spectroscopic Identification
(a) Bulk material. — IR spectrum of KBr dispersion of sam-
ple, previously dried 3 h at 105°, exhibits max. at same wave-
lengths as that of similar prepn of USP Ref . Std Hydrocorti-
sone.
(b) Tablets. — Powder tablets equiv. to ca 50 mg hydro-
cortisone and digest 5 min with 15 mL hexane. Decant hexane,
ext residue with 15 mL peroxide-free ether in same manner as
before, and discard ext. Digest residue with 25 mL dehydrated
alcohol for 5 min, filter, and evap. alcohol ext on steam bath
to dryness. Add 10 mL H 2 0, mix, let residue settle, and decant
H 2 0. Dry residue at 105°. Dissolve USP Ref. Std Hydrocor-
tisone in alcohol and treat as sample prepn, beginning with
"evap. alcohol ext . . .". The IR spectrum of KBr dispersion
of sample residue exhibits absorbance maxima at the same
wavelengths as that of similar USP Ref. Std Hydrocortisone
prepn .
Thin Layer Chromatographic identification
G. Apparatus and Reagents
(a) Thin layer plate. — 20 X 20 cm, coated with 0.25 mm
thick layer of chromatographic silica gel with fluorescent in-
dicator.
(b) Developing solvent.— CHCl v MeOH-H 2 (180 + 15 +
1).
(c) Spray reagent.— H 2 S0 4 -EtOH (1 + 4).
(d) Std soln.— 0.2 mg USP Ref. Std Hydrocortisone/mL
CH 2 C1 2 .
H. Procedure
Apply 10 |xL portions of sample prepn from LC Determi-
nation and std soln (d) to TLC plate 2 cm from bottom edge.
Develop plate in suitable tank equilibrated with developing sol v.
Let plate dry and examine under shortwave UV light. Spray
plate with H 2 S0 4 -EtOH (1 + 4), heat 5 min at 120°, and ex-
amine under longwave UV light. Hydrocortisone has R ( of ca
0.2, appearing as dark spot under UV before spraying and as
bright yellow spot in final step.
Ref.: JAOAC67, 218(1984).
CAS-50-23-7 (hydrocortisone)
974.44* Dexamethasone Phosphate
in Drugs
Spectrophotometry Method
First Action 1974
Surplus 1988
See 39.056-39.060, 14th ed.
988.27 Dexamethasone
in Drug Substance and Elixirs
Quantitative and Identification Methods
First Action 1988
A. Principle
Dexamethasone content in drug substance and elixir is detd
by normal phase LC using quaternary mobile phase with con-
trolled H 2 content, UV detection at 254 nm, and cortisone
as internal std. Identity is confirmed in bulk drug substance
and elixir by TLC and in drug substance by IR spectroscopy
and relative LC retention time ratios. Alcohol content in elixir
is detd by GC on porous polymer column using internal std
and flame ionization detector.
Liquid Chromatographic Method
B. Apparatus
(a) Liquid chromato graph. — Model 8100 (Spectra-Physics,
3333 N First St, San Jose, CA 95134-1995) equipped with
Model 100-10 photometric detector (Hitachi/NSA, 460 E
Middlefield Rd, Mountain View, CA 94043), 15-30 uX in-
jection valve (Valco Instruments Co., Inc., PO Box 55603,
Houston, TX 77255), and Model CR1A integrator (replace-
ment Model C-R6A) (Shimadzu Scientific Instruments, Inc.,
7102 Riverwood Rd, Columbia, MD 21046). Equiv. LC sys-
tem, UV detector, auto-sampler, and strip chart recorder may
be used. LC pumping system in which bubbles develop in mo-
bile phase is unsuitable. 1 |xg dexamethasone should produce
50% full scale response with appropriate detector and recorder
or integration settings at 254 nm. Mobile phase flow rate 1.2
mL/min at ambient temp.
(b) Chromatographic column. — Stainless steel, 25 cm X
4.6 mm id, packed with 5 |xm Zorbax-Sil (E.I. du Pont de
Nemours and Co.), or equiv. meeting appropriate LC system
suitability requirements. Stainless steel guard column, 3 cm X
4.6 mm id, packed with 10 (xm silica particles may be used.
If necessary, dry silica column by eluting with 20 mL CH 2 C1 2 -
HOAc-2,2-dimethoxypropane (90 + 2 + 2 v/v/v).
C. Reagents
(a) Solvents.— Glacial HOAc (J.T. Baker, Inc., No. 9508,
or equiv.), UV grade MeOH and CH 2 C1 2 (Burdick and Jackson
Laboratories, Inc., or equiv.), and distd-in-glass H 2 0.
(b) Methanol soln. — Pipet 5.0 mL H 2 into 100 mL vol.
flask and dil. to vol. with MeOH.
(c) Mobile phase. — Pipet 1.0 mL glacial HOAc and 45.0
mL MeOH soln into 1 L vol. flask, and dil. to vol. with CH 2 C1 2 .
Degas mixt. Adjust MeOH content to obtain retention times
of approx. 6 and 9 min for cortisone and dexamethasone, resp.
Cortisone retention time should be used for mobile phase com-
position adjustments; increased MeOH content decreases re-
tention time.
(d) Sodium bicarbonate soln. — 1M. Dissolve 8.4 g Na-
HCO3 in 100 mL H 2 0.
(e) Internal std soln. — Dissolve 30 mg cortisone (Sigma
Chemical Co. or equiv.) in 4.0 mL MeOH and dil. to 100.0,
mL with CH 2 C1 2 .
(f) Dexamethasone std soln. — 4.0 mg/100 mL. Transfer
ca 25 mg accurately weighed USP Ref. Std Dexamethasone
(previously dried 30 min at 105°) to 25 mL vol. flask, and
dissolve in and dil. to vol. with MeOH. Transfer 2.0 mL ali-
quot of this soln to 50 mL vol. flask contg 6.0 mL internal
std soln, and dil. to vol. with CH 2 C1 2 . Do not filter thru mem-
brane filter.
AOAC Official Methods of Analysis (1990)
Adrenocortico Steroids
615
D. Sample Preparation
(a) Drug substance. — Prep, as directed for Dexamethasone
std soln, using 25 mg dexamethasone. Do not filter thru mem-
brane filter.
(b) Elixir. — Transfer accurately measured 10 mL portion of
Dexamethasone Elixir, contg 1 mg dexamethasone, to 125 mL
separatory funnel, add 5 mL 1M NaHC0 3 soln, and ext with
four 20 mL portions of CH 2 CJ 2 . Collect exts in 250 mL sep-
aratory funnel contg 5 mL H 2 0. Back- wash combined exts and
filter thru cotton wet with CH 2 C1 2 into suitable beaker. Rinse
H 2 back-wash and 125 mL separatory funnel consecutively
with 10 mL CH 2 C1 2 . Filter this rinse into beaker. Evap. filtrate
on steam bath under jet of air to approx. 10 mL and quant,
transfer with CH 2 C1 2 to 25 mL vol. flask contg 1.0 mL MeOH
and 3.0 mL internal std soln. Dil. to vol. with CH 2 C1 2 . Do not
filter thru membrane filter.
E. Determination
Equilibrate column with mobile phase at 1.2 mL/min. Mon-
itor response at 254 nm. Make 3 replicate injections of dex-
amethasone std soln. Using either peak area or peak ht mea-
surements for each injection, calc. coefficient of variation (CV)
of peak response ratios of dexamethasone to internal std. In
suitable system, CV should be <2.5% and resolution factor,
/? s , for dexamethasone peak and internal std peak should be
>3. Make duplicate injections of std and sample solns and det.
response ratio for each. Relative retention ratios of dexameth-
asone to internal std should agree within ±2.0%. If relative
retention ratios differ by >2.0%, then dry silica column as
described in Apparatus (b).
F. Calculations
Calc. content of dexamethasone as follows:
Drug substance:
Dexamethasone, mg = 625 x C X (RR/RR r )
Elixirs:
Dexamethasone, mg/5 mL - 12.5 x C x (RR/RR')
where C - final concn of std soln (mg/mL), and RR and RR'
= av. response ratio for peak ht or area of analyte to that of
internal std for sample and std solns, resp.
Thin Layer Chromatographic Identification
G. Apparatus, Reagents, and Test Solutions
(a) Thin layer plates. — Glass, 20 X 20 cm, coated with 250
fxm layer of silica gel G with fluorescent indicator (Analtech,
Inc., Cat. No. 02011 or equiv.).
(b) Developing solns. — (J) Drug substance. — CHCl 3 -di-
ethylamine (2 + 1). (2) Elixir. — CHCl 3 -acetone-glacial HO Ac
(80 + 40 + 1).
(c) TLC std test solns. — (1) Drug substance.— Prep. 1 mg/
mL soln of USP Ref. Std Dexamethasone in CH 2 Cl 2 -MeOH
(1 + 1). (2) Elixir. — Evap. 10 mL of Dexamethasone std soln
(f ) just to dryness on steam bath. Dissolve residue in 1 mL
CH 2 Cl 2 -MeOH (1 + 1). Prep, individual 400 |xg solns of dex-
amethasone (Sigma Chemical Co., or equiv.) and cortisone in
CH 2 Cl 2 -MeOH (1 + 1) to serve as chromatgc identification
stds.
(d) TLC sample test solns. — (/) Drug substance. — Prep,
as directed for TLC std test soln. (2) Elixir.— Evap. 10 mL
of elixir sample prepn (b) just to dryness on steam bath. Dis-
solve residue in 1 mL CH 2 CI 2 -MeOH (1 + 1).
H. Chromatography
Equilibrate suitable chromatgc tank with appropriate devel-
oping solv. Spot 5 (xL of each test soln ca 2.5 cm from bottom
of coated plate. Let spots dry and develop chromatogram until
solv. front has moved 10 cm from origin. Remove plate, mark
solv. front, air-dry plate, and locate spots under shortwave UV
light. For drug substance, R f of major spot in sample test soln
corresponds to that for std test soln. For elixir, relative R { of
dexamethasone to cortisone for TLC sample test soln corre-
sponds to that for TLC std test soln.
/. Infrared Spectroscopic Identification
Drug substance. — Prep. KBr dispersions from previously
dried sample and std material. Scan spectra between 2.5 and
15.0 fxm. Compare sample and std spectra. If difference ap-
pears, dissolve portions of both sample and std in CH 3 CN,
evap. solns to dryness, and repeat test on residues. Sample and
std prepns exhibit maxima at same wavelength.
J. Identification by Relative Retention Times
Drug substance. — Compare retention ratios of main peak to
internal std peak obtained for dexamethasone std soln and for
assay sample prepn as directed in LC assay. Ratios that do not
differ by >2.0% confirm identity.
Alcohol in Elixir
Gas Chromatographic Method
K. Apparatus and Reagents
(a) Gas chromato graph. —Model 5830A, with flame ioni-
zation detector and electronic integrator (Hewlett-Packard), or
equiv. Operating conditions: column temp. 165° and N gas
flow adjusted so that 2-propanol elutes in 3-5 min.
(b) Chromatographic column. — Glass, 6 ft X 4 mm id,
packed with 80-100 mesh copolymer of ethylvinylbenzene and
divinylbenzene that has nominal surface area of 500-600 sq
m/g and av. pore diam. of 0.0075 jxm. This material has been
washed with org. solvs and acids and then preconditioned in
bulk in O-free atm. Super-Q (Alltech Associates Cat. No. 2735)
has been found to be suitable.
(c) Internal std soln. — Dil. 5.0 mL 2-propanol with H 2 to
250 mL.
(d) Alcohol std soln. — Dil. 5.0 mL absolute alcohol with
H 2 to 250 mL. Pipet 10 mL of this soln and 10 mL internal
std soln into 100 mL vol. flask and dil. to vol. with H 2 0.
L. Preparation of GC Column
With small plug of silanized glass wool in end of column,
apply vac. to exit of column and add packing in small amts
to inlet end. With aid of gentle vibration, pack column firmly.
Condition column overnight at 235° with slow N flow. Check
column for voids after conditioning. Gently vibrate column to
remove voids. Check column performance by injecting alcohol
std soln and calcg following: resolution >3; RSD <1.5% for
alcohol peak area relative to 2-propanol peak area with 6 rep-
licate injections; and tailing factor <2.0 for alcohol.
M. Sample Preparation
Pipet 4 mL elixir and 10 mL std soln into 100 mL vol. flask
and dil. to vol. with H 2 0.
N. Determination and Calculation
Inject ca 5 \xL each of sample and std solns in duplicate,
Calc. % alcohol in elixir as follows:
% alcohol (v/v) = (RR/RR r ) x C X D
616
Drugs: Part IV
AOAC Official Methods of Analysis (1 990)
where RR and RR f = av. response ratio for peak area of an-
alyte to that of internal std for sample and std, resp.; C = %
alcohol in std soln; and D = sample diln factor.
Ref.: J AOAC 70, 967(1987).
CAS-50-02-2 (dexamethasone)
988.26 Dexamethasone Acetate
in Bulk Drug and Suspensions
Liquid Chromatographic Method
First Action 1988
A. Principle
Bulk drug or suspension is dissolved in CH 3 CN-0.025M
phosphate pH 6 buffer (1 + 1) and analyzed by external std
method. Dexamethasone acetate is resolved from extraneous
components by reverse phase liq. chromatgy and detected at
254 nm.
B. Apparatus
(a) Liquid chromatograph. — Equipped with isocratic pump
system with UV detector (254 nm) and suitable recorder. Op-
erate at ambient temp.
(b) Column. — Reverse phase octadecylsilane, 10 jxm.
(c) Ultrasonic bath.
C. Reagents
Use LC grade reagents.
(a) Potassium phosphate pH 6 buffer. — Mix 3 mL IN NaOH,
138 mL 0.5N KC1, and 50 mL 0.5M KH 2 P0 4 in 1 L vol. flask.
JDil. vol. with H 2 (0.025M and 0.1 (jl soln).
(b) Diluent. — Mix CH 3 CN and 0.025M phosphate pH 6
buffer (1 + 1). Let mixt. equilibrate to room temp.
(c) Mobile phase. — Degas mixt. of 450 mL CH 3 CN and
550 mL H 2 0. Adjust vol. of CH 3 CN as needed to obtain suit-
able retention time.
D. Preparation of Standard Solutions
Dry USP Ref. Std 2 h in 105° oven. Accurately weigh ca
30 mg dried std and transfer to 100 mL vol. flask. Add diluent,
sonicate until std is dissolved, and dil. to vol. with diluent.
Further dil. soln 10 mL to 50 mL, 10 mL to 25 mL, and 5
mL to 10 mL to prep. 3 std solns.
E. Preparation of Samples
Bulk drug. — Accurately weigh ca 25 mg bulk drug that has
been dried 2 h in 105° oven and transfer to 250 mL vol. flask.
Dissolve in and dil. to vol. with diluent. Use an aliquot, concn
ca 0.1 mg/mL, for LC analysis.
Suspension. — Measure sample vol. of 1 or more vials as
follows: Shake vial vigorously until product is homogeneous
(but S:15 s). Remove sample immediately by successive use
of clean, dry hypodermic syringes of appropriate size. Deliver
samples into same stdzd cylinder graduated to contain. Read
vol. Transfer contents of cylinder to vol. flask. Rinse all glass-
ware twice with diluent, and add rinses to vol. flask. Concn
of soln should not exceed 0.8 mg/mL. Dil. soln as needed to
concn of ca 0. 1 mg/mL. Filter soln thru 0.45 (mm filter before
LC analysis if necessary.
F. System Suitability Tests
Condition column with mobile phase until baseline is ac-
ceptable. Dexamethasone acetate peak should fulfill following
performance specifications: column efficiency, >1500 theo-
retical plates; asymmetry or tailing factor (at 5% peak height),
<2; capacity factor, k' >2; relative std deviation <1% for 5
replicate 20 |xL injections.
G. Determination
Inject each of 3 std solns before and after all samples. Use
peak area to calc. amt of each sample, u,g/mL, with respect
to stds. Curve-fit samples and calc. results mathematically or
by calculator.
H. Calculations
(1) Calc. concn, (xg/mL, of dexamethasone acetate in each
std soln (C std ) as follows:
C std - (HW^std) x (VjV df ) x (P/IQ0) x 1000
where W sxd = wt of std, mg; V sld - vol. of std, mL; V d = vol.
of aliquot transferred for diln, mL; V df - vol. of flask used
for diln, mL; P ~ purity of std as %.
(2) Use linear regression procedure of PA sld vs C std to prep,
std curve mathematically or by computer. Correlation coeffi-
cient (r) should be >0.999 and intercept <±3.0.
Substitute calcd values for consts m and c, and variable X
in following equation to calc. individual Y values:
Y = mX + c
where Y - std concn, fxg/mL; and X = av. of peak areas for
stds injected before and after samples.
Enter PA sam (av. peak areas for samples = X) on std curve
and obtain value for C sam (concn of sample, u-g/mL = Y)
(J) Calc. dexamethasone acetate and dexamethasone equiv.
in bulk drug and suspension, resp. as follows:
Bulk drug:
Dexamethasone acetate, mg = (C sam x £>)/1000
Suspension:
Dexamethasone equiv., mg/mL
= fAa m x (D/N) x /n/1000
where D - diln factor; N - vol. for vials sampled, mL; F -
factor to convert acetate to free base = 0.903.
Ref.: JAOAC 71, 534(1988).
CAS-558 12-90-3 (dexamethasone acetate monohydrate)
CAS-50-02-2 (dexamethasone)
977.33 Prednisolone or Prednisone
in Drugs
Semiautomated Method
First Action 1977
Final Action 1979
A. Principle
Alcoholic soln of drug is extd with CHC1 3 and reacted with
tetramethylammonium hydroxide and blue tetrazolium. A of
resulting complex is read in flowcell at 525 nm.
B. Apparatus
(a) Automatic analyzer. — Include following modules: Sam-
pler with 30/hr (3:1) cam; proportioning pump; colorimeter,
equipped with 15 mm tubular flowcell and matched 525 nm
filters; recorder compatible with colorimeter; manifold (see Fig.
977.33, or equiv.).
(b) Shaker. — Wrist-action
(c) Ultrasonic generator. — 150 watt.
C. Reagents
(a) Blue tetrazolium (BT) reagent. — 0.15%. Dissolve 1.5 g
BT in 50 mL MeOH and dil to 1 L with alcohoL Store in light-
resistant bottle.
AOAC Official Methods of Analysis (1990)
Adrenocortico Steroids
617
All transmission tubing is Acidflex
a 0.034" Polyethylene tubing.
WASTE
FIG.
I
PROPORTIONING
1 5 mm flowcell PUMP
525 nm
Si = silicone, RA = Red Acidflex
1 T = Tvaon,S = Soivaflex,A = Acidflex
COLORIMETER RECORDER Yy
977.33— Flow diagram for semiautomated analysis for prednisolone or prednisone
(b) Tetramethylammonium hydroxide {TMAH) reagent. —
0.15%. Dil. 15.0 mL 10% TMAH soln to 1 L with alcohol.
(c) Prednisolone std soln. — (]) Stock soln. — 0.4 mg/mL.
Accurately weigh ca 40 mg USP Prednisolone Ref. Std into
100 mL vol. flask, dissolve in 50% alcohol, and dil to vol.
(2) Working soln. — 0.10 mg/mL. Pipet 25 mL stock soln into
100 mL vol, flask and dil. to vol. with 50% alcohol.
(d) Prednisone std soln.— (I) Stock soln. — 0.2 mg/mL.
Accurately weigh ca 20 mg USP Prednisone Ref. Std into 100
mL vol. flask, dissolve in 50% alcohol, and dil. to vol. (2)
Working soln. — 0.05 mg/mL. Pipet 25 mL stock soln into
100 mL vol. flask and dil. to vol. with 50% alcohol.
D. Preparation of Sample
Disintegrate individual tablet or disperse weighed composite
in accurately measured vol. 50% alcohol to give prednisolone
concn of 0.1 mg/mL or prednisone concn of 0.05 mg/mL.
Use ultrasonic generator until tablet is disintegrated and shake
mech. 15 min. Let soln settle >2 hr.
E Analytical System
Sample is withdrawn and extd with air-segmented stream of
CHC1 3 in double mixer, and org. phase is sepd in BO fitting.
BT and TMAH reagents are added to org. phase and mixed.
Soln is passed thru delay coil and A is detd at 525 nm in 15
mm flowcell.
F. Start-Up and Shut-Down Operations
Pump alcohol thru CHC1 3 line 10 min; then pump CHC1 3
thru line 5 min. Place remaining tubes in their resp. solns and
let system equilibrate 20-30 min or until steady baseline is
obtained. To shut down system, place CHC1 3 , BT, and TMAH
lines in alcohol and remove all other lines from their solns.
After 15 min, remove remaining lines from alcohol soln and
pump system dry.
G. Determination
Fill sample cups in following order: 3 cups std soln, 5 cups
sample soln, 1 cup std soln, 5 cups sample soln, etc. Place 2
cups std soln at end of each series. (First 2 cups of std solns
are used to equilibrate system, but are not included in calcns.)
Start Sampler II. After last cup has been sampled, let system
operate until steady baseline is obtained. Draw tangent to ini-
tial and final baselines. Subtract baseline to det. net A and A'
for each sample and std peak, resp. Discard values for first 2
and last std peaks and calc. av. std A'.
mg Drug in portion taken = (A /A') x C x D
where C = concn of std in mg/mL and D = diln factor.
Ref.: J AOAC 60, 27(1977).
CAS-50-24-8 (prednisolone)
CAS-53-03-2 (prednisone)
986.38 Prednisolone in Tablets
and Bulk Drugs
Liquid Chromatographic Method
First Action 1986
Final Action 1989
A. Principle
Prednisolone is detd by normal phase liq. chromatgy, using
silica column and UV detection.
B. Apparatus
(a) Liquid chromatography — Equipped with sol v. delivery
system, UV detector, and data module. Operating conditions:
flow rate 1.5 mL/min; 254 nm detector, 0.20 AUFS; temp,
ambient; 10-15 uX, injection.
(b) LC column. — 25 cm x 4.6 mm id, packed with 5-6
jxm porous spherical silica particles, that passes system suit-
ability tests.
(c) Filters. — Polytetrafluoroethylene membrane filters pore
sizes 0.5 and 5.0 (xm, resp.
618
Drugs: Part IV
AOAC Official Methods of Analysis (1990)
C. Reagents
(a) Solvents. — Distd in glass, LC grade.
(b) LC mobile phase.— Mix 60 mL 95% MeOH with 1.0
mL acetic acid and dil. to 1 L with H 2 0- washed 1,2-dichlo-
roethane. Filter thru Type FH filter and degas 5 min.
(c) Internal std soln. — 1.0 mg/mL. Transfer 100 mg
fluoxymesterone to 100 mL vol. flask. Dissolve in 5 mL MeOH
and dil. to vol. with CH 2 C1 2 .
(d) Prednisolone std soln. — (1) Stock std soln. — 0.5 mg/
mL. Accurately weigh ca 25 mg USP Ref. Std Prednisolone
into 50 mL vol. flask. Add 2 mL MeOH and dil. to vol. with
CH 2 C1 2 . (2) Working std soln.— 0.05 mg/mL. Pipet 10.0 mL
stock std soln and 5.0 mL internal std soln into 100 mL vol.
flask. Add 6 mL MeOH and dil. to vol. with CH 2 C1 2 .
D. Preparation of Samples
Bulk drugs.— Accurately weigh ca 50 mg sample (dried in
vac. 3 h at 105°) into 100 mL vol. flask. Add 4 mL MeOH
and dil. to vol. with CH 2 C1 2 . Pipet 10.0 mL sample soln and
5.0 mL internal std soln into 100 mL vol. flask. Add 6 mL
MeOH and dil. to vol. with CH 2 CI 2 .
Tablets. — Grind tablets to pass No. 60 sieve. Transfer ac-
curately weighed portion of powder contg 5 mg prednisolone
to 100 mL vol. flask. Add 6 mL MeOH and place flask in
ultrasonic bath 2 min. Add ca 50 m CH 2 CJ 2 and return to ul-
trasonic bath 1 min. Add 5.0 mL internal std soln and dil. to
vol. with CH 2 C1 2 . Shake flask vigorously and filter portion of
soln thru Type LS filter into 25 mL g-s flask.
Individual tablet assay {content uniformity). — Place tablet
in 125 mL g-s flask and add 200 |xL H 2 0. Let stand until tablet
disintegrates and add 1 mL MeOH for each mg prednisolone
declared. Place flask in ultrasonic bath until tablet is dispersed.
Add 1.0 mL internal std soln for each mg prednisolone de-
clared and dil. with CH 2 C1 2 to ca 0.05 mg/mL. Shake flask
vigorously and filter portion of soln thru Type LS filter into
25 mL g-s flask.
E. Determination
Let LC system equilibrate with 1.5 mL/min flow rate. In-
ject 10-15 (xL prednisolone working std soln. Retention times
of fluoxymesterone and prednisolone should be ca 6 and 9 min,
resp., with R (resolution) value >6. Inject 5 replicate aliquots
and prednisolone working std soln and calc. response ratios.
CV will be <2.0% in suitable system. Proceed with sample
analysis, injecting same amt of sample soln.
F. Calculations
Calc. results, using response ratios (A and A') relative to
internal std:
Bulk drugs:
Prednisolone, % = (A/A') x (C/S) X 100
Tablets (composite):
Prednisolone, mg/tab. = (A/A') x C x (W/S) x 100
Tablet (individual):
Prednisolone, mg/tab. = (A/A f ) X C X (T/D)
where A and A' — response ratios for sample and std solns,
resp.; C = mg prednisolone/mL working std soln.; W - av.
tablet wt (g); S = sample wt (g); T ~ labeled amt (mg) of
prednisolone in tablet; and D = concn (mg/mL) of predni-
solone in tablet soln, based on labeled amt/tab. and diln.
Ref.: J AOAC 67, 674(1984).
CAS-50-24-8 (prednisolone)
THYROID
982.39 Iodine in Thyroid Drug Tablets
Differential Pulse Polarographic Method
First Action 1982
Final Action 1984
A. Apparatus
Polarograph. — With dropping Hg electrode. Typical op-
erating parameters: scan rate 5 mV/s; scan direction ** — "; po-
tential scan range 1.5 V; initial potential -0.9 V; modulation
amplitude 50 mV; differential pulse operating mode; display
direction " + "; drop time, 1 s; low pass filter off; push-button,
initial; offset, off; current range 1-10 jxamp, or as needed.
B. Reagents
Use anal, reagents and glass-distd H 2 thruout.
(a) Bromine water. — Br-satd H 2 0. Prep, fresh daily.
(b) Potassium carbonate. — If reagent grade K 2 C0 3 gives
high blank, purify as follows: Dissolve ca 200 g K 2 C0 3 in 400
mL H 2 0, add 50 g 20-50 mesh Amberlite IRA-400 ion ex-
change resin (Mallinckrodt Chemical Works), and agitate 30
min. Filter thru glass wool plug into porcelain crucible, evap.
to dryness on hot plate, and heat at 675° in muffle 25 min.
Cool to room temp., and grind to fine powder with mortar and
pestle.
(c) Reagent blank. — Dissolve 8 g K 2 C0 3 in ca 70 mL H 2
in 100 mL vol. flask. Add 1 mL Br-satd H 2 and 20 mg
Na 2 S0 3 . Mix, dil. to vol. with H 2 0, and mix.
(d) Standard solns. — (7) 1 mg I/mL: Dissolve 1.686 g KI0 3
in ca 200 mL H 2 in 1 L vol. flask. Dil. to vol. and mix. (2)
32 |xg I/mL: Pipet 8 mL std soln (7) into 250 mL vol. flask,
dil. to vol., and mix.
(e) Working soln. — Pipet aliquot (V) of std soln (2) contg
same amt off contained in one tablet (see below) into 100 mL
vol. flask contg 8 g K 2 C0 3 dissolved in 70 mL H 2 0.
Tab. strength,
gr. thyroid
I content,
Std soln 2,
mL
32.4
128.6
259.2
643.0
1
4
8
20
Add 1 mL Br-satd H 2 and mix. Add Na 2 S0 3 (ca 20 mg) until
soln becomes colorless; mix. Dil. to vol. with H 2 and mix.
C. Sample Preparation
(a) Composite assay. — Weigh and finely powder tablets.
Weigh portion of powder equiv. to 1 tablet into porcelain cru-
cible that has been washed with HN0 3 (1 + I), rinsed with
H 2 0, and wiped dry. Mix with 4 g K 2 C0 3 and overlay with
addnl 4 g K 2 C0 3 . Place crucible in preheated 675° muffle 25
min. Cool, add 30 mL H 2 0, carefully heat on hot plate to
dissolve residue, and filter thru funnel with glass wool plug
into 100 mL vol. flask. Repeat heating with 2 addnl 30 mL
portions of H 2 0, and add these exts to vol. flask. Add 1 mL
Br-satd H 2 0, mix, add Na 2 S0 3 (ca 20 mg) until soln becomes
colorless. Dil. to vol. with H 2 and mix.
(b) Individual tablet assay. — Crush 1 tablet in porcelain
crucible with glass rod. Remove any sample adhering to glass
rod with spatula, and add to crucible. Proceed as in Composite
Assay, (a), beginning "Mix with 4 g K 2 C0 3 ..."
AOAC Official Methods of Analysis (1990)
Thyroid
619
D. Determination
Add ca 10 mL working soln to dry polarographic cell. Bub-
ble N thru cell 5 min; then direct stream of N above soln.
Using typical operating parameters as guide, switch selector
to external cell and wait until pen becomes stationary; then
depress scan button. Similarly, using same settings, analyze
sample soln followed by reagent blank. From baseline estab-
lished by reagent blank, measure peak hts of std and sample
solns at ca -1.18 V vs SCE. Calc. as follows:
I as % of declared thyroid
= (PH x V x W t X 3.2)/(PH' x W, X TH)
where PH and PH' = peak ht of sample and std, resp.; V =
mL of 32 (jtg/mL std used to prep, working std soln; W t and
W s - av. wt of tablet and wt of sample, g, resp.; and TH =
declared thyroid per tablet, mg.
Ref.: JAOAC65, 1059(1982).
CAS-7553-56-2 (iodine)
Common and Chemical Names of Drugs in this Chapter
Common Name
Chemical Name
Beta-Estradiol
Cortisone acetate
Dexamethasone (acetate, phosphate)
Dienestrol
Diethylstilbestrol
Dimethisterone
Equilin
Estradiol valerate
Estrone (sodium sulfate)
Ethinyl estradiol
Ethisterone
Hexestrol
Hydrocortisone
Medroxyprogesterone (acetate)
Mestranol
Norethindrone (acetate)
Norethynodrel
Norgestrel
Prednisolone
Prednisone
(1 70)-Estra-1 ,3,5(1 0)-triene-3,1 7-diol
1 7,21 -Dihydroxypregn-4-ene-3,1 1 ,20-trione 21 -acetate
9-Fluoro-1 1 p, 1 7,21 -trihydroxy-1 6a-methylpregna-1 ,4-diene-3,20-dione
4,4'-(1 ,2-Diethylidene-1 ,2-ethanediyl)-bisphenol
4,4'-(1 ,2-Diethyl-1 ,2-ethenediyl)-bisphenol
1 7p-Hydroxy-6a-methyl-1 7-(1 -propynyl)-androst-4-en-3-one
3-Hydroxyestra-1 ,3,5(1 0),7-tetraen-17-one
(173)-Estra-1 ,3,5(1 0)-triene-3,17-diol-17-pentanoate
3-Hydroxyestra-1,3,5(10)-trien-17-one
(17a)-19-Norpregna-1,3,5(10)-trien-20-yne-3,17-diol
1 7-Ethynyl-1 7p-hydroxyandrost-4-en-3-one
4,4'-(1,2-Diethylethylene)diphenol
(11,1 7,21 -Trihydroxy-pregn-4-ene-3,20-dione
6a-17-(Acetyloxy)-6-methyl-pregn-4-ene-3,20-dione
3-Methoxy-19-norpregna-1,3,5(10)-triene-20-yn-17-ol
17a-Hydroxy-19-norpregn-4-en-20-yn-3-one acetate
1 7-Hydroxy-1 9-nor-1 7a-pregn-5(1 0)-en-20-yn-3-one
1 3-Ethyl-1 7«-hydroxy-1 8, 1 9-dinorpregn-4-en-20-yn-3-one
1 1 ,17,21-Trihydroxypregna-1 ,4-diene-3,20-dione
17,21-Dihydroxypregna-1,4-diene-3,11,20-trione
Source: USAN and the USP Dictionary of Drug Names (1983; 1989), U.S. Pharmacopeial Convention, Rockville, MD.
22. Drugs: Part V
Linda L. Ng, Associate Chapter Editor
Merck Sharp & Dohme Research Laboratories
Diacetylmorphine
(Heroin) in Drug Powder
Microchemical Tests
Final Action
See 930.40.
Lysergic Acid Diethylamide
Optical Crystallographic Tests
Final Action
See 960.57 (lysergic acid diethylamide (LSD) tartrate, 4-
methyl-2,5-dimethoxyamphetamine.HCl (STP.HC1, DOM), and
psilocybin).
978.29 Cocaine Hydrochloride
in Drug Powders
Gas Chromatographic Method
First Action 1978
Final Action 1983
A. Principle
Cocaine is extd from weakly basic, aq. soln with CHC1 3
contg internal std, and then sepd by GC from other amines,
org. -sol. neutrals, and internal std.
B. Apparatus and Reagents
(a) Gas chromatography — With H flame detector and 1.8
m (6') x 4 (id) mm glass column packed with 3% OV-1 on
100-120 mesh Chromosorb W(HP) (Applied Science). Typi-
cal operating conditions: temps (°): column 225, detector and
injector 240; N carrier gas flow rate, 60 mL/min, Adjust col-
umn temp, to elute cocaine in 3 ± 0.5 min. Adjust H and air
flow rates and electrometer sensitivity so that 4 |ulL cocaine std
soln gives 40-60% full scale deflection. Retention time of co-
caine relative to internal std is ca 0.65.
(b) Dibasic potassium phosphate. — 10%. Dissolve 5 g
K 2 HP0 4 in 50 mL H 2 0, and mix well
(c) Dilute hydrochloric acid. — AN. Dil. 7.0 mL HC1 to
1 L with H 2 0, and mix well.
(d) Internal std soln. — 0.8 mg/mL. Dissolve 80 mg tetra-
cosane in 100 mL CHC1 3 , and mix well.
(e) Cocaine hydrochloride std soln. — 1 mg/mL. Accu-
rately weigh ca 10 mg cocaine. HC1 into 10 mL vol. flask, and
dil. to vol. with internal std soln. Prep, fresh every 3 months.
Store in refrigerator.
C. Preparation of GC Column
Plug column exit with silanized glass wool. Apply vac. to
exit end, and slowly add packing material thru column inlet
while gently tapping column. Fill to within 1 cm of column
inlet, and plug with silanized glass wool. Condition column
overnight at 260° with slow stream of N. Sat. column with
cocaine by making successive 4 jxL injections of cocaine std
soln until cocaine: internal std ratio differs by <3% from pre-
ceding std injection.
D. Determination
Accurately weigh ca 250 mg finely ground sample and dil .
quant, with 0.17V HC1 to estd cocaine.HCl concn of 1 mg/
mL. Mix well and let any insol. material settle. Pipet 2 mL
sample prepn, 2 mL internal std soln, and 1 mL 10% K 2 HP0 4
into test tube; stopper, and shake vigorously. Let layers sep.
Pipet 2 mL cocaine std soln, 2 mL 0.1 TV HC1, and 1 mL 10%
K 2 HP0 4 soln into another test tube, stopper, and shake vig-
orously. Let layers sep. Inject 4 |jlL CHC1 3 (bottom) layer of
sample and std solns, each in duplicate, using 10 uX syringe.
% Cocaine. HC1 - (P/P') x (B'/B) x (C/W) x D x 100
where P and P r - av. areas or peak hts of sample and std
cocaine.HCl, resp.; B and B' = av. areas or peak hts of in-
ternal std in sample and std, resp.; C — mg cocaine. HCl/mL
in std soln; W = mg sample; and D = diln factor for sample.
mg Cocaine. HCl/tablet
= % cocaine.HCl
av. tablet wt in mg/100
Refs.: JAOAC 61, 473, 683(1978).
CAS-53-21-4 (cocaine hydrochloride)
921.13* Diacetylmorphine (Heroin)
in Drug Tablets
Titrimetric Method
First Action
Surplus 1970
See 36.022, 11th ed.
955.56* Diacetylmorphine and Quinine
in Drug Powders
Spectrophotometric Method
Final Action 1965
Surplus 1973
See 40.003-40.005, 12th ed.
985.50 Diazepam in Drug Tablets
Liquid Chromatographic Method
First Action 1985
Final Action 1988
A. Principle
Diazepam content of tablets is detd by reverse phase liq.
chromatgy using MeOH-H 2 mobile phase, UV detection at
254 nm, and /?-tolualdehyde as internal std.
B. Apparatus
(a) Liquid chromato graph. — Tracor Model 950 sol v. pump
with Model 970A variable wavelength detector capable of
monitoring elution at 254 nm (Tracor Instruments Inc. (re-
placement models 951 and 971, resp.)), injection valve with
620
AOAC Official Methods of Analysis (1990)
Methaqualone
621
20 jjlL sample loop (Valco Instruments, Inc., Houston, TX
77055), and suitable strip chart recorder, or equiv. LC system.
Operating conditions: mobile phase flow rate ca 1.2 mL/min,
temp, ambient, sensitivity adjusted to give 60-90% FSD for
sample and std injections. Retention times for /?-tolualdehyde
and diazepam ca 5 and 10 min, resp.
(b) Chromatographic column. — Stainless steel, 30 cm X
3.9 mm id, packed with C t8 (xBondapak, 10 |mm (Waters As-
sociates, Inc.) or equiv. column meeting LC system suitability
requirements.
C. Reagents
(a) p-Tolualdehyde. —9S% (cat. no. 89850, Fluka Chemi-
cal Corp., 980 S. Second St, Ronkonkoma, NY 11779, or
equiv.).
(b) Solvents. — LC grade MeOH.
(c) Mobile phase.— MeOH-H 2 (65 4- 35), degassed be-
fore use.
(d) Internal std soln. — Prep, fresh daily as follows: Pipet
-1 mL p-tolualdehyde into 50 mL vol. flask, dil. to vol. with
MeOH, and mix. Pipet 4 mL of this soln into 250 mL vol.
flask, dil. to vol. with MeOH, and mix.
(e) Diazepam std soln.— USP Diazepam Ref. Std (RS),
previously dried in vac. over P2O5 4 h at 60°. Dissolve ac-
curately weighed amt in MeOH, and dil. quant, with MeOH
to ca .1 mg/mL. Pipet 5.0 mL of this soln and 5.0 mL internal
std soln into 25 mL vol. flask, dil. to vol. with MeOH, and
mix.
D. LC System Suitability Test
Make 5 replicate injections of std soln and record peak ht
or peak area responses. System is suitable if relative std dev.
(5 r ) is <2.0%, using the equation:
S T , %
= 100 pi {:
x |_ n
2 (* - xf
where x ~ mean of set of n measurements, and x ; = individual
measurement.
Resolution factor, R, between p-tolualdehyde and diazepam
should be ^3.5, using the equation:
R = \2(t' - t))/(PW + PW)
where t and /' - mm retention of diazepam and p-tolualde-
hyde, resp.; and PW and PW = mm peak widths measured
at baseline obtained by extrapolating rel. straight sides of peaks
to baseline of diazepam and />tolualdehyde, resp.
Tailing factor ratio, T, should be <2.5, using the equation:
T = Wo.os/2/
where W (X03 - distance from leading edge to trailing edge of
peak; and/ = distance from peak max. to leading edge of
peak, both measured at point 5% of peak ht from baseline.
B. Sample Preparation
Weigh and finely powder >20 tablets. Transfer accurately
weighed portion of powder, equiv. to ca 10 mg diazepam, into
50 mL vol. flask. Pipet 10 mL internal std soln into flask, add
ca 25 mL MeOH, mech. shake 30 min, dil. to vol. with MeOH,
and mix. Filter soln thru 0.5 |iim membrane filter, discarding
first 10 mL filtrate.
F. Determination
Introduce equal vols (10-20 jjlL) of sample prepn and std
soln into Jiq. chromatograph by means of suitable syringe or
sampling device. Measure responses and det. response ratios
(diazepam/internal std peaks) for sample and std solns.
Diazepam, mg/tablet = 50C x (R/R f ) x (T/W)
where C = concn diazepam in std soln, mg/mL; R and R r —
ratios of peak responses of diazepam to p-tolualdehyde for
sample prepn and std soln, resp.; T = av. tablet wt, mg; and
W — sample wt taken for assay, mg.
Ref.: J AOAC 68, 545(1985).
CAS-439-14-5 (diazepam)
969.52* Lysergic Acid Diethylamide
in Drug Powders
Paper Chromatographic-Spectrophotometric Method
First Action 1969
Surplus 1977
See 40.008-40.011, 13th ed.
962.24 Cannabinol (Marihuana)
in Drug Powders
Duquenois-Levine Qualitative Test
First Action 1962
Final Action 1965
A. Reagent
Duquenois reagent. — Dissolve 12 drops acetaldehyde (fresh)
and 1 g vanillin in 50 mL alcohol.
B. Test
Ext ca 100 mg sample with 25 mL pet ether, filter into white
porcelain dish, and evap. to dryness on steam bath. Add 2 mL
Duquenois reagent and stir to dissolve residue. Add 2 mL HO,
stir, and let stand 10 min. Note color, transfer soln to test tube,
add 2 mL CHC1 3 , and shake. Let sep. and note color in CHC1 3
layer; purple color is pos. test.
Ref.: JAOAC 45, 597(1962).
CAS-52 1-35-7 (cannabinol)
977.34 Methaqualone in Drug Powders
Gas Chromatographic Method
First Action 1977
Final Action 1980
A. Apparatus and Reagents
(a) Gas chromatograph. — With flame ionization detector
and 1.8 m x 4 (id) mm glass column packed with 3% OV-1
on 100-120 mesh Chromosorb W(HP). Typical operating con-
ditions: temps (°): column 235, detector and injector 260; flow
rates (mL/min): N carrier gas 60, H 30, air 300; set column
temp, and flow rate to give methaqualone retention time of
2.5 ± 0.5 min.
(b) Sodium bicarbonate soln. — \M. Dissolve 7 g NaHC0 3
in 100 mL H 2 0.
(c) Internal std soln. — Dissolve tetraphenylethylene (East-
man Kodak Co.) in CHC1 3 to give concn of 4 mg/mL. Each
analysis requires 25 mL.
(d) Methaqualone hydrochloride std soln. — 4 mg/mL. Ac-
curately weigh methaqualone. HC1 and dil. with internal std
soln to give concn of 4 mg/mL.
B. Determination
Condition new column overnight at 270° with slow stream
of N. Sat. column immediately before analysis by making three
3 [xL injections of methaqualone. HC1 std soln.
622
Drugs: Part V
AOAC Official Methods of Analysis (1990)
Accurately weigh sample contg ca 100 mg methaqua-
lone.HCl and transfer quant, to 50 mL erlenmeyer. Pipet in
25 mL internal std soln and add ca 10 mL \M NaHC0 3 soln.
Heat on steam bath 5-8 min, cool to room temp., stopper,
and shake. Let sep. and inject 1-2 uX of CHC1 3 (bottom) layer
into gas chromatograph.
% Methaqualone.HCl - (P/P') x (B' /B) x (C/W) x 2500
where P and P' = areas or peak hts of sample and std meth-
aqualone.HCl, resp.; B and B' = areas or peak hts of sample
and std internal std, resp.; C = mg methaqualone.HCl/mL in
std soln; and W - mg sample.
Ref.: JAOAC 60, 935(1977).
CAS-72-44-6 (methaqualone)
983.30 Oxazepam in Drug Tablets
and Capsules
Liquid Chromatographic Method
First Action 1983
A. Principle
Oxazepam is extd into MeOH and detd by liq. chromatgy
with UV (254 ntn) detector.
B. Apparatus
(a) Liquid chromatograph. — Model 204 equipped with 2
Model 6000 A pumps, Model 660 sol v. programmer, Model
440 UV (254 nm) detector, Model U6K injector (all Waters
Associates, Inc.), and Model 3380A integrator (Hewlett-Pack-
ard), or equiv.
(b) LC column. — Bondapak C 18 , 3.9 mm id x 30 cm long
(Waters Associates, Inc.) at ambient temp., or equiv.
(c) Filter. — Millipore type EG (replacement Model GV)
(pore size 0.2 \im) (Millipore Corp., Bedford, MA 01730) or
0.45 |xm MeOH compatible equiv.
C. Reagents
(a) Methanol. — UV quality, LC grade.
(b) Mobile phase.— MeOH-H 2 0-glac. HOAc (60 + 40 +
1) at flow rate of 1.0 mL/min. MeOH concn and flow rate
may be varied to give approx. retention time of 6-8 min for
oxazepam.
(c) System suitability std soln. — Dissolve 10 mg USP
Oxazepam and J 5 mg USP 2-Amino-5-chlorobenzophenone
in 250.0 mL MeOH.
(d) Oxazepam std soln. — Transfer 25 mg USP Oxazepam,
accurately weighed, to 250 mL vol. flask. Add 5 mL H 2 and
dil. to vol. with MeOH. Soln is stable 90 min.
D. Sample Preparation
(a) Tablets. — Det. av. wt/tablet and grind tablets to pass
No. 60 mesh sieve. Transfer accurately weighed portion of
powder contg 25 mg oxazepam to 250 mL vol. flask. Add 5
mL H 2 and 25 mL MeOH. Mix thoroly. Add 75 mL MeOH
and place in ultrasonic bath 10 min. Dil. to vol. with MeOH.
Stir 30 min. Filter portion of soln thru type EG filter into small
g-s flask. Soln is stable 90 min.
(b) Capsules. — Det. av. wt of capsule contents. Transfer
accurately weighed portion of capsule contents contg 25 mg
oxazepam to 250 mL vol. flask. Proceed as in (a).
E. System Suitability Check
(a) Resolution. — Inject 10.0 \lL system suitability std soln.
Retention times for oxazepam and 2-amino-5-chlorobenzo-
phenone should be ca 6 and 14 min, resp. Resolution factor,
/?, for the 2 peaks should be >5.0, using following formula:
R = 2{t' ~ t)/(PW + PW)
where t and t' = mm retention of oxazepam and 2-amino~5-
chlorobenzophenone peaks, resp.; and PW and PW - mm
peak widths measured at baseline of oxazepam and 2-amino-
5-chlorobenzophenone, resp. Adjust MeOH concn if resolu-
tion is unsatisfactory.
(b) Repeatability. — Make five 10.0 jjlL injections of oxa-
zepam std soln and measure peak areas. In suitable system,
coefficient of variation is not >2.0%.
F. Determination
Make duplicate 10 |xL injections each of sample soln and
std soln, alternating sample and std solns. Calc. results by us-
ing peak areas:
mg Oxazepam /tablet or capsule
= {PA/PA') x C x (T/S)
where PA and PA' = peak area for sample and std solns, resp.;
C - mg oxazepam/250 mL std soln; T - av. wt, g, of tablet
or capsule contents; S - sample wt, g.
Ref.: JAOAC 66, 864(1983).
CAS-604-75-1 (oxazepam)
988.28 Enantiomers of Amphetamine
in Bulk Drugs, Syrups, and Capsules
Liquid Chromatographic Method
First Action 1988
A. Principle
Samples are dissolved in CH 2 C1 2 , and 2-naphthoy.l amide
derivatives are formed by adding 2-naphthoyl chloride. Iso-
mers are detd by liq. chromatgy on chiral stationary phase LC
column, with hexane-isopropyl alcohol-CH 3 CN (97 + 3 + 0.5)
mobile phase and detection at 254 nm.
B. Apparatus
(a) Liquid chromatograph. — Spectra-Physics Model 8000
equipped with Valco 7000 psi injection valve with 10 |ulL in-
jection loop, temp. -controlled oven or column H 2 jacket, and
printer plotter. Equiv. app may be used.
(b) Detector. — Spectra- Physics Model 770 UV-vis, or
equiv., set at 254 nm, 0.04 AUFS, and time constant at 4 s.
(c) Data integration system. — Set peak width to peak
threshold ratio at 1 :60 for Spectra-Physics Model 8000 or at
appropriate settings for equiv. chromatgc data system.
(d) LC column. — Pirkle covalent o-phenyl glycine analyt-
ical column (J.T. Baker, Inc. (Bakerbond Chiral Phase
[DNBPG] column No. 711.3) or Regis Chemical Co., 8210 N
Austin Ave, PO Box 519, Morton Grove, IL 60053), or equiv.
C. Reagents
(a) Solvents.— Use UV quality, LC grade H 2 0, hexane,
isopropyl alcohol, CH 3 CN, and CH 2 C1 2 .
(b) LC mobile phase. — Hexane-isopropyl alcohol -CH 3 CN
(97 + 3 + 0.5 v/v/v).
(c) 2-Naphthoyl chloride .—9%% (Aldrich Chemical Co.).
(d) Reagent solns.— 20% NaOH soln; 0.0 1M soln of 2-
naphthoyl chloride in CH 2 C1 2 ; 0.01M H 2 S0 4 soln.
D. Preparation of LC Standard and Sample Solutions
(a) Standard.— Dissolve 10 mg USP Ref. Std in 5 mL
CH 2 C1 2 , and add 5 mL 20% NaOH soln. Continue with (e).
(b) Bulk drug. — Dissolve 10 mg bulk drug in 5 mL CH 2 C1 2 ,
and add 5 mL 20% NaOH soln. Continue with (e).
(c) Syrup. — Mix 10 mL syrup with 5 mL CH 2 C1 2 , and add
5 mL 20% NaOH soln. Continue with (e).
AOAC Official Methods of Analysis (1990)
Phencyclidine
623
FIG. 988.28— Chromatogram of system suitability standard
composed of 50:50 mixture of d: /-amphetamine: 1, /-amphet-
amine; 2, d-amphetamine
(d) Capsules. — Place l capsule in 5 mL 20% NaOH soln,
ultrasonicate 45 min or until dissolved. Filter resulting soln.
Add 5 mL CH 2 Cl 2 to filtrate. Continue with (e).
(e) Working solns. — Transfer soln from (a), (b), (c), or (d)
to 30 mL separatory funnel. Add 10 mL 0.0 1M soln of 2-
naphthoyl chloride in CH 2 C1 2 , and shake mixt. 1 min. Transfer
org. phase to another 30 mL separatory funnel. Wash aq. phase
with 5 mL CH 2 C1 2 , and combine org. layers. Wash combined
org. layers with 5 mL 0.01 M H 2 S0 4 , and filter washed org.
layer thru syringe (10 mL plastic syringe with plunger re-
moved) contg glass cotton plug and anhyd. Na 2 S0 4 . Discard
aq. layers.
E. Liquid Chromatography
(a) Column preparation. — Equilibrate overnight chiral sta-
tionary phase LC column with mobile phase at flow rate of
0.25 mL/min with temp, controlled at 20 ± 1°. Circulate sol v.
during analysis without interruption. Use flow rate of 2 mL/
min during analysis.
(b) System suitability test. — After derivatization, inject 10
\xL aliquots of 50:50 mixt. of d: /-amphetamine (system suit-
ability std; available from Sigma Chemical Co.) into chro-
matgc column. Fig. 988.28 shows LC resolution of this mixt.
Repeat analysis in triplicate. Calc. mean and coefficient of
variation as follows:
mean, X = (X, + X 2 + . . . X n )/n
Coeff. of var., % = (std dev./mean) x 100
Efficiency is optimum when resolution is ^1.2 and max. rel-
ative std dev. is <2.0.
(c) Procedure. — Inject 10 jjlL aliquots of working solns of
std, bulk drug, syrup, or capsules. Make each injection in du-
plicate.
F. Calculations
Calc. % area of /-amphetamine (percent /-amphetamine) and
^/-amphetamine (percent d- amphetamine) in std and samples
as follows:
/-Amphetamine, % = [PAJ(PA X + PA d )] x 100
^/-Amphetamine, % = \PAj(PA, + PA d )] x 100
where PA\ and PA d — peak areas for /- and ^-amphetamines,
resp.
Ref.: JAOAC 71, 530(1988).
CAS- 156-34-3 (/-amphetamine)
CAS-5 1-64-9 (J-amphetamine)
979.27 Phencyclidine
in Drug Powders
Gas Chromatographic Method
First Action 1979
Final Action 1983
A. Principle
Phencyclidine is extd from weakly basic, aq. soln with CHC1 3
contg internal std. Phencyclidine is sepd by GC from other
amines, org. sol. neutral compds, and internal std.
B. Reagents and Apparatus
(a) Internal std soln. — Dissolve 80 mg eicosane (C X) H 42 ) in
100 mL CHC1.,.
(b) Phencyclidine hydrochloride std soln. — Weigh 10.0 mg
phencyclidine. HC1 (USP Authentic Substance) and dissolve in
10.0 mL internal std soln. Store in refrigerator and replace
every 3 months.
(c) Gas chroma to graph. — With H flame detector. Typical
operating conditions: temps (°): column 190, detector 240, in-
jection port 240; N carrier gas flow rate 60 mL/min. Adjust
column temp, to elute phencyclidine in 3 ± 0.5 min. Adjust
H and air flow rates and electrometer sensitivity so that 4 |ulL
phencyclidine std soln gives 40-60% full scale deflection. Re-
tention time of phencyclidine relative to internal std is ca 0.75.
(d) Column. — 1.8 m (6') x 4 mm (id) glass column, packed
with 3% OV-1 on 100-120 mesh Chromosorb W HP (Applied
Science Laboratories, Inc.). Plug column exit with plug of si 1~
anized glass wool. Apply vac. to exit end and slowly add
packing thru inlet end while tapping gently. Fill to within 1
cm of inlet and plug with silanized glass wool. Condition col-
umn overnight at 260° with slow flow of N while disconnected
from detector. Sat. column by making successive phencycli-
dine injections until phencyclidine/internal std ratio differs by
<3% from that of preceding injection.
C. Determination
Accurately weigh ca 250 mg finely ground sample, dissolve
in 0. \N HC1, and quant, dil. to estimated phencyclidine.HCl
concn of 1 mg/mL. Let any insol. material settle. Pipet 2 mL
aliquots sample soln and 2 mL std soln into sep. test tubes.
Add 2.0 mL internal std soln and 1 .0 mL 10% K 2 HP0 4 soln
to sample tube, stopper, and shake vigorously. Add 2.0 mL
0AN HC1 and 1.0 mL 10% K 2 HP0 4 to std tube, stopper, and
shake vigorously. Let sep. Inject duplicate 4 uX aliquots of
lower CHCI3 layers into gas chromatograph from 10 (xL sy-
ringe.
% Phencyclidine.HCl
= (/////') (C'/VV) (B'/B) X DF X 100
where H and H' — av. peak ht or area of sample and std, resp.;
C - mg std phencyclidine. HCI/mL in std soln; W = mg sam-
ple; B and B' = av. peak ht or area of internal std in sample
and std, resp.; and DF = diln factor for sample.
mg Phencyclidine. HO /tab let
- % Phencyclidine.HCl x av. tablet wt (mg)/100
Ref.: JAOAC 62, 560(1979).
CAS-956-90-1 (phencyclidine hydrochloride)
624
Drugs: Part V
AOAC Official Methods of Analysis (1990)
Common and Chemical Names for Drugs in this Chapter
Common Name
Chemical Name
Amphetamine
Cannabinol
Cocaine (hydrochloride)
Heroin
Diazepam
Lysergic acid diethylamide
Methaquaione
Morphine (hydrochloride, sulfate, diacetate)
Oxazepam
Phencyclidine (hydrochloride)
Quinine ethylcarbonate
(±)-a-Methylphenethylamine
6,6,9-Trimethyl-3-pentyl-6tf-dibenzo(fc>,d)pyran-1-ol
3-(Benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester
Diacetylmorphine
7-Chloro-1 ,3-dihydro-1 -methyl-5-phenyl-2H-1 ,4-benzodiazepin-2-one
9,10-Didehydro-A/,A/-diethyl-6-methylergoline-8p-carboxamide
2-Methyl-3-(2-methylphenyl)-4(3H)-quinazolinone
7,8-Didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol
7-Chloro-1,3-dihydro-3-hydroxy-5-phenyl-2H-1,4-benzodiazepin-2-one
1 -(1 -Phenylcyclohexyl)piperidine
6'-Methoxycinchonan-9-ol ethyl carbonate (ester)
Source: USAN and the USP Dictionary of Drug Names (1983; 1989) U.S. Pharmacopeial Convention, Rockville, MD.
23. Drugs and Feed Additives in Animal Tissues
Charlie X Barnes, Associate Chapter Editor
Food and Drug Administration
961.23 ANOT Residues
in Animal Tissues
Spectrophotometric Method
Final Action
A. Principle
ANOT, metabolite of zoalene, is liberated from ground tis-
sue by enzymatic digestion with ficin. Digest is treated with
NaHC0 3 and extd with acetone. CHC1 3 is added to sep. soln
into 2 layers. Org. layer is coned and passed thru alumina col-
umn. Adsorbed ANOT is washed with CHC1 3 and eluted with
80% alcohol. Alcohol soln is passed thru cation exchange resin
and ANOT is eluted with 4N HCl. Colored compd formed by
diazotization and coupling with /V-1-naphthylethylenediamine
is measured at 540 nm.
B. Apparatus
(a) Chromatographic tube. — 600 X 16 mm id.
(b) Ion exchange columns for Dowex resin. — 180 X ] 1 mm
id.
(c) Mixer. — High, speed, high shear mixer with explosion-
proof motor, and ca 1 L container.
(d) Spectrophotometer. — Beckman Model DU, 24, or 25
(replaced by Models DU-64), or equiv.
C. Reagents
(a) Alumina. — Activated, Alcoa grade F-20, 80-200 mesh.
(b) 3-Amino-5 '-nitro-o-toluamide . — ANOT, anal. std.
Available from Dow Chemical Co.
(c) Ammonium sulfamate soln. — 1 .0%. Prep, fresh weekly.
(d) Coupling reagent. — 0.25% aq. soln of /V- 1 -naphthyl-
ethylenediainine.2HCl. Prep, fresh weekly and store in dark
bottle.
(e) Dowex 50W-X8 cation exchange resin. — Hydrogen form,
200-400 mesh (Bio-Rad Laboratories).
(f) Ficin. — ICN Pharmaceuticals, Inc., Life Sciences Group.
{Caution: Ficin is potent proteolytic enzyme which attacks liv-
ing tissues. Avoid contact with skin and eyes and breathing
dust.)
(g) Sodium nitrite soln. — 0.25%. Prep, fresh daily.
D. Preparation of Alumina Column
Insert small plug of glass wool into chromatgc tube and
compress in lower end of tube. Add 60 g alumina and pack
by gently tapping tube on rubber stopper to ht of ca 30 cm.
Add 100 mL CHC1 3 and drain to just above level of alumina.
Do not drain CHCl 3 below level of alumina.
E. Preparation of Ion Exchange Column
Heat 100 g Dowex 50W-X8 on steam bath with 400 mL 67V
HCl 2-3 hr. Filter on buchner and wash with H 2 until wash-
ings are acid-free. Wash resin with 100 mL 80% alcohol. Then
mix resin with 250 mL 80% alcohol. Pour enough resin slurry
into ion exchange column to give bed ht of ca 5 cm after set-
tling. Wash resin with 25 mL 80% alcohol. Slight air pressure
can be used to increase flow of liq. thru resin. Do not let liq.
level drain below top of resin bed.
F. Preparation of Standard Curve
Accurately weigh 100 mg ANOT into 1 L vol. flask, dis-
solve in 50 mL acetone, and dil. to vol. with H 2 0. Dil. 10
mL of this stock soln to 100 mL with H 2 to give working
soln of 10 |mg/mL. Pipet 0, 2, 4, 6, 8, and 10 mL aliquots of
this soln into sep. 50 mL vol. flasks. Dil. each to ca 40 mL
with 47V HCl. Proceed as in 961. 23G, beginning "Add 1 mL
0.25% NaNO-2 • ■ ■"• Plot A at 540 nm against u,g ANOT.
G. Determination
(Caution: See safety notes on distillation, toxic solvents, and
chloroform.)
Collect tissue, freeze with solid C0 2 , and keep frozen until
analyzed. Grind tissue while at least partially frozen and weigh
50 g into 1 qt (1 L) Mason jar. Add 125 mL H 2 0, 15 mL \N
HC1, and 5 g ficin, and mix with mixer ca 5 min. Cover jar
loosely and keep 24 hr at 30°. Then keep 30 min in bath at
70-80°, remove, and cool.
Weigh ca 10 g NaHC0 3 and slowly add to jar with stirring,
taking care that sample does not foam over top of jar. When
foaming has subsided, add 500 mL acetone and mix 5 min
with mixer.
Filter on buchner into I L filter flask, using 11 cm paper
and ca 5 g Super-Cel as filter pad. Wash residue with 200 mL
acetone, collecting washings in same flask. Transfer filtrate to
2 L separator and add 1 L CHC1. 3 . Shake ext in separator vig-
orously and let stand until layers sep. Drain CHC1 3 layer into
2 L beaker. Ext aq. layer with 200 mL CHC1 3 and combine
CHC1 3 washing with original ext. Evap. CHC1 3 ext to ca 50
mL under heat lamp with air current. Add 100 mL CHC1 3 and
again evap. to 50 mL. If soln is not clear, repeat addn and
evapn of CHC1 3 to remove H 2 0.
Add clear CHC1 3 soln to alumina column and drain to level
of alumina. Wash with four 50 mL portions CHC1 3 . Discard
washings. Add 90 mL 80% alcohol to column to elute ANOT.
Discard first 30 mL effluent and collect 60 mL in 100 mL
beaker. Transfer this soln to ion exchange column. Slight air
pressure may be used to increase flow of soln. After soln has
drained to top of resin bed, wash with 50 mL 80% alcohol
followed by 50 mL H 2 0. Discard washings. Add 45 mL 4A r
HC1 and collect effluent in 50 mL vol. flask.
Add 1 mL 0.25% NaN0 2 , mix, and let stand 5 min. Add
1 mL 1% NH 4 sulfamate soln, mix, and let stand 5 min. Add
1 mL coupling reagent, mix, dil. to vol. with AN HC1, and
mix thoroly. Let stand 15 min and read A at 540 nm, using 1
cm cells, against H 2 as ref.
H. Calculations
Obtain |xg of ANOT corresponding to A from std curve.
ppm ANOT in sample - p,g ANOT/g sample
Refs.: J. Agric. Food Chem. 9, 201(1961). JAOAC 49, 708
(1966).
CAS-3572-44-9 (ANOT)
625
626
Drugs and Feed Additives in Animal Tissues
AOAC Official Methods of Analysis (1990)
973.78 Arsenic (Total) Residues
in Animal Tissues
Spectrophotometry Method
First Action 1973
Final Action 1975
(Complete analysis in 1 day; otherwise stop after ashing
step.)
A. Reagents and Apparatus
(a) Silver diethyldithiocarbamate. — Chill 200 mL AM
AgN0 3 soln (3.4 g/200 mL) and 200 mL 0AM Na diethyldi-
thiocarbamate soln (4.5 g/200 mL) to 10° or lower. Add car-
bamate soln to AgN0 3 soln slowly with stirring. Filter thru
buchner, wash with chilled H 2 0, and dry under reduced pres-
sure at room temp. Dissolve salt in pyridine (reagent grade)
with stirring, chill, and add cold H 2 slowly until completely
pptd. Filter thru buchner, and wash with H 2 to remove all
pyridine. Dry pale yellow crystals under reduced pressure (mp
185-187°; recovery 85-90%). Store in amber bottle in refrig-
erator. (Second recrystn may be necessary to obtain correct
mp.)
(b) Silver diethyldithiocarbamate soln. — Dissolve 0.5000 g
salt, (a), in colorless pyridine in 100 mL vol. flask, and dil.
to vol. with pyridine. Mix, and store in amber bottle. Reagent
is stable several months at room temp.
(c) Arsenic std solns. — (1) Stock soln. — 500 jixg/mL. Ac-
curately weigh 0.660 N1ST SRM As 2 3 , or equiv., dissolve
in 25 mL 2N NaOH, and dil. to 1 L with H 2 0. (2) Working
solns. — 0-2 ppm. Just before use, prep, by dilg stock soln
with H 2 0.
(d) Zinc.— Shot, contg <0.00001% As (Fisher Scientific
Co., No. Z-12, or granules of equiv. purity).
(e) Cellulose powder. — Whatman CF-11 fibrous.
(f) Distillation apparatus. — (7) Flask. — 250 mL erlen-
meyer. (2) Connecting tube. — L-shaped 8 mm od glass tube
with 1 1 and 7 cm sides. Plug shorter end with 2 pieces of glass
wool satd with 10% Pb(OAc) 2 soln and dried (replace plugs
when discolored). (3) Delivery tube. —L-shaped 6 mm od glass
tube with 22 and 5 cm sides. Constrict end of longer side to
1 mm opening. (4) Receiver. — 8 cm length of 15 mm glass
tube sealed to open end of 100 x 10 mm od test tube.
Connect flask thru 1-hole rubber stopper with 1 1 cm side of
connecting tube. Attach connecting tube to 5 cm side of de-
livery tube with rubber tube sleeve. Fit constricted end of de-
livery tube into bottom of receiver.
B. Dry Ashing
Blend liver and kidney in high-speed blender. Pass fibrous
tissues such as muscle and skin thru meat grinder, and divide
and quarter. Weigh 10 g tissue into 100 mL Coors crucible.
Add 3 g MgO and 20 mL cellulose powder (10 mL beaker is
convenient measure) to liver, kidney, and skin samples and 10
mL cellulose powder to muscle samples. Mix thoroly and char
cautiously over open flame until evolution of smoke ceases.
(Caution: Rapid rise in temp, will cause crucibles to crack;
avoid overheating samples to prevent loss of As.)
Cool, add 3 g Mg(N03) 2 .6H 2 O s and place in cold furnace
preset at 555°. After furnace reaches operating temp., ash 2
hr. Cool, moisten ash with .10 mL H 2 0, and transfer quant, to
250 mL erlenmeyer with 90 mL 6N HC1. Dil. to 175 mL with
H 2 0. (Presence of black carbonaceous particles does not in-
terfere.)
C. Distillation
Add 2 mL 15% Kl soln, and swirl. Add 1 mL SnCl 2 soln,
963,21 A(g), and swirl. Cool in freezer or ice bath 45 min or
until sample reaches 4°. Prep, blank contg 90 mL 6N HCJ and
85 mL H 2 0, and treat similarly.
Prep, trapping soln by pipetting 3 mL AgDDC reagent into
receiving tube and place in ice bath. Attach delivery tube to
connecting tube and insert delivery tube into AgDDC soln.
Add 10 g Zn shot or granules to cooled erlenmeyer, imme-
diately connect flask to connecting tube, and let distn proceed
1 hr at room temp. Det. A against reagent blank in 1 cm cell
at 540 nm; det. As content from std curve.
Calc. As concn in sample by multiplying A at 540 nm by
reciprocal slope of std curve, disregarding y intercept term.
D. Preparation of Standard Curve
Add As working std solns (but <2 mL soln) to 10 g tissue
to provide curve over desired range (usually 0-2 ppm As).
Carry these samples thru ashing and distn. Det. best fitting
straight line from S:4 sets of detns for each tissue by method
of least sqs, Definitions of Terms and Explanatory Notes.
Ref.: J AOAC 56, 793(1973).
CAS-7440-38-2 (arsenic)
974.45 CSopicJol Residues
in Animal Tissues
Gas Chromatographic Method
First Action 1974
Final Action 1977
(Diazomethane is toxic, can cause specific sensitivity, and is
potentially explosive. Prep, diazomethane reagent, methylate,
and evap. in hood. Avoid metal, ground glass joints, etched
or scratched glassware, and sharp edges. Store diazomethane
solns in freezer; do not expose to direct sunlight or strong
artificial light.)
A. Principle
Tissues and eggs are extd with MeOH; ext is filtered and
cleaned up on alumina and anion exchange columns. Eluate is
methylated with diazomethane, producing Me ether of clopidol
(3,5-dichloro-4-methoxy-2,6-lutidine), which is detd by elec-
tron capture GC. Applicable to >0.1 ppm in chicken tissues
and ^0.05 ppm in eggs.
B. Apparatus
(a) Centrifuge. — Clinical (Model CL, International Equip-
ment Co.), or equiv., with head and cups to accommodate 13
x 100 mm tubes.
(b) Flask.— 500 mL r-b 1 29/42 neck. Make 6 irregularly
spaced 6 mm projections into flask by heating spot ca 2 cm
diam. with torch and pushing spot in with blunt instrument.
(c) Gas chromatograph . — With electron capture detector.
Operating conditions: temps (°) — column 155, injection port
220, detector 220; flow rates — N carrier gas 120 mL/min;
sensitivity 3 X 10" l0 amp; and chart speed 20"/hr.
(d) Gas chromatographic column.— 25% DC-200 silicone
oil (Dow Corning Corp.) on 80-100 mesh Chromosorb W (AW)
(Applied Science). Prepd column available from Applied Sci-
ence, or prep, as follows: Weigh 12 g Chromosorb W (AW),
from which fines have been removed on No. 100 sieve, into
specially modified r-b flask, (b), contg 100 mL CHC1 3 and 3
g DC-200 fluid. Dry on rotary evaporator under vac. Use heat
lamp or hot H 2 to aid evapn. Sieve and discard fines passing
No. 100 sieve. Pack 1.9 m (74") x 3 mm id U-shaped bo"-
rosilicate glass column and condition >18 hr at 200° with N
flow of 75-100 mL/min before use. Add packing to column,
tapping on floor to settle. Insert glass wool plug at effluent
AOAC Official Methods of Analysis (1990)
Clopidol 627
end. Level of packing in injection arm should be few mm be-
low depth of needle point at inlet.
(e) Liquid chromatographic columns. — (/) Alumina col-
umn. — Add 6 g (1 heaping 5 mL beaker) alumina, 974.45C(a),
to 300 X 18 mm id column, with coarse fritted disk and 30
x 5 mm id stem. (2) Anion exchange column. — Place 1 cm
(after settling) AG1-X8 resin, 974.45C(b), in 170 x 10 mm
id column, with coarse fritted disk and 30 x 5 mm id stem,
using MeOH to transfer resin. Rinse column with 2 mL MeOH,
applying air pressure from squeeze bulb.
(f ) Homogenizer. — See 961.236(c), or equiv., for use with
Brockway 4 oz (125 mL) sq powder jar (No. 72 G1333, with
38 mm Polyseal caps, Brockway Plastics, inc., 9211 Forest
Hill Ave, PO Box 351 10, Richmond, VA 23235-01 10) and pt
(500 mL) or qt (1 L) Mason jars.
(g) Meat grinder. — With stainless steel attachment.
(h) Shaker. — Wrist-action (Model BT, Burrell Corp., or
equiv.).
(i) Culture tubes. — 13 X 100 mm (Corning Glass Co., No.
9825), with 13 mm rubber- lined plastic screw caps.
C. Reagents
(a) Alumina. — Alcoa F-20, 80-200 mesh (Fisher Scientific
Co.).
(b) Anion exchange resin.— Bio-Rad AG1-X8, 100-200
mesh, acetate form (Bio-Rad Laboratories).
(c) Diazomethane reagent. — Approx. 18 mg/mL in Et ether.
Add 35 mL 2-(2-ethoxy-ethoxy) ethanol (Aldrich Chemical Co.,
Inc.) and 10 mL ether to soln of 6 g KOH in 10 mL H 2 in
125 mL long-neck distg flask. Place mag. stirring bar in flask
and mount above H 2 bath on top of hot plate mag. stirrer.
Attach dropping funnel and efficient condenser connected in
series to 250 and 50 mL erlenmeyers. Place 25 mL ether in
second flask and place inlet tubing below surface of ether. Cool
both receiving flasks in ice. Place soln of 21.5 g N-methyl-jV-
nitroso-p-toluenesulfonamide (Diazald; Aldrich Chemical Co.,
Inc.) dissolved in 140 mL ether in dropping funnel. Heat FLO
bath to 55° and raise it to heat distn flask. Stir contents of flask
while adding Diazald soln over 20 min. Interrupt distn when
distillate is nearly colorless. Combine contents of the 2 re-
ceivers and store at 0° in culture tubes with screw caps or as
in JAOAC 65, 273-274(1982). Reagent is stable several weeks
if kept in freezer in full, closed tubes. Diazomethane reagent
may also be prepd as in Anal Chem. 32, 1412-1414(1960).
(d) Methanolic hydrochloric acid. — 5%. Add 25 mL HC1
to 475 mL MeOH.
D. Standard Solutions
(Caution: See safety note on benzene.)
(a) Clopidol std solns. — (7 ) Stock soln. — 100 |ULg/mL. Ac-
curately weigh ca 100 mg Clopidol Anal. Std (available from
Dow Chemical Co.) in weighing bottle, transfer to 1 L vol.
flask with MeOH, to total vol. of ca 950 mL. Stir mag. to
dissolve clopidol (may take 2-3 hr). Dil. to vol. at room temp.,
and mix well. (2) Intermediate soln. — 10 |JLg/mL. Pipet 10
mL stock soln into 100 mL vol. flask, dil. to vol. with MeOH,
and mix. (3) Working soln. — 1 (xg/mL. Pipet 10 mL inter-
mediate soln into 100 mL vol. flask, dil. to vol. with MeOH,
and mix.
(b) 3 ,5-Dichloro-4~methoxy-2 ,6-lutidine (clopidol methyl
ether) std solns. — (/ ) Stock soln. — 1 mg clopidol equiv. /mL.
Weigh 107.3 mg 3,5-dichloro-4-methoxy-2,6-lutidine anal, std
(available from Sampling Coordinator, Ag-Organics Dept, Dow
Chemical USA) in weighing bottle. Transfer to 100 mL vol.
flask with benzene, dil. to vol. with benzene, and mix well.
(2) Intermediate soln I. — 10 |xg clopidol equiv. /mL. Pipet 1
mL stock soln into 100 mL vol. flask, diL to vol. with ben-
zene, and mix. (3) Intermediate soln II. — 1.0 |xg clopidol
equiv. /mL. Pipet 10 mL intermediate soln /into 100 mL vol.
flask, dil. to vol. with benzene, and mix. (4) Working solns. —
Prep, series of std solns contg 0.01 to 0.20 \xg clopidol equiv./
mL by dilg portions of intermediate soln // with benzene.
E. Chromatography of Standards
Fill syringe needle with benzene, avoiding entrapped air,
draw 3 |jlL sample aliquots of clopidol methyl ether working
soln into syringe, and inject onto column. Measure peak fits
(PH) in terms of % full-scale deflection, and plot PH against
|jLg/mL. Prep, std curve daily and check by injecting std soln
after every 1 or 2 samples.
F. Determination
(a) Muscle, liver, and kidney. — Homogenize by grinding
thru meat grinder. Accurately weigh ca 20 g tissue into 4 oz
(125 mL) jar. Add 50 mL MeOH and 3 g HyFlo Super-Cel
filter aid for muscle; use 12 g filter aid for liver and kidney.
Attach jar to homogenizer and blend 3 min at max. speed.
Filter thru 2 g pad of filter aid in 60 mL coarse fritted glass
buchner mounted on filter assembly. Collect filtrate in 100 mL
graduate, and wash jar and filter cake with MeOH to nearly
100 mL. If filter cake goes dry, break up with spatula during
addn of more MeOH to prevent channeling. Dil. to 100 mL
at room temp., stopper, and mix well.
Place anion exchange column under alumina column. Pipet
20 mL ext onto alumina column and let elute thru both col-
umns into beaker. Wash columns with 10 mL MeOH added
to alumina column, rinsing sides. Remove alumina column and
beaker. Place 25 mL vol. flask under anion exchange column
and elute clopidol with two 10 mL portions 5% HC1 in MeOH.
Dil. eluate to vol. with MeOH, and mix well. Pipet 1 mL
aliquot into 13 x 100 mm tube and evap. to dryness (4-5 min)
by mounting tube in 70° H 2 bath so H 2 level is at same ht
as soln in tube. Direct very small jet of air down tube using
rubber tubing and medicine dropper tip, or equiv. Remove tube,
add 0.2 mL 80% MeOH, and heat briefly to redissolve resi-
due. Add 1 mL diazomethane reagent, seal with screw cap,
and heat gently 2 min by mounting tube as before in 70° H 2
bath. Remove tube, and let cool 5 min before removing cap.
Add small SiC boiling chip and evap. reagents gently by
mounting tube with only extreme rounded bottom portion
touching H 2 of 70° bath. Continue heating 2-3 min until ether
is evapd. Add 0.1 mL \N NaOH, 5 mL H 2 0, and 1.0 mL
benzene, cap tube, and vigorously shake mixt. 1 min. Centrf.
3 min. Dil. further by adding more benzene, if necessary. In-
ject 3 fJuL of benzene layer as in 974. 45E. Det. PH and in-
terpolate (xg/mL clopidol in benzene ext from std curve.
(b) Eggs. — Accurately weigh ca 20.0 g sample into 4 oz.
(125 mL) jar, and add ca 12 g Hyflo Super-Cel. Shake mech.
15 min. Proceed as in (a), beginning "Filter thru 2 g pad ..."
Elute with 9.5 mL 5% HC1 in MeOH and collect eluate in 10
mL vol. flask.
(c) Recovery factor . — Accurately weigh 20.0 g samples of
homogenized clopidol-free tissue, and add equiv. of 0.0, 0. 1,
and 0.5 ppm clopidol working std soln (0.0, 2.0, and 10.0
mL, resp.). Proceed as in (a) and calc. av. recovery factor, R
- (ppm found from std curve) /(ppm added). For eggs, use 20
g samples, add equiv. of 0.00, 0.05, and 0.2 ppm (0.0, 1.0,
and 4.0 mL working std soln, resp.), proceed as in (b), and
calc. av. recovery factor, R.
ppm Clopidol in tissues - 6.25 (G - G')/R
ppm Clopidol in eggs = 2.5 (G — G')/R
628
Drugs and Feed Additives in Animal Tissues
AOAC Official Methods of Analysis (1990)
where G - |xg/mL from std curve of sample, and G' - |utg/
mL from std curve of blank.
Refs.: JAOAC 57, 914(1974); 59, 476(1976).
CAS-297 1-90-6 (clopidol)
973.79 Decoquinate Residues
in Animal Tissues
Fluorometric Method
First Action 1973
Final Action 1974
(Applicable to chicken tissues at <2.5 ppm level)
A. Principle
Tissue is homogenized in MeOH-CHCl 3 . After addn of
metaphosphoric acid, decoquinate is extd into CHC1 3 and sepd
from interfering materials by chromatgy on Florisil. Deco-
quinate is eluted from column with CaCl 2 -MeOH and detd by
tluorometry against std treated similarly. Range 0.1-2.2 ppm;
sensitivity 0.1 ppm.
B. Apparatus
(a) Fluorometer. — Aminco-Bowman SPF, or equiv.
(b) Chromatographic columns. — Draw 30 cm length of 9
mm tubing (7 mm id) to drip tip. Insert small glass wool plug
to support adsorbent. Close drip end with short piece of tubing
and pinch clamp. Add 5 mL CHC1 3 to column, then 0.4 ±
0.02 g Florisil. Add 2 mL addnl CHC1 3 and stir with thin glass
rod to settle adsorbent. Remove tubing and wash down sides
of tube with CHCI3. Prep, just before use.
C. Reagents
(a) Methanol- chloroform soln. — Mix 4 parts MeOH, re-
distd in all-glass app., and 1 part CHC1 3 , spectral grade.
(b) Decoquinate std so Ins. — (7) Stock soln. — 200 [xg/mL.
Weigh 20 mg Decoquinate Ref. Std (available from Hess &
Clark Laboratories). Dissolve and dil. to 100 mL with CHC1 3 .
(2) Working soln. — 10 |ig/mL. Pipet 5 mL stock soln into
100 mL vol. flask and dil. to vol. with CHC1 3 . (3) Fluores-
cence reference soln. — 0.2 fjig/mL. Pipet 2 mL working soln
into 100 mL vol. flask and dil. to vol. with elution solv., (c).
Solns are stable £:]. month.
(c) Elution solvent. — Dissolve 10 g anhyd. CaCl 2 in 1 L
redistd MeOH. Let stand 24 hr. Decant from any insol. resi-
due.
(d) Metaphosphoric acid soln. — 5%. Dissolve 50 g meta-
phosphoric acid (J. T. Baker, Inc., No. 0252) in 1 L H 2 0.
Refrigerate at 5° and use cold.
(e) Florisil. — 100-200 mesh (Fisher Scientific Co., No. F-
101).
D. Preparation of Standard Curve
Add 55 mL (50 g) MeOH-CHCl 3 (4 + 1) soln to each of
four 250 mL separators. Add 0.0, 0.1 , 0.3, and 0.5 mL work-
ing soln contg 0, 1, 3, and 5 |xg decoquinate, resp. Proceed
with Determination, beginning "Add 100 mL 5% metaphos-
phoric acid, . . .", adding entire CHC1 3 ext to column. Con-
struct std curve by plotting fluorescence against |xg decoqui-
nate /mL.
E. Determination
Weigh 20 g tissue into high-speed blender. Add 80 ± 1 g
MeOH-CHCl 3 (4+1) (weigh on top-loading balance). Blend
1 min. Transfer to centrf. bottle and centrf. 5 min at ca 2000
rpm. Decant and weigh 50 g supernate (equiv. to 10 g tissue)
into 250 mL separator. Add 100 mL 5% metaphosphoric acid,
invert 50 times, let phases sep. 10 min, and drain and retain
CHCI3 layer. Add 10 mL addnl CHC1 3 to separator, shake,
and let sep. as before. Combine CHC1 3 exts, add 2 mL MeOH,
and dil. to 25 mL with CHC1 3 .
Depending on expected decoquinate content, add 5, 10, or
25 mL ext (2, 4, or 10 g tissue, resp.) to chromatgc column.
Normally use 10 mL for liver, kidney, skin, and fat, and 25
mL for muscle samples. Wash column with 10 mL MeOH.
Elute with 15 mL elution solv., (c), collecting in tube marked
at 15 mL. Mix and transfer to fluorometer cell. Set activation
wavelength at 270 nm and emission wavelength at 390 nm.
With fluorescence ref. std in cell, adjust microphotometer con-
trols to give reading of 80 on rel. intensity scale. Det. fluo-
rescence of samples, and calc. jxg decoquinate from std curve.
Ref.: JAOAC 56, 71(1973).
CAS- 18507-89-6 (decoquinate)
968.48 Ethoxyquin Residues
In Animal Tissues
Photofluorometric Method
First Action 1968
(Applicable to chicken tissues and eggs)
A. Apparatus
(a) Photofluorometer . — {Caution: See safety notes on pho-
tofluorometers.) Instrument with primary filter passing only
365 nm Hg line and secondary filter passing light between 410
and 580 nm (but not below 410 nm).
(b) Separators . — 250 mL with Teflon stopcocks.
B. Reagents
(a) Isooctane. — Fluorescence <2% that of soln contg 0.020
|xg quinine sulfate /mL 0.1/V H 2 S0 4 . If necessary, purify
isooctane by passing thru 30 X 2 cm activated alumina col-
umn.
(b) Sulfuric acid-sodium sulfate soln. — 0.37V H->S0 4 contg
2% Na 2 S0 4 .
(c) Ethoxyquin std solns. — (7) Stock soln. — 10 jxg/mL. Place
10 mg com. grade ethoxyquin in 1 L vol. flask; dissolve and
dil. to vol. with isooctane. Store in refrigerator. (2) Working
solns.— 0. 010, 0.020, 0.030, and 0.050 pg/mL. Transfer 1,
2, 3, and 5 mL aliquots stock soln to 1 L vol. flasks and dil.
to vol. with isooctane. Prep, fresh on day of use.
C. Preparation of Standard Curve
Prep, std curve at time of analysis of final ethoxyquin exts.
Read ethoxyquin stds with photofluorometer set at with shut-
ters closed and at 100 with most coned std. Plot instrument
reading against fxg ethoxyquin on linear graph paper.
D. Preparation of Sample and Extraction
(All glassware must be free of stopcock grease.)
(a) Egg yolk. — Carefully break egg to avoid rupturing yolk
and sep. as much of egg white from yolk as possible. Wash
yolk in running H 2 to remove most of remaining egg white.
Dry yolk on absorbent paper, break yolk sac, pour yolk into
bottle, and stopper.
Weigh bottle contg yolk and pour ca 5 g yolk into mortar
contg 25 g anhyd. granular Na 2 S0 4 and 3 g anhyd. powd
Na 2 C0 3 . Reweigh bottle and record wt yolk added. (Several
AOAC Official Methods of Analysis (1990)
Melengestrol Acetate
629
samples may be prepd from same yolk.) Grind mixt. in mortar
until uniform and dry 1 hr in desiccator contg Drierite.
Transfer dried mixt. to 4 oz (125 niL) screw-cap bottle and
shake 30 min with 50 mL isooctane. Centrf. and filter super-
nate thru Whatman No. 1 paper into 250 mL separator. Repeat
extn with second 50 mL isooctane and add ext to separator.
Gently shake isooctane ext 1 min each with two 50 mL por-
tions 0.37V PLSCVNa^SC^ soln. Combine acid exts and add 10
mL 6N NaOH. Ext alk. soln with two 50 mL portions isooc-
tane. Combine isooctane exts and dry 15 min over anhyd.
Na 2 S0 4 ; decant, and dil. to 100 mL with isooctane.
(b) Tissue (muscle and liver). — Accurately weigh ca 5 g
muscle or 1 g liver and add to 15 g anhyd. Na 2 S0 4 and 2 g
anhyd. Na 2 C0 3 in mortar. Grind until uniform and place in
desiccator 1 hr.
Shake dried mixt. 30 min in 4 oz (125 mL) screw-cap bottle
with 100 mL isooctane. Centrf., and filter into 250 mL sep-
arator. Continue as in (a), 4th par., beginning "Gently shake
isooctane ext ..."
(c) Fat. — Accurately weigh ca 1 g frozen fat and add to 10
g granular, anhyd. Na 2 S0 4 and 1 g anhyd. Na 2 C0 3 in glass
mortar. Grind mixt. thoroly. Add 20 mL isooctane and con-
tinue grinding several min. Decant isooctane into 4 oz (125
mL) screw-cap bottle. Repeat grinding with isooctane 3 times.
Transfer isooctane ext to bottle, shake, and centrf.
Decant supernate isooctane layer into 250 mL separator and
continue as in (a), 4th par., beginning "Gently shake isooctane
ext ..."
E. Determination
Det. fluorescence of isooctane soln and calc. ethoxyquin
content from std curve.
Ethoxyquin, ppm
- (|mg ethoxyquin /mL) X (mL ext/g sample)
Refs.: JAOAC 50, 844(1967); 51, 453, 537(1968).
CAS-9 1-53-2 (ethoxyquin)
976.36 yelengestrol Acetate Residues
in Animal Tissues
Gas Chromatographic Method
First Action 1976
Final Action 1978
A. Principle
Melengestrol acetate (MGA) is extd from lean tissue with
CH 3 CN and ext is partitioned with hexane. MGA in fatty tis-
sues is extd with hexane and then transferred into CH 3 CN.
Residue from either ext, after evapn of soJv., is chromatgd on
Florisil to remove interfering lipid materials with hexane and
hexane-acetone (95 + 5). MGA is eluted with hexane-acetone
(80 + 20). Residue is dissolved in hexane-acetone, and detd
by GC.
For those liver samples where MGA is poorly resolved on
chromatogram, hexane-acetone is evapd, partitioned with aq.
70% MeOH-hexane, transferred into CHCI 3 , and evapd. Dry
residue is dissolved in hexane-acetone and reinjected onto GC
column.
B. Apparatus
(a) Adapters.— 1 24/40, Nos. 5225-10 and 5205 (Ace Glass,
Inc., or equiv.).
(b) High-speed blender. — Waring Blendor Model 702-B with
1 L glass bowl having polyethylene gaskets (see (i)), or equiv.
(c) Chromatographic tubes. — Glass, 400 X 19 mm id, fit-
ted with medium porosity fritted glass disks. Teflon stopcocks,
and I 24/40 tops.
(d) Containers. — Plastic, with lid. For storage of frozen
tissues.
(e) Flasks.— R-b, 50, 500, and 1000 mL.
(f) Funnels. — Medium porosity fritted glass funnels, 350
rnL.
(g) Gas chromato graph. — F&M Model 402, replaced by
HP 5890A series, (available from Hewlett-Packard Co.,
Avondale Div.), or equiv., with all-glass on-column injection
system, 63 Ni electron capture detector, and 1 mv strip chart
recorder. Operating conditions: temps (°) — column 240-250,
injection port 240-250, detector 270-275; flow rates — He
carrier gas 60-80 mL/min (40 psi, 3.0-3.5 rotameter setting),
Ar-CH 4 purge gas (95 + 5) 135-150 mL/min (40 psi); atten-
uation I6x or 32 X; pulse interval 150; electrometer sensitivity
1 x JO" 12 amp full scale deflection with 1 mv recorder. Ap-
prox. retention time of MGA under these conditions is 5-6
min.
(h) Gas chromatographic column. — Use borosilicate glass
tubing, 0.2362 ± 0.013" (6.00 ± 0.33 mm) od and 0.118 ±
0.01" (3.00 ± 0.25 mm) id. Bend 0.9 m (3') piece of tubing
into proper design for instrument. Pack column with 1% OV-
17 on 100-120 mesh Gas Chrom Q (max. operating temp.,
350°, Applied Science Laboratories, Inc., or equiv.), and plug
both ends with 0.5 cm loosely packed silanized glass wool.
Pack far enough from ends so that no part of column packing
or glass wool is inside injection port or detector inlet fittings.
Connect column to injection port and cap detector inlet. Con-
dition column 1 hr at 240° with He carrier gas at 40 mL/min,
and then 16 hr at 275° with He carrier gas at 80 mL/min.
Remove cap and connect column to detector.
(i) Gaskets.— Polyethylene, cut from 1 qt (1 L) freezer con-
tainers .
(j) Nitrogen pressure manifold for columns. —(Optional).
Adapters No. 5205 (Ace Glass, Inc., or equiv.) connected thru
manifold regulated at 3 psi (20.7 kPa), with individual control
valve.
(k) Pipets. — Transfer pipets, 9" Dispo-pipettes (Scientific
Products, Inc., or equiv.).
(!) Reservoirs.— 250 mL I" 24/40 r-b flasks with 24/40 male
joint in bottom, or equiv.
(m) Rotary evaporator . — 4-6 small size Rinco evaporators
(Valley Electromagnetics, One Wolfer Park, Spring Valley, IL
61362), or equiv., controlled with 4 mm bore stopcocks con-
nected to manifold that leads to 2 condensation traps connected
in series to vac. pump with free air capacity of 140 L/min.
Cool traps with solid C0 2 -alcohol mixt. Connect each sample
in r-b flask with 2 adapters in series to evaporator, and heat
in thermostatically controlled H 2 bath at 45°.
(n) Separators .—With Teflon stopcocks, 500 and 1000 mL.
(o) Silanized glass wool. — Applied Science, or equiv.
(p) Syringe. — 10 (ulL, Hamilton No. 701 N, or equiv.
C. Reagents
(All solvs must show no impurities when processed thru entire
detn in absence of tissues.)
(a) Argon-methane, 95 + 5. — Purge gas (Matheson Gas
Products, 30 Seaview Dr, PO Box 1587, Seacaucus, NJ 07096,
or equiv.).
(b) Diatomaceous earth. — Celite 545 (Manville Filtration
and Minerals, or equiv.).
(c) Florisil. — 60-100 mesh (available from Floridin Co.).
Activated by manufacturer at 650° (1225-1250°F). Heat in oven
at 130° >48 hr before use.
630
Drugs and Feed Additives in Animal Tissues
AOAC Official Methods of Analysis (1 990)
(d) Glassware cleaner. — Haemo-Sol (Scientific Products,
Inc., or equiv.).
(e) Helium. — 99.5% min. purity (Matheson Gas Products,
or equiv.).
(f) Solid carbon dioxide.
(g) Solvents. — Acetone, CH 3 CN, benzene, CHC1 3 , hexane,
and MeOH. Distd-in-glass grade (Burdick & Jackson Labo-
ratories, Inc., or equiv.).
(h) Solvent mixtures .— (v /v) . (/) Hexane -acetone. — (8 +
2). (2) Hexane-acetone.— (95 + 5). (3) 70% Methanol.
(i) Anhydrous sodium sulfate. — Mallinckrodt Chemical
Works, or equiv. Wash with CHC1 3 , dry in 110° oven, and
store in g-s bottle until used.
D. MGA Standard Solutions
(a) Stock solns. — (7) A. — 1 mg/mL; 1000 ppm. Dissolve
100.0 mg melengestrol acetate (99.5% purity, Upjohn Co.) in
100 mL acetone. Soln is stable 2-3 months. (2)B. — 100 ppm.
Oil. 10.0 mL soln A to 100 mL with MeOH. Prep, soln fresh
daily. (3) C — 10 ppm. Oil. 10.0 mL soln B to 100 mL with
MeOH. Prep, soln fresh daily.
(b) Intermediate solns. — (7) D. — 0.5 ppm. Dil. 5.0 mL soln
C to 100 mL with MeOH. (2) £.—1.0 ppm. Dil. 10.0 mL
soln C to 100 mL with MeOH. (3) F.—1.5 ppm. Dil. 15.0
mL soln C to 100 mL with MeOH.
(c) Working solns.— 0.25, 0.50, 0.75 ppm. Transfer 5.0
mL solns D, E, and F into sep. 50 mL r-b flasks and evap.
on rotary evaporator. Dissolve residues in 10.0 mL portions
hexane-acetone (8 + 2).
E. Extraction
(Wash all glassware in detergent and rinse in H 2 to remove
traces of cleaning agent. Then rinse with MeOH, acetone, or
CHCI3. Caution: See safety notes on blenders, acetonitrile,
acetone, and cyclohexane. Store samples in freezer.)
(a) From fat. — Transfer 25.0 g sample to 250 mL beaker.
Add 150 mL hexane and warm on steam bath in fume hood
without boiling. Stir with spatula until fat dissolves. Place 20
g diat. earth (2 heaping tablespoons) in fritted funnel and wash
with 100 mL CH^CN. Discard wash. Filter warmed fat soln
thru cake on funnel with vac. into 1 L filter flask. Rinse beaker
with <50 mL hexane to remove solids, and transfer to funnel.
Remove top 3 mm diat. earth cake and transfer to blender bowl.
(Some diat. earth is left in funnel for next filtration.)
Add 150 mL hexane and homogenize 3 min at low speed.
Filter soln thru diat. earth cake into filter flask. Rinse blender
bowl with enough hexane to remove solids, and transfer to
funnel. Adjust combined filtrates to ca 400 mL with hexane
in filter flask. Rinse beaker and blender bowl with two 50 mL
portions CH 3 CN, and transfer to funnel. (Rinse cake thoroly,
since MGA may adsorb onto diat. earth from hexane.) Warm
filter flask on steam bath in hood and transfer filtrate to 1 L
separator. Rinse flask with 5-15 mL CH 3 CN, and transfer to
separator. Shake vigorously 1 min. Let layers sep. 30 min.
Drain lower layer into 1 L r-b flask. Add 100 mL CH 3 CN to
separator. Repeat extn and sepn twice. Add 50 mL benzene
to r-b flask and evap. on rotary evaporator.
(b) From muscle, liver, and kidney. — Transfer 25.0 g fro-
zen tissue to blender bowl. Let thaw 5-10 min at room temp.
Add 150 mL CH 3 CN, 20 g diat. earth (2 heaping tablespoons),
and 50 g anhyd. Na?S0 4 (2 tablespoons). Homogenize at low
speed 3 min. Place 20 g diat. earth into fritted funnel and wash
with 100 mL CH 3 CN. Discard wash. Filter soln thru cake with
vac. into 1 L filter flask. Rinse blender bowl with <50 mL
CH3CN to remove remaining solids. Sep. tissue cake from fil-
ter pad and transfer to blender. (Do not disturb diat. earth be-
low tissue cake. Household fork is good transfer tool.)
Add 10 g diat. earth, 25 g anhyd. Na 2 S0 4 , and 150 mL
CH 3 CN to blender bowl. Homogenize 3 min at low speed,
filter, and rinse. Transfer combined filtrate to 1 L r-b flask
and add 50 mL benzene. Evap. to dryness in rotary evaporator.
(Caution: Bumping may occur. See safety note on benzene.)
To dry residue, add 200 mL hexane and 100 mL CH 3 CN thru
adapter. Remove adapter, and transfer sol v. mixt. to 1 L sep-
arator. Add another 200 mL portion hexane to r-b flask and
transfer to separator. Shake vigorously 1 min. Let layers sep.
30 min. Drain lower layer into 1 L r-b flask. Add 100 mL
CH 3 CN to separator. Repeat extn and sepn twice. Add 50 mL
benzene and evap. on rotary evaporator.
F. Column Chromatography
Before analysis of samples, confirm, using MGA std soln,
that hexane-acetone (8 + 2) elutes MGA completely, as fol-
lows: Pipet 1 mL 1 ppm MGA std soln into 50 mL r-b flask
and evap. solv. on rotary evaporator. Chromatograph on Flor-
isil column as indicated below. Det. recovery of MGA. If re-
covery is <95%, det. new elution vol. or obtain new batch of
Florisil.
To 19 mm id glass tube, add cooled Florisil to ht of 10 cm
with tapping. Push small wad of glass wool into tube until it
touches Florisil. Place reservoir on top of column. Consecu-
tively prewash column with 100 mL hexane, 100 mL acetone,
and 100 mL hexane. (N pressure may be used to speed up this
washing.) Remove reservoir.
Dissolve sample residue in 20 mL hexane and transfer to
top of column. Replace reservoir and consecutively wash flask
with 20 mL hexane, 200 mL hexane, and 300 mL hexane-
acetone (95 + 5), and add each washing to column; if N pres-
sure is used, add adapter. When last of solv. has reached top
of column, place 500 mL r-b flask under column, wash sample
residue flask with 150-170 mL hexane-acetone (8 + 2), and
transfer to column for MGA elution. Elute sample until col-
umn goes dry, using N pressure to blow out last of solv. Evap.
to dryness on rotary evaporator. Quant, transfer dried residue
with five 2 mL portions acetone to 50 mL r-b flask and evap.
on rotary evaporator. Dil. sample to 1.0 mL with hexane-ace-
tone (8 + 2).
MGA gives poorly resolved chromatogram with some liver
samples. Following addnl cleanup is necessary to remove in-
terferences: Evap. remainder of 1 mL hexane-acetone soln on
rotary evaporator. To dried residue, add three 20 mL portions
hexane and transfer to 500 mL separator. Add 50 mL 70%
MeOH, shake vigorously 1 min, let sep. 15 min, and drain
lower layer into second 500 mL separator. Add 50 mL 70%
MeOH to first separator. Repeat extn and sepn twice. To MeOH
layer in second separator, add 1.0 mL satd Na 2 S0 4 soln, 100
mL deionized H 2 0, and 50 mL CHC1 3 . Shake vigorously 1
min. (Caution: Vent frequently.) Let layers sep. 15 min and
drain lower layer into 500 mL r-b 11 ask. Add 50 mL CHC1 3
to separator and repeat extn and sepn twice. Evap. CHC1 3 on
rotary evaporator. Quant, transfer dried residue with five 2 mL
portions acetone to 50 mL r-b flask and evap. on rotary evap-
orator. Add 1 .0 mL hexane-acetone (8 + 2) and reinject on
column.
G. Gas Chromatography
Alternately inject 2-4 |ulL aliquots sample blank and 0.25
ppm MGA std soln until reproducible peak hts are obtained
for std. Inject 1-4 \xL 0.25 ppm MGA std soln. Adjust gas
flow and attenuation until 20-25 mm peak ht is obtained. Use
this std soln for measurement and calcn of samples at ca 10
ppb (ng/g) level, 0.5 ppm std soln for samples at ca 20 ppb,
and 0.75 ppm std soln for samples at 30 ppb.
Inject same sample vol. as used for std soln to obtain 20-
AOAC Official Methods of Analysis (1990)
Nalidixic Acid 631
25 mm (or suitable) response. Measure peak ht of std, H' , and
sample, H, at retention time of MGA by baseline technic.
ppb MGA - (H/H') x C x (V//)/g sample
where C = ng MGA std injected on column; V - total mL
soln (sample + solv.) in r-b flask (1.0 mL); and / = mL sam-
ple soln injected onto column.
Ref.: JAOAC 59, 507(1976).
CAS-29 19-66-6 (melengestrol acetate)
970.84 Nalidixic Acid Residues
in Animal Tissues
Spectrofluorometric Method
First Action 1970
Final Action 1974
(Applicable to chicken liver and muscle contg >100 ppb
nalidixic acid)
A. Principle
Nalidixic acid is extd from aq. tissue homogenate with EtOAc.
EtOAc is collected, coned, and passed thru alumina column
which retains nalidixic acid. Nalidixic acid is removed from
column with borate buffer, acidified, and re-extd with CHCl 3 .
After CHCl 3 removal, residual nalidixic acid is made to flu-
oresce with H 2 S0 4 and resultant fluorescence is measured with
spectrofluorometer.
B. Apparatus
(a) Spectrofluorometer. — (Caution: See safety notes on
photofluorometers.) Aminco-Bowman 4-8202, or equiv., with
Xe lamp, IP 28 photomultiplier tube, and operated with man-
ufacturer's slit arrangement No. 3. Precise wavelength settings
for excitation and emission may vary slightly between instru-
ments. Det. optimal wavelengths (ca 325 and 408 nm) after
evapn of 2 mL working std soln (I (xg nalidixic acid) and soln
of residue in 10 mL 21. 5N H 2 S0 4 , 970.84C(c)(/).
(b) Chromatographic tubes. — 160 x 11.5 (id) mm (Kontes
Glass Co., No. K -420000, or equiv.).
(c) Shaker. — Reciprocating (Sargent-Welch Scientific Co.,
No. S-74070, or equiv.).
C. Reagents
(a) Phosphate buffer soln. — pH 6.0. Weigh 28 g Na-
H2PO4.H2O into 1 L beaker, add ca 600 mL H 2 0, and adjust
pH potentiometrically with aq. NaOH. Dil. to 1 L.
(b) Borate buffer soln.— pH 10.0. Dissolve 30 g H 3 B0 3 in
ca 600 mL H 2 and adjust pH potentiometrically with aq.
NaOH. Dil. to 1 L.
(c) Dilute sulfuric acid. — (Caution: See safety notes on sul-
furic acid.) (J) 21. 5N. —Measure 200 mL H 2 into 1 L flask
and gradually add, with cooling, 300 mL H 2 S0 4 . Use soln at
room temp. (2) 7N. — Dil. 1 vol. (J) with 2 vols. H 2 0.
(d) Alumina. — Neut. (Fisher Scientific Co., No. A-950, or
equiv.).
(e) Nalidixic acid std solns. — (7) Stock soln. — 500 p,g/mL.
Dissolve 50.0 mg nalidixic acid anal, std (available from Ster-
ling Organics, 33 Riverside Ave, Rensselaer, NY 12144) in
100 mL MeOH. (2) Intermediate soln. — 5.0 |xg/mL. Dil. 2.0
mL stock soln to 200 mL with MeOH. (3) Working soln. —
0.5 jxg/mL. Dil. 1 0.0 mL intermediate soln to 100 mL with
MeOH.
D. Determination
(Caution: See safety notes on centrifuges, flammable solvents,
chloroform, and ethyl acetate.)
Transfer 10 g chicken liver or muscle to high-speed blender.
Add 100 mL phosphate buffer and blend 2-3 min. Transfer
homogenate to 500 mL g-s extn bottle and add 300 mL EtOAc.
Add 100 mL phosphate buffer to each of five 500 mL g-s
extn bottles. Transfer 0, 1.0, 2.0, 3.0, and 4.0 mL working
soln contg 0.0, 0.50, 1 .0, 1 .5, and 2.0 jxg nalidixic acid, resp.
Add 300 mL EtOAc to each. Mech. shake all bottles contg
sample and std 10-15 min and centrf. ca 5 min at 2500 rpm.
Withdraw 250 mL EtOAc supernate from each and transfer to
sep. 600 mL beakers. Evap. each under air current on steam
bath to ca 60 mL.
Prep, adsorption column for sample and each std as follows:
Place glass wool plug at bottom of chromatgc tube and add
alumina to depth of 3 cm (ca 3 g). Place another glass wool
pad at top of column. Wash each column with 25 mL EtOAc.
Transfer tissue and std exts from beakers to respective col-
umns. Rinse each beaker with 25 mL EtOAc followed by two
25 mL portions ether and two 25 mL portions MeOH. Transfer
each solv. rinse to corresponding column and discard all eluates.
Add two 25 mL portions borate buffer and collect eluate in
50 mL graduate. Transfer eluate from graduate to 125 mL sep-
arator with Teflon stopcock. Ext with 25 mL ether and discard
ether. Acidify aq. soln with 10 mL 7N H 2 S0 4 . Thoroly ext
with 25 mL and 10 mL CHC1 3 . Withdraw each CHC1 3 ext and
combine in 100 mL beaker. (Do not introduce any aq. phase.)
Evap. solv. just to dryness on steam bath.
Add 10.0 mL 21.5 N H 2 S0 4 to each beaker. Mix thoroly
>10 min. Det. relative fluorescence (product of linear scale
meter reading and meter multiplier setting) of processed blank,
stds, and tissue sample in 1 cm cell at excitation 325 nm and
emission, 408 nm. Subtract relative fluorescence of reagent
blank from relative fluorescence of all std and sample prepns.
Prep, std curve with reagent blank-corrected relative fluo-
rescence values of processed stds as ordinate and correspond-
ing |xg nalidixic acid as abscissa. From std curve, det. amt
nalidixic acid (jc) which corresponds to reagent blank-corrected
relative fluorescence of processed tissue sample.
ppb (ng/g) Nalidixic acid = (x X 1000)/10 g (tissue wt)
Ref.: JAOAC 53, 464(1970).
CAS-389-08-2 (nalidixic acid)
982.40 Sulfamethazine Residues
in Swine Tissues
Gas Chromatographic-Mass Spectrometric Method
First Action 1982
Final Action 1984
(Applicable to residues at 0.05-0.20 ppm)
A. Principle
Sulfamethazine is extd from tissue with CHCl 3 -acetone. Ext
is filtered and solv. is removed by evapn. Residue is redis-
solved in hexane and partitioned against IN HO. Acid phase
is neutzd and sulfamethazine is extd with CH 2 C1 2 . Solv. is re-
moved and residue is methylated using diazomethane. Sulfa-
methazine is identified and quantitated using electron impact
gas chromatgy/mass spectrometry (E1GC/MS) in selected ion
mode. Six ions, m/z 92, 98, 227, 228, 233, and 234 are mon-
itored. Ion current from each is accumulated thruout GC run,
stored on mag. tape, and plotted as ion current vs time. Peaks
appearing in ion current profiles are identified and retention
times and areas for each peak are calcd. Sulfamethazine is
quantitated from std curve prepd by least squares linear regres-
sion using data from analysis of known std solns. Identity of
632
Drugs and Feed Additives in Animal Tissues
AOAC Official Methods of Analysis (1990)
sulfonamide residues is confirmed by presence of significant
ions appearing at proper retention time in proper relative abun-
dances,
Procedure gives quant, results as well as data for confir-
mation of residues detected. Procedure is accurate at 0.1 ppm
level with expected coefficient of variation of 4.6%. Min. de-
tectable level is 0.002 ppm. To accommodate residues >0.2
ppm, reconstruct std curve as follows:
Expected
Concn Range
Use Stds (in ppm)
B
-0.2 ppm
0.2-2.0 ppm
2.0-20.0 ppm
0.05
0.50
5.0
0.10
1.0
10.0
0.20
2.0
20.0
B. Apparatus
(a) Blender. — Virtis Model 45, or equiv., with 500 mL
flasks .
(b) Evaporator, — N-Evap Model III (Organomation As-
sociates, PO Box 159, South Berlin, MA 01549).
(c) Gas chromato graph— mass spectrometer. — Hewlett-
Packard Model 5992 quadrupole operated in multiple ion mon-
itoring under following conditions: electron energy, 70 eV;
electron multipler, 2000-2800 eV; source temp., 140°; inte-
gration time, 200 ms/mass monitored. Column: 2 mm id X 3
ft glass, packed with 3% OV-.17 on 80-100 mesh Gas-Chrom
Q (or equiv. packing). GC conditions: injection port 230°; col-
umn 220°, He flow 30 mL/min; GC/MS interface, silicone
membrane separator. Total analysis time is ca 17 min. Sul-
famethazine retention time is 9-12 min.
C. Reagents
(a) Solvents. — Distd in glass, or equiv.: acetone; CH 2 C1 2 ;
ether; MeOH (shake with and store over anhyd. Na 2 S0 4 ); CH 3 C1
(no preservatives).
soln. — Add 720 g tri sodium citrate
(b) Trisodium citrate
dihydrate to 1 L H 2 0.
(c) Diazomethane kits.-
1 and Zl 0,025-0. Prep.
— Aldrich Chemical Co. No. Z10,159-
according to manufacturer's instruc-
tions. Caution: Prep, diazomethane in hood behind protective
screen or shield. Wear gloves to prevent skin contact with re-
agents. Diazomethane is toxic and under some conditions ex-
plosive. Freshly made diazomethane soln is golden yellow. Store
in freezer at <0°. Storage time may vary with method of prepn.
See J AOAC 65 , 273-274(1982).
(d) Sulfamethazine stock std soln. — 100 (ULg/mL. Accu-
rately weigh 10.0 mg sulfamethazine into 100 mL vol. flask.
Dissolve in and dil. to vol. with anhyd. MeOH.
(e) Fortification std soln. — 5 (xg/mL. Pipet 5.0 mL stock
std soln into 100 mL vol. flask and dil. to vol. with H 2 0. Prep,
std fresh weekly.
(f) GC/MS std soln.— 50 jJLg/mL. Accurately weigh 10.0
mg unlabeled sulfamethazine into 200 mL vol. flask. Dissolve
in and dil. to vol. with anhyd. MeOH.
(g) n C -Labeled sulfamethazine std soln. — 50 jxg/mL. Ac-
curately weigh 10.0 mg 13 C-JabeIed sulfamethazine into 200
mL vol. flask. Dissolve in and dil. to vol. with anhyd. MeOH.
D. Extraction and Cleanup
Weigh 25 g (± 0.1 g) ground, frozen tissue into 250 mL
centrf. bottle. Select blank sample as control. Fortify second
blank sample at 0.2 ppm with 1 mL fortification std soln (5
jxg/25 g — 0.2 ppm). Analyze both blanks in parallel with
each set.
Add 100 mL acetone, washing any adhering tissue off walls
of centrf. bottle; then blend 1 min with tissue grinder. Centrf.
10 min at 2000 rpm. Filter acetone ext thru 24 cm Whatman
2V fluted paper into 500 mL r-b flask. Add 10 mL 5N HCI
to acetone ext and place on rotary evaporator in 55 ± 5° bath.
Evap. until acetone odor is absent.
Transfer remaining acid phase to 125 mL separator with two
25 mL portions of ether. Gently rotate separator 1 min and let
phases sep. 30 min. Transfer acid phase to 100 mL beaker -and
add 50 mL satd aq. trisodium citrate soln. Adjust pH to 6.0-
6.5, using ION NaOH. Transfer contents of beaker to 250 mL
separator, add 30 mL CH 2 C1 2 , and shake 2 min. Let phases
sep. and transfer 10 mL CH 2 C1 2 phase to 15 mL centrf. tube.
Evap. to dryness under stream of N at 45°.
E. Thin Layer Chromatographic Screen
Dissolve residue in 0.1 mL MeOH. Aspirate entire sample
into 100 pT syringe and place on automatic TLC spotter. Spot
sample on TLC plate, along with 10 |xL TLC spotting std soln
(100 ng). Develop 10 cm at room temp, with CHCl 3 -tert-bu-
tanol (80 + 20). Remove plate from tank and let air-dry 10
min at room temp. Spray plate with 1.0% sodium nitrite in
1.0N HO. Dry with blow dryer or in 100° oven. Spray with
0.4% NEDA in MeOH. Dry plate as above to produce pink
spots. Compare R { values with std for identification. If TLC
results are pos., continue thru GC/MS below.
F. GC/MS Quantitation /Confirmation
Weigh 50.0 g (± 0.1 g) ground, frozen tissue into 500 mL
blender flask. Select blank tissue as control. Fortify second
blank tissue sample at 0.1 ppm level with unlabeled sulfa-
methazine (100 |xL GC/MS std). Spike all samples at 0.10
ppm level with n C- labeled sulfamethazine std soln (100 \xL).
Add 100 mL CHC] 3 -acetone (1 + I) to .flask. Blend 1 min
at low speed. Decant and filter liq. (no vac.) thru 24 cm What-
man 2V fluted paper into J L r-b flask.
Repeat extn and filtering twice more. Transfer all tissue to
filter paper after third extn.
Rinse flask with ca 25 mL CH0 3 -acetone (1 + 1), transfer
rinsing to filter paper, and let drain. Rinse filter paper and
contents with three 20 mL aliquots of CHCi 3 ~-acetone (I + 1).
If combined filtrates are not clear, ref liter and wash filter paper
with ca 20 mL CHCl 3 -acetone (1 + 1).
Evap. on rotary evaporator at 55° (± 5°) to oily residue (ca
1-2 mL). Remove from rotary evaporator promptly. Quant,
transfer residue to 250 mL separator using, in order, four 25
mL portions of hexane, two 3 mL portions of acetone, and
two 25 mL portions of hexane. Add 10 mL IN HC1 to sepa-
rator. Shake gently 2 min and let phases sep. Emulsions may
be eliminated by placing separator in 60° H 2 bath. Repeat
extn 3 times with 5 mL portions of IN HO, drawing off acid
phase and combining filtrates in the 125 mL separator. Add
3.0 mL ION NaOH to 125 mL separator and mix. Det. pH,
using pH paper. If pH is not 12-13, add addnl ION NaOH
with mixing to attain this pH.
Add 25 mL CHCI 3 to basic soln and shake 1 min. Let phases
sep. completely, and discard CHCI 3 . Repeat CHC1 3 extn a sec-
ond time, discarding CHC1 3 . Quant, transfer aq. phase to small
beaker (ca 100 mL). Buffer by adding 25 mL satd aq. triso-
dium citrate. Adjust pH with pH meter to 5.55-5.65 by adding
NaOH or HO as required.
Quant, transfer contents of beaker to 125 mL separator, add
15 mL CH 2 C1 2 , and shake 1 min. Let phases sep. and transfer
CH 2 C1 2 to 50 mL conical centrf. tube. Check pH of aq. phase
and re-adjust to 5.55-5.65 if necessary. Repeat CH 2 C1 2 extn
twice .
Evap. contents of centrf. tube to dryness at 45° under stream
of N on N-Evap. Dissolve residue in 1 mL anhyd. MeOH.
Add 1 mL diazomethane soln, mix with vortex mixer, and let
stand at room temp. 5 min. Transfer soln from tube to 15 niL
AOAC Official Methods of Analysis (1990)
Sulfamethazine 633
or smaller concentrator tube and evap. to dryness at 45° under
stream of N on N-Evap. Dissolve methylated residue in 200
(xL anhyd. MeOH.
G. Preparation of Standard Curve
Add 100 |xL '^-sulfamethazine std soln (50 fxg/mL) to each
of three 15 mL concentrator tubes. Label tubes A, B, and C.
Add 50 [xL GC/MS std soln (50 u.g/mL) to tube A (equiv.
to 0.05 ppm ,2 C). Add 100 \xL GC/MS std soln (50 |xg/mL)
to B (equiv. to 0.10 ppm 12 C). Add 200 \lL GC/MS std soln
(50 |xg/mL) to tube C (equiv. to 0.20 ppm ,2 C). Add J mL
freshly prepd diazomethane soln to each concentrator tube and
mix with vortex mixer, let stand at room temp. 5 min. Evap.
to dryness at 45° under stream of N on N-Evap. Dissolve each
methylated residue in 200 jxL anhyd. MeOH.
Set selected ion monitor (SIM) data acquisition program area
as follows:
a Mass 1 =227, dwell time = 200 ms
b Mass 2 = 228, dwell time = 200 ms
c Mass 3 = 233, dwell time = 200 ms
d Mass 4 = 234, dwell time - 200 ms
e Mass 5 = 92, dwell time = 200 ms
f Mass 6-98, dwell time = 200 ms
g Solvent elution time = 1 . 1 min
Make injections in order given below.
a Inject 2.0 \xL std A
b Inject 2.0 |jlL std B
c Inject 2.0 jxL std C
For each std injection, det. ratio of areas of m/z 227 peak to
area of m/z 233 peak. Using method of least squares, calc.
std curve for 227/233 mass ratio vs amt of unlabeled sulfa-
methazine added to each std (amt may be expressed as ppm
based on 50 g sample). Similarly for each std, calc. following
confirmation ratios: 228/227 and 234/233. Mean 228/227 and
234/233 ratios computed from 3 std injections will be used
for confirmation of identity of sulfamethazine detected in pro-
cessed samples.
H. Determination
With SIM program set as for Preparation of Standard Curve,
inject 2.0 uJL from each sample to be analyzed. Plot recon-
structed ion current profiles at end of each run and calc. 227/
233 ion mass ratio. Read sulfamethazine content of sample
from std curve.
For identification purposes: (a) Sample unlabeled sulfa-
methazine must co-elute with added I3 C-labeled sulfametha-
zine, (b) The m/z 92, 277, and 228 from unlabeled sulfa-
methazine and m/z 98, 233, and 234 from 13 C-labeJed
sulfamethazine must all be present, (c) Ratios of 228/227 and
234/233 ions in sample should be within 10% of mean ratio
detd for stds.
Refs.: JAOAC64, 1386(1981).
CAS-57-68-1 (sulfamethazine)
982.41 Sulfamethazine Residues
in Swine Tissues
Gas Chromatographic Method
First Action 1982
Final Action 1984
(Applicable to residues >0.1 ppm)
A. Principle
Tissue is extd with acetone-CHCl 3 , IN HCl is added, and
solv. is evapd. Aq. soln is washed with hexane, pH is adjusted
to 5.55-5.65, and sulfamethazine is extd with CH 2 Cl 2 , meth-
ylated with diazomethane, and detd by electron capture GC.
B. Reagents and Apparatus
Rinse all clean glassware thoroly with MeOH and let dry
Use distd in glass solvs suitable for pesticide analyses (Burdick
& Jackson Laboratories, Inc., or equiv.).
(a) Sulfamethazine std solns. — Prep, std solns contg 1,2,
and 10 |mg sulfamethazine USP/mL acetone.
(b) Diazomethane derivatizing reagent. — Caution: Diazo-
methane is toxic, can cause specific sensitivity, and is poten-
tially explosive. Prep, diazomethane reagent, methylate, and
evap. in hood. Avoid ground glass joints, etched or scratched
glassware, and sharp edges. Store diazomethane solns in freezer;
do not expose to direct sunlight or strong artificial light. Prep.
diazomethane by etheral basic distn of 21.5 g N- methyl -/V-ni-
tro-p-toluenesulfonamide (Diazald, Aldrich Chemical Co.), with
200 mL ether as described in Diazald kit, Cat. No. Z10, 025-
0. (Note: Read Diazald kit instructions carefully for safe han-
dling of diazomethane.) After distn of second portion (40 mL)
of ether, transfer ether soln of diazomethane thru funnel to
narrow-mouth bottle and cap tightly with a poly seal cap; store
in freezer (see JAOAC 65, 273-274(1982)). When stored in
freezer, soln retains its efficiency as methylating agent >1
month.
(c) Gas chromato graph. — Tracor Model 222, or equiv., with
63 Ni linearized electron capture detector and 6 f t x 2 mm id
glass column packed with 5% OV-7 on 100-120 mesh Gas-
Chrom Q (or equiv. packing). Operating conditions: injector
290°, column oven 260°; detector 290°; argon-methane (90 +
10) carrier gas at 30 mL/min; detector purge flow 20 mL/
min. Retention time for methylated sulfamethazine is 4-4.5
min. Alternative column: 6 f t x 4 mm id, packed with 5%
OV-25.
(d) Food chopper. — Model 84142D (Hobart Manufactur-
ing Co., 71 1 Pennsylvania Ave, Troy, OH 45374), or equiv.
(e) Flasks. — 100 mL pear-shaped, 24/40 joint (Kontes Glass
Co. No. K-608700).
C. Procedure
Cut tissue into 1.5 cu. in. pieces and freeze in plastic bags.
Pulverize enough dry ice in bowl of Hobart food chopper to
chill bowl and grater thoroly. Slowly add small portions (ca
50 g) of sample and continue chopping until complete sample
is chopped. If necessary, add more dry ice to maintain sample
in frozen state during chopping procedure. Store sample in a
freezer (— 20°F) until dry ice has dissipated.
Transfer 15 g sample to blender and blend 5 min at medium
speed with 150 mL acetone~-CHCl 3 (1 + 1). Filter thru glass-
fiber paper, collecting first 100 mL filtrate in 100 mL grad-
uate. Transfer 100 mL aliquot (equiv. to 10 g sample) to 250
mL r-b flask with 24/40 joint and add 10 mL IN aq. HCl.
Evap. org. solvs on rotary vac. evaporator with flask sub-
merged in 32 ± 4° H 2 G bath. (For muscle and fat, some extd
fat will prevent complete removal of solvs.) Add 50 mL n-
hexane to IN HCl phase and quant, transfer both phases to
125 mL separator. Rinse evapn flask with addnl 5 mL IN HCl
and transfer rinse to 125 mL separator. Shake contents of sep-
arator gently by inverting funnel and returning to upright po-
sition once a second for 50 s. Let funnel sit until phases sep.
(ca 10 min). Draw off lower phase (IN HCl) into second 125
mL separator. (Centrifugation may be required to avoid trans-
fer of emulsified solv. which tends to bump during hydrolysis
step.) Rinse r-b flask with 5 mL IN HCl, transfer to first sep-
arator, contg hexane, mix, and sep. as above. Drain HCl phase
into second separator and discard hexane. For high fat samples
only, add 15 mL CH 2 C1 2 to HCl phase in second separator,
634
Drugs and Feed Additives in Animal Tissues
AOAC Official Methods of Analysis (1990)
shake 30 s, and let phases sep. Draw off and discard lower
CH 2 C1 2 phase.
Buffer aq. HC1 ext in separator by adding 25 mL satd aq.
trisodium citrate; then adjust pH with pH meter to 5.55-5.65
by adding 3N NaOH (ca 2.5 mL). Add 15 mL CH 2 CI 2 to sep-
arator and shake vigorously for 90 s. Let phases sep. and trans-
fer lower CH 2 C1 2 layer to 100 mL pear-shaped flask. After first
extn, check pH of aq. phase and re-adjust to 5.55-5.65 if nec-
essary. In similar manner, ext with 3 addnl portions of CH 2 C1 2 ,
combining exts in 100 mL pear-shaped flask. Evap. solv. in
rotary vac. evaporator with H 2 bath at 25-30°. Do not ex-
ceed 30°. Sample may be kept overnight at this stage.
Dissolve residue in 1.0 mL acetone. Swirl flask to dissolve
any residue on walls of flask. In fume hood, add 1 mL dia-
zomethane derivatizing reagent and Jet stand 15 min with in-
termittent gentle swirling. Evap. solv. under gentle stream of
N. Dissolve residue in 1.0 mL acetone.
Prep, methylated stds by pipetting 1.0 mL aliquots of each
sulfamethazine std soln into sep. 100 mL pear-shaped flasks.
Add 1 mL diazomethane derivatization reagent and treat in
same manner as sample.
D. Gas Chromatography
Inject 2-8 |xL methylated 1 fxg/mL sulfamethazine std into
gas chromatograph. (Resulting peak ht should be 30-40% FSD.)
Inject up to 3 samples followed by std which approx. matches
sample. If sample peak goes off scale, quant, dil. methylated
sample soln with acetone to give response that is 30-60% FSD.
Correct results for diln.
Sulfamethazine, ppm
= 1.5 (A x C x V')/{A f x W XV)
where A, A' = peak area of sample and std, resp.; V, V =
GC injection vol. (jxL) of sample and std, resp.; C = concn
of std ((xg sulfamethazine/mL); W = wt sample (g); 1.5 —
150/100 to correct for 100 mL aliquot of 150 mL sample ext
taken for analysis. Method reliably quantitates sulfamethazine
at 0.1 ppm and above.
Ref.: JAOAC 64, 1386(1981).
CAS-57-68-1 (sulfamethazine)
983.31 Sulfonamide Residues
in Animal Tissues
Thin Layer Chromatographic Screening Method
First Action 1983
Final Action 1984
(Applicable to swine, turkey, and duck tissues)
A. Principle
Sulfonamides are extd with ethyl acetate after addn of sul-
fapyridine as internal std. Exts are cleaned up by partitioning
between org. and aq. solvs, and chromatographed on silica gel
TLC plates. Developed plates are treated with fluorescamine
and scanned by fluorescence densitometer.
B. Reagents
(a) Ethyl acetate, hexane, methylene chloride, and metha-
nol. — Distd in glass (Burdick & Jackson Laboratories, Inc.).
(b) Glycine buffer soln.— Prep, glycine (Fisher Scientific
Co.) as 0.2M aq. soln and adjust pH to 12.25 with NaOH.
(c) Fluorescamine derivatizing soln. — Dissolve 25 mg flu-
orescamine (Pierce Chemical Co.) in 250 mL acetone. Replace
soln after treating 8-9 plates.
(d) Sulfonamide stds. — Com. sulfamethazine (SMZ), sul-
fadimethoxine (SDM), sulfaquinoxaline (SQX), sulfathiazole
(STZ), sulfabromomethazine (SBR), and sulfapyridine (SPY).
(e) Stock std solns. — Dissolve 100 mg sulfonamide in 100
mL acetone. Store in refrigerator.
(f ) Fortification std solns. — Combine sulfonamides of in-
terest and dil. to 5.00, 2.50, and 1.25 |xg/mL (equiv. to 0.2,
0.1, and 0.05 ppm in tissue), using 0.05M pH 7.5 phosphate
buffer. All solns should contain 2.50 \xg SPY/mL. Store for-
tification stds in refrigerator and prep, weekly.
(g) Internal std soln. — Using stock std soln (e), prep. 2.50
jjig SPY/mL 0.05M pH 7.5 phosphate buffer.
C. Apparatus
(a) Densitometer.— CAMAG TLC/HPTLC scanner (Ap-
plied Analytical Industries, Rt 6, PO Box 55, Wilmington, NC
28405) equipped with 400 nm interference filter on excitation
source. Replace std 400 nm cutoff filter on photomultiplier by
500 nm interference filter. Slit dimensions 7.8 X 0.3 mm. Scan
plates at either 1 or 2 mm/s.
(b) TLC plates. — 20 x 20 cm channeled plates, channels
8 mm wide with 0.25 mm silica gel layer and pre-adsorbent
spotting zones (No. 4865-821, Whatman, Inc.).
(c) TLC spotting capillaries. — 20 jjiL glass capillary tubes
(Corning Glass Works).
(d) Heat strip. — Automatic spotter heat strip set at 85° (An-
alytical Instrument Specialties, Inc., PO Box 596, Liberty-
ville, IL 60048). Any equiv. heating device with temp, control
can be substituted.
(e) TLC tank. — Std 2-trough tank (AUtech Associates, 2015
Waukegan Rd, Deerfield, IL 60015), atm. fully satd by lining
with satn pads.
(f) Derivatization tank. — Stainless steel (Thomas Scien-
tific).
(g) Evaporator. — N-£vap (Organomation Associates, Inc.,
PO Box 159, South Berlin, MA 01549).
(h) Homogenizer. — Tekmar SDT Tissumizer (Tekmar Co.,
PO Box 371856, Cincinnati, OH 45222).
(i) Shaker. — Horizontal reciprocating shaker set at ca 240
cycles/min (Eberbach Corp., PO Box 1024, Ann Arbor, MI
48106-1024).
(j) Centrifuge. — Set at 2500 rpm for 5 min (Model PR-
7000, International Equipment Co., or equiv.).
(k) Polypropylene centrifuge tubes. — 50 mL capacity
(Corning Glass Works).
D. Sample Extraction
Accurately weigh ca 2.5 g homogenized liver or muscle into
50 mL centrf. tube. Add 100 |xL internal std soln. Prep. 3
control samples (using tissue known to be free of sulfon-
amides) and fortify with internal std (0. 1 ppm) and each sul-
fonamide of interest, one control each at 0.05, 0.10, and 0.20
ppm. Wait 15 min and then add 25 mL ethyl acetate. Blend
muscle samples 1 min with Tissumizer, and centrf. For liver
samples, cap tube tightly, shake 20 min on horizontal shaker,
and centrf. Transfer ethyl acetate to clean tube and discard
tissue. Add 10 mL glycine buffer to ext, mech. shake 5 min,
and centrf. Vac. -aspirate and discard org. phase. Adjust pH
of aq. phase to 5.2-5.3 by adding 2 mL (1 + 1) mixt. of 2M
pH 5.25 phosphate buffer and 1.7M HO. Check pH and make
final adjustments with either addnl buffer or 0. IN NaOH. Add
5 mL hexane, mech. shake 5 min, and centrf. Aspirate and
discard hexane phase. Remove any solid or emulsified mate-
rial remaining at interface. Add 10 mL CH 2 C1 2 , shake 5 min,
centrf., and aspirate and discard aq. phase. Add 10 |jlL diethyl-
amine to CH 2 C1 2 ext and cone, just to dryness under stream
of N at 40°. During evapn, occasionally rinse tube walls with
CH 2 C1 2 . Redissolve residue in 100 |xL MeOH and mix 30 s
AOAC Official Methods of Analysis (1990)
Zoalene
635
on vortex mixer. Let stand 5 min before chromatgy to let in-
soluble oils settle.
E. Chromatography
Apply 20 |xL portion of sample to pre-adsorbent spotting
zone of TLC plate. Avoid using lane at each side of plate, and
spot the 3 fortified control samples at intervals across plate to
minimize effects of across-plate variation. Develop plate 1 cm
in MeOH followed by 2 developments, 6 cm and 12 cm, in
CHCI 3 -ter*-BuOH (80 + 20). Between each development, dry
plate 1 min at 1 10°. If STZ is suspected or present, pre wash
CHCl 3 -terf-BuOH with H 2 0. Maintain development tank temp.
at 30-33° for optimum resolution of multiple sulfonamides.
Derivatize compds by quickly dipping plate in fluorescamine
soln. Bands are visible after 15-30 min. Scan each lane and
obtain its response scanning curve. For each sample and std
(each lane), det. ratio of response for sulfonamide of interest
to response for internal std (SPY).
F. Calculations
For each sulfonamide, calc. slope and intercept of std curve,
concn vs response ratio, using linear regression and results for
the 3 fortified samples. Plotting sulfonamide concn on j-axis
results in std error of est. (S y . x ) having dimension of ppm, thus
simplifying estn of confidence intervals. For quality assurance
purposes, S yK should be <0.02 ppm, and correlation coeffi-
cient, r, should be ^0.995. Use linear regression slope and
intercept to calc. concn of samples from their respective sul-
fonamide/internal std peak ht ratios.
Ref.: J AOAC 66, 884(1983).
CAS- 1 16-45-0 (sulfabromomethazine)
CAS-122-1 1-2 (sulfadimethoxine)
CAS-57-68-1 (sulfamethazine)
CAS- 144-83-2 (sulfapyridine)
CAS-59-40-5 (sulfaquinoxaline)
CAS-72-14-0 (sulfathiazole)
966.26 Zoalene Residues
in Animal Tissues
Spectrophotometric Method
First Action 1966
Final Action 1967
A. Principle
Ground tissue is extd with acetone, and benzene is added to
sep. soln into 2 layers. Org. layer is coned, and passed thru
alumina column. Absorbed zoalene is washed with CHC1 3 and
eluted with 80% alcohol. Ale. soln is evapd just to dryness
and residue dissolved in ale. DMF soln. Colored complex
formed by addn of 1 ,3-diaminopropane is measured at 560 nm.
B. Apparatus
See 961.236(a), (b), and (d).
C. Reagents
(a) Acetone-benzene soln. — Mix 35 parts acetone with 65
parts benzene.
(b) Alumina.— See 961.23C(a).
(c) 1 ,3-Diaminopropane . — Aldrich Chemical Co.
(d) Dimethylformamide-alcohol soln. — Mix 4 parts DMF
with 1 part absolute ethanol.
(e) Zoalene. — Anal. std. (Available from Agricultural Dept,
Dow Chemical Co.)
D. Preparation of Alumina Column
See 961. 23D.
E. Preparation of Standard Curve
Accurately weigh 100 mg zoalene into 1 L vol. flask, dis-
solve in 50 mL acetone, and dil. to vol. with FLO. Dil. 10
mL of this stock soln to 100 mL with H 2 to give working
soln of 10 |xg/mL. Pipet 0, 2, 4, 6, 8, and 10 mL aliquots
working soln into sep. 100 mL beakers and evap. just to dry-
ness under heat lamp. Add 5 mL ale. DMF soln to each beaker
and stir 1-2 min. Add 5 mL 1,3-diaminopropane. After 10
min, measure A of soln at 560 nm, using 1 cm cells against
H 2 as ref. Prep, std curve by plotting A against fxg zoalene.
F. Determination
(Caution: See safety notes on blenders, distillation, flammable
solvents, toxic solvents, benzene, and acetone.)
Collect tissue, freeze with solid C0 2 , and keep frozen until
analyzed. Grind tissue while at least partially frozen and weigh
50 g into 1 qt (1 L) Mason jar. Add 250 mL acetone and mix
with high-speed mixer ca 5 min. Filter on buchner into 1 L
filter flask, using 1 1 cm paper and ca 5 g Super-Cel as filter
pad. Wash residue with 100 mL acetone, collecting washings
in same flask. Transfer filtrate to 1 L separator and add 500
mL benzene. (Caution: See safety note on benzene.)
Vigorously shake ext in separator and let stand until layers
sep. Swirl funnel and let stand again until layers sep. Drain
aq. layer into 250 mL centrf. bottle. Transfer org. layer to 1
L beaker. Rinse separator with 100 mL acetone-benzene soln
and add to centrf. bottle. Stopper, shake vigorously, and centrf.
20 min at ca 1700 rpm. Remove lower layer with suction tube
and transfer org. layer to 1 L beaker.
Evap. to 10 mL under heat lamp with air current. Add 100
mL CHC1 3 and evap. to 50 mL. If soln is not clear, repeat
addn and evapn of CHC1 3 to remove H 2 0.
Add clear CHC1 3 soln to alumina column and drain to level
of alumina. Wash with four 50 mL portions CHC1 3 . Discard
washings. Add 90 mL 80% alcohol to column to elute zoalene.
Discard first 30 mL effluent and collect 60 mL in 100 mL
beaker. Evap. soln under heat lamp with air current until res-
idue no longer flows. Do not heat residue after beaker is dry.
Add 5 mL ale. DMF soln to beaker and warm with stirring
to ca 45° to dissolve residue. When completely in soln, add 5
mL L3-diaminopropane to develop color. Filter thru small fluted
paper. After 10 min, measure A of soln at 560 nm, using 1
cm cells against H 2 as ref.
G. Calculations
Obtain |xg zoalene corresponding to A from std curve.
ppm Zoalene in sample - jxg zoalene/g sample
Refs.: J. Agric. Food Chem. 9, 201(1961). JAOAC 49, 708
(1966).
CAS- 148-0 1-6 (zoalene)
636
Common Names
AOAC Official Methods of Analysis (1990)
Common and Chemical Names of Drugs in this Chapter
Common Name
Chemical Name
Clopido!
Decoquinate
Ethoxyquin
Melengestrol (acetate)
Nalidixic acid
Sulfabromomethazine
Sulfadimethoxine
Sulfamethazine
Sulfapyridine
Sulfaquinoxaline
Sulfathiazole
Zoalene
3,5-Dichloro-2,6-dimethyl-4-pyridinol
6-(Decyloxy)-7-ethoxy-4-hydroxy-3<iuinolinecarboxylic acid ethyl ester
6-Ethoxy-1,2-dihydro-2,2,4-trimethyl-quinoline
1 7-Hydroxy-6-methyl-1 6-methylene-pregna-4,6-diene-3,20-dione
1 -Ethyl-1 ,4-dihydro-7-methyl-4-oxo-1 ,8-naphthyridine-3-carboxy!ic acid
4-Amino-W-(5-bromo-4,6-dimethyl-2-pyrimidinyl)-benzenesulfonamide
4-Amino-/V-(2,6-dimethoxy-4-pyrimidinyl)-benzenesulfonamide
4-Amino-A/-{4,6-dimethyl-2-pyrimidinyl)-benzenesulfonamide
4-Amino-N-2-pyridinyl-benzenesulfonamide
4-Amino-A/-2-quinoxalinyl-benzenesulfonamide
4-Amino-A/-2-thiazolyl-benzenesulfonamide
2-Methyl-3,5-dinitrobenzamide
Sources: USAN and the USP Dictionary of Drug Names (1983) U.S. Pharmacopeial Convention, Rockville, MD; The Merck index (1983) 10th ed., Merck &
Co., Inc., Rahway, NJ.
24. Forensic Sciences
Stanley M. Cichowicz, Associate Chapter Editor
Bureau of Engraving & Printing
974.35 Detection of Fingerprints
(Latent) on Objects
Powder Brushing Method
First Action 1974
Final Action 1975
(Applicable to development of latent images deposited on non-
porous surfaces within 120 hr and not subjected to extreme
temp, or humidity changes)
Pour small amt fingerprint powder (Hi-Fi Volcano, Sirchie
Fingerprint Laboratories, Inc., Gravelly Hollow Rd, Medford,
NJ 08055, or equiv.) into shallow dish or onto piece of paper.
Pick up small amt with end of camel hair brush. Hold brush
over surface to be dusted, and tap handle lightly to permit
powder to drift onto surface. Brush surface lightly until image
begins to appear. Continue with light strokes, following ridge
direction in pattern as it forms. Apply addnl powder, if nec-
essary, to obtain good contrast, but retain ridge detail. When
sufficient detail and contrast are obtained, remove excess pow-
der with ostrich feather duster. Preserve image by photograph-
ing and then taping over, leaving tape in place if practical. If
not, pull off tape slowly and evenly, and place on 3 X 5" card
of contrasting color.
Refs.: JAOAC 57, 662(1974); 58, 126(1975).
976.28 Detection of Fingerprints
(Latent) on Papers
Chemical Development Method
First Action 1976
(Applicable to forced development of latent images deposited
on bond and newsprint papers)
A. Apparatus and Reagent
(a) Steam iron.-— With heat indicator.
(b) Ninhydrin soln. — 0.5%. Dissolve 0.5 g 1,2,3-indan-
trione.H 2 in 100 m.L acetone, mix, and let stand 15 min.
B. Pretreatment
(If evidence contains handwriting, printing, or typewriting to
be examined by document examiner, protect area by brushing
pretreatment, allowing for migration of ninhydrin soln. In
general, spray large objects.)
Perform one of following pre treatments on papers:
(a) Dipping. — Pour small vol. ninhydrin soln into flat dish,
pick up paper with tweezers, submerge paper in soln until satd,
hold paper above dish, letting excess liq. drip into dish, and
place paper on clean blotter to air dry.
(b) Brushing. — With paint brush or cotton swab on wooden
stick, pick up small vol. ninhydrin soln from flat dish, paint
paper until surface is coated, transfer to clean blotter, and let
air dry.
(c) Spraying. — Pour ca 30 m'L ninhydrin soln into spraying
cannister. Use com. compressed air or inert gas, or com-
pressed air from laboratory line as propel 1 ant. Place papers in
637
exhaust hood or similar cabinet. Hold spraying cannister up-
right ca 8-10" (20-25 cm) from paper and spray until paper
is coated. Transfer paper to blotter and let air dry.
Discard ninhydrin soln in flat dish. Return ninhydrin spray-
ing soln to supply bottle for future use.
C. Development
Fill steam iron with H 2 and turn heat indicator to "Steam".
Place papers on clean blotter. When steam is being ejected
from sole plate holes, hold iron ca 1" (2.5 cm) above papers
and move iron around to distribute heat and steam evenly. Vary
ht above paper with rate steam is being projected beneath iron.
Steam should just reach papers before rising to sole plate. Wipe
sole plate frequently to prevent condensate from dripping on
paper. Images will begin to develop in ca 45 sec and continue
to intensify until plum or purple color is obtained. Move paper
to clean blotter and let air dry; as alternative, after steam de-
velopment of prints, shut off steam, wipe bottom of iron free
of moisture, and then lightly pass iron over developed prints
to enhance images. (Use caution not to burn or scorch papers.)
Continue with each paper until all have been treated. When
papers are dry, place each in appropriately labeled envelope
or plastic protector, handling with tweezers.
Ref.: JAOAC 59, 1003(1976).
973.65 Characterization and Matching
of Glass Fragments
Dispersion Microscopy (Double Variation Method)
First Action 1973
Final Action 1974
A. Principle
Refractive indices (n) of glass and stdzd liqs are matched at
different wavelengths by varying temp, of mixt. The n of glass
remains relatively const with temp, change; those of liqs de-
crease with increasing temp. Plot of wavelength, where n of
glass and liq. match, against temp, for specific stdzd liq., is
characteristic of particular glass.
B. Apparatus
(a) Microscope. — Compd, transmitted light type, with il-
lumination system capable of restricted substage aperture or,
preferably, phase contrast optics which permits easier match-
ing, with provision for long working distance.
(b) Hot stage. — Capable of attaining, holding, and indi-
cating temp, accurately (±0. l c ) and permitting use of required
illumination (Model FP-5 (replacement Model FP-82), Mettler
Instrument Corp., or equiv.).
(c) Monochromatic light source. — Accurately calibrated (± 1
nm at 486) with sufficient intensity in small beam of low an-
gular aperture over range at least 460-680 nm (continuous in-
terference filter, 400-700 nm, band width ca 15 nm, No. 50
09 00, Carl Zeiss West Germany, PO Box 1369/1380, D-
7082, Oberkochen, West Germany, is satisfactory).
(d) Graph paper. — Hartmann net (linear temp., nonlinear
wavelength resulting in straight line) preferable; available from
Walter C. McCrone Associates, Inc., 2820 S Michigan Ave,
Chicago, IL 60616. If unavailable, use ordinary graph paper.
638
Standard Solutions and Certified Reference Materials
AOAC Official Methods of Analysis (1990)
C. Reagents
(a) Refractive index liquids. — Calibrated immersion liqs,
range I.. 50- 1.65 (R. P. Cargille Laboratories, Inc., 55 Com-
merce Rd, Cedar Grove, NJ 07009), or N1ST SRM 1823 sil-
icone liqs. Use high dispersion set with most glasses.
(b) Collodion so In. — Dil. I mL 3% collodion, flexible
(Fisher Scientific Co., No. C-409), to 100 mL with amyl ace-
tate.
D. Preparation of Samples
(All particles examined must be ^100 |mm in major dimension.)
(a) Single small flake. — Arrange 3 small (2-3 mm) cover
slip flakes at corners of ca 1 cm triangle near end of 5 /s x 3"
microscope slide and cement in place by placing drop of dild
collodion soln outside of triangle and drawing soln to each
fragment in turn with fine tungsten needle. With very small
drop of dild collodion soln, cement test fragment on slide within
triangle, near 1 of fragments as locator. Avoid touching col-
lodion cementing cover slip fragments. Second fragment can
be cemented close by for simultaneous and direct comparison.
Excessive collodion interferes with index readings; if neces-
sary, thin collodion around test fragment with amyl acetate.
Place cover slip on the 3 supports, and place small drop of
liq. of known n at edge to be drawn around fragments by cap-
illary attraction. After n measurement, clean slide, if necessary
to change liq., by removing cover slip and rinsing tilted slide
held over waste containers with drops of benzene added from
dropper.
(b) Many small flakes. — Obtain many small flakes by
crushing in anvil-striker type hammer mill. Place few flakes
on slide without cementing, cover with cover slip, and add
small drop of liq. at edge of cover slip. New prepn may be
used for each liq. or slide may be cleaned by removing cover,
pushing particles to side with razor cut edge of filter paper,
adding drop of new liq., pushing again to side, and adding
drop of fresh liq.
E. Determination
Choose immersion liq. which matches n of glass at far red
end of spectrum at temp, slightly above room temp., ca 30°.
With such match, all subsequent temp. -wavelength matches
will be <60°, requiring small temp, corrections. Use narrow
beam of light parallel to optic axis passing thru closed down
substage aperture, but open enough to see Becke lines. (There
will be best iris setting for each wavelength.) Place mounted
sample on hot stage set at lowest even degree temp, at which
wavelength match is observed (ca 660 nm). Darkened room
and intense illumination are advantageous. Record av. of sev-
eral matching wavelengths for that temp. Set temp, at succes-
sively higher even degree intervals, let equilibrate 30 sec, and
read wavelength. Repeat match several times and det. av.
Continue increasing temp, until no match can be obtained (ca
450 nm). Let hot stage cool, and recheck 1-2 of lower temp.
matches. Difference indicates change in liq. at high temp, and
requires repeat of second half of data at higher temp, with
fresh liq. Rate should be 1 data point/min. Plot data on Hart-
man n net or ordinary graph paper.
F. Calculations
From graph, read temp, corresponding to 486.1 (F), 589.3
(D), and 656.3 (C) nm. From Table 973.65, obtain n of liq.
used at these wavelengths and temp, coefficient to be used in
correcting table values at 25° to actual matching temp. Report
matching temp, and corrected refractive index (n x ) for each of
the 3 wavelengths specified.
Table 973.65 Refractive Indices and Temperature Coefficients
of Cargille Liquids
fif
"0
dn/dt
(25-35°)
A Series
1 .46594
1.460
1.45762
0.00037
1 .47666
1.470
1 .46735
0.00037
1 .48739
1.480
1 .47709
0.00037
1.49812
1.490
1 .48682
0.00038
1 .50884
1.500
1 .49656
0.00038
1.51957
1.510
1 .50629
0.00038
1.53030
1.520
1.51603
0.00038
1.54103
1.530
1 .52576
0.00038
1.55175
1.540
1 .53550
0.00039
1.56248
1.550
1 .54523
0.00039
1.57321
1.560
1.55497
0.00039
1.58393
1.570
1 .56470
0.00039
1 .59466
1.580
1 .57444
0.00040
1 .60539
1.590
1.58417
0.00040
1.61611
1.600
1 .59391
0.00040
High Dispersion Series
1.5134
1.500
1.4943
0.00045
1 ,5247
1.510
1.5039
0.00046
1.5360
1.520
1.5134
0.00046
1.5451
1.530
1.5242
0.00047
1.5557
1.540
1 .5339
0.00047
1 .5664
1.550
1.5437
0.00047
1.5770
1.560
1.5534
0.00047
1 .5877
1.570
1.5632
0.00047
1 .5983
1.580
1.5729
0.00047
1.6090
1.590
1.5827
0.00047
1.6196
1.600
1.5924
0.00047
1 .6303
1.610
1.6021
0.00047
1.6410
1.620
1.6119
0.00047
1.6516
1.630
1.6216
0.00047
1 .6623
1.640
1.6314
0.00047
Reprinted with permission of Ann
Arbor Science
Publishers Inc.,
Ann
Arbor, Ml.
Example: Using
Cargille liq.
1 .520:
n at this
Matching
wavelength
Calcd n at
temp, from
(Table 973:65)
matching
Wavelength
graph
at 25°
temp.
486
42.8
1.53030
1 .52354
589
33.6
1.52000
1.51673
656
29.6
1.51603
1.51428
Calcd n is obtained by using dn/dt — 0.00038/° in equa-
tion:
n { = n 25 - (matching temp. - 25)(d«/dt)
n 4S6 = 1 .53030 - (42.8 - 25)(0. 00038)
= 1.53030 - 0.00676 - 1.52354
Repeat for 589 and 656 nm.
Refs.: JAOAC 56, 1223(1973); 57, 668(1974). McCrone, W.
C, and Delly, J. G., "The Particle Atlas/' 2nd ed.,
Vol. 4, Ann Arbor Science Publishers, PO Box 1425,
Ann Arbor, Ml 48106 (1973), Table: Optical Constants
for Cargille Refractive Index Liquids.
981.23
Mineral Wool Insulation
Comparison of Properties
First Action 1981
A. Apparatus and Reagents
(a) Microscopes. — (7) Phase contrast, with provision for
long working distances; (2) Low power inspection type.
AOAC Official Methods of Analysis (1990)
Glass Fragments
639
(b) Illuminators. — White and UV (253.7 nm) incident light
sources.
(c) Monochromator. — See 973.65B(c); or for transmitted
light illumination.
(d) Hot stage.— See 973.650(b).
(e) Graph paper. — See 973.65B(d)
(f) Annealing oven. — 650°. Controlled temp, muffle fur-
nace.
(g) Refractive index liquids. — See 973.65C(a).
B. Microscopic Examination
Compare known and unknown source materials under low
power microscope in incident light to det. color of resin, if
any, and disposition of resin on fibers (e.g. evenly coated, in
globs, etc). Compare diams of fibers, and relative abundance
of slugs and shot.
C. Annealing
Place known source and unknown source fibers and slugs
in porcelain crucibles with covers and heat to 650° (1200° F)
in controlled temp, muffle furnace. Hold at 650° >=10 min;
then lower temp, at rate of ca 28° (50° F)/30 min to ca 365°
(690° F) when rate of cooling becomes immaterial.
D. Comparison of Properties
(a) Fluorescence. — If enough material is available, com-
pare fluorescence of annealed fibers under UV light, (b). Note
color and brightness of fluorescence.
(b) Solubility in acid. — Place annealed fibers on glass slide
under low power microscope, add drop HC1 (1 + 3), and note
solubility. Repeat, if indicated, with coned HC1.
(c) Optical properties. — Place representative fibers and slugs
from annealed known and unknown source materials on mi-
croscope slides under cover slips. Introduce refractive index
liq. , (g), under cover slip. Insert on hot stage and adjust temp,
until fibers essentially disappear when viewed thru phase mi-
croscope and monochromator set near red end of visible spec-
trum (near 656 nm). Note temp, and monochromator setting.
Increase temp, in 5° (9° F) increments to find >3 match points
of glass and oil within 656-486 nm range. Plot results on graph
paper, (e), on which oil dispersion curves have been plotted
and calibrated. If variations along fibers or among fibers and
slugs have been observed, curve for material is best expressed
by band covering all match points observed.
Because of wide variation of n among mineral wool insu-
lations, it may be necessary to make several trials before ap-
propriate oil is found. If fibers are clearly visible in all wave-
lengths of light at temps between 35 and 90°, choose different
oil. Repeat trial-and-error procedure until oil is found in which
fibers match oil at 3 different wavelengths beween 656 and
486 nm.
Compare properties of known and unknown source mate-
rials.
Refs.: JAOAC 60, 772(1977); 62, 792(1979).
975.52 Voice Print Identification
Sound Spectrograph^ Method
First Action 1975
Final Action 1976
A. Principle
Voice print method of speaker identification consists of aural
and visual comparison of one or more known voices to un-
known or questioned voice. Aural examination dets if acoustic
properties of known and unknown voices exhibit preponder-
ance of similarities or differences. Visual analysis with speech
spectrograms compares spectrograph ic features of similar sounds
in both known and unknown voices.
0. Apparatus
(a) Sound spectrograph. — Model 4691 or 700 from Voice
Identification, Inc., PO Box 714, Somerville, NJ 08876, or
equiv.
(b) Tape recorders. — (1) Model 110 Sony cassette (ac ca-
pability, 115-120 v, 60 Hz; frequency response, 50-10,000
Hz), or equiv. (2) Multitrack, for aural evaluation (optional).
(c) Patch cord.
(d) Spectrograph paper.
C. Determination
Record unknown and known voices onto spectrograph. Use
patch cord to record speech samples from cassette recorder to
spectrograph. Adjust signal peaks to zero volume units (VU)
for proper record level, and monitor recording.
Listen to known and questioned voices until familiar with
context of each call and each speaker's voice. Set spectrograph
modes for normal (bar), wide-band, expanded linear frequency
scale with high shaping. Adjust scan playback level to zero
VU for production of each spectrogram. Label unknown spec-
trogram by call number. Label known spectrogram by name
given for each speaker. Properly label all speech sounds pro-
duced on each spectrogram.
Aurally compare questioned and known voices, using mul-
titrack recorder, or 2 recorders. Visually examine similar sounds
between unknown and known voices as displayed in speech
spectrograms. Conduct aural and visual examinations simul-
taneously in arriving at conclusion, without limitations on time
or restrictions on number of speech samples necessary.
Make 1 of 5 alternative judgments: (1) positive identifica-
tion; (2) positive elimination; (3) probable identification; (4)
probable elimination; (5) unable to arrive at conclusion. Base
all positive judgments on >10 pairs of like sounds in known
and unknown voices.
Refs.: JAOAC 58, 453(1975); 59, 927(1976).
appendix: Standard Solutions and Certified Reference
Materials
Robert Alvarez, Associate Chapter Editor
National Institute of Standards and Technology
942.25
Standard Solutions
and Materials
Use accurately calibrated equipment, which meets NIST
specifications. Because alk. and other corrosive solns dissolve
glass, to avoid vol. errors do not store such solns in calibrated
app. Burets used continuously with such solns should be re-
calibrated periodically.
Working temp, of std soln should approximate that of its
temp, during stdzn. If temp, corrections are necessary, suffi-
cient accuracy may be obtained by use of Table 942.25.
Ref.: JAOAC 25, 650(1942).
942.26 Standard Solutions
of Ammonium and Potassium Ttiiocyanates
Final Action
A. Reagents
(a) Purified silver nitrate. — Dissolve 50 g AgN0 3 in 20 mL
boiling FLO contg ca 5 drops HN0 3 . Heat to dissolve, filter
while still hot thru fritted glass filter, using suction, and collect
filtrate in clean Pyrex beaker. Wash beaker and filter with ca
5 mL hot H 2 0, adding washings to filtrate. Cool in ice bath,
stirring to induce crystn, and place in refrigerator at ca 10°
until equilibrium is reached. Decant liq. thru fritted glass filter
and transfer crystals to filter. Cover filter with watch glass and
draw air thru filter to remove adhering liq. Transfer crystals
to small, clean Pyrex beaker. Cover beaker with watch glass
and place inside larger covered Pyrex beaker. Dry at 105° and
fuse at 220-250° (mp 208°), holding at this temp, ca 15 min
after crystals are melted. Protect from dust during prepn. Cool
in desiccator, remove product from beaker, powder in mortar,
dry 0.5 hr at 105°, and store in brown g-s bottle in dark over
good desiccant
(b) Reference soln.— To mixt. of 5 mL HNQ 3 (1 + 1), 2
mL Fe alum soln, 941.18D (a), and 1 15 mL H 2 0, add ca 0.02
mL 0..W thiocyanate, 942. 26B, noting exact vol. used.
B. Preparation of Standard Solution
Prep, ca 0.1 JV soln from reagent that shows no CI, using
7.612 g NH 4 SCN or 9.718 g KSCN/L.
C. Standardization
Accurately weigh, on tared watch glass, enough purified
AgN0 3 to give titrn of ca 40 mL (ca 0.7 g for 0. IN soln) and
transfer with H 2 thru glass funnel to 250 mL g-s erlenmeyer.
Dissolve in ca 75 mL H 2 (halogen-free), and add 5 mL HN0 3
(1 + 1) and 2 mL Fe alum soln, 941.18D(a). Titr. with thi-
ocyanate soln until titrd soln is reddish brown, which remains
after shaking vigorously J min. Record buret reading and set
flask aside 5 min, shaking occasionally and maintaining end
point color by addn of thiocyanate soln as required. Then add
addnl thiocyanate soln, if necessary, to produce permanent end
point color, matching with color of ref. soln, 942.26A(b). From
total vol. thiocyanate soln used in titrn, subtract vol. contained
in ref. soln.
Normality - g AgN0 3 X 1000/mL titer x 169.87
Refs.: JAOAC 25, 661(1942); 30, 105, 496(1947).
939.12
Standard Solution
of Arsenious Oxide
Final Action
A. Reagent
Arsenious oxide.— Use NIST SRM 83. Dry 1 hr at 105°
immediately before using.
B. Preparation of Standard Solution
Accurately weigh As 2 3 by difference from small g-s
weighing bottle (use ca 4.95 g/L for 0. IN). Dissolve in IN
NaOH (50 mL/5 g As 2 3 ) in flask or beaker by heating on
steam bath. Add ca same vol. IN H 2 S0 4 . Cool, quant, transfer
mixt. to vol. flask, and dil. to vol. (Soln must be neut. to
litmus, not alk.)
Normality = g As 2 3 X 4000/mL final vol. X 197.84
Refs.: JAOAC 22, 568(1939); 24, 100, 639(1941).
964.24 Buffer Solutions
for Calibration of pH Equipment
First Action 1964
Final Action 1965
Use H 2 with pH of >6.5 but <7.5, obtained by boiling
H 2 15 min and cooling under C0 2 -free conditions. Store std
buffer solns except Ca(OH) 2 in bottles of chem. resistant glass.
Protect phosphate, borax, and Ca(OH) 2 buffers from C0 2 . pH
values as function of temp, are given in Table 964.24.
(a) Potassium tetroxalate buffer soln. — 0.0496M; 0.05m.
Transfer 12.61 g KHC 2 4 .H 2 C 2 O d .2H 2 (air wt) (NIST SRM
189) to 1 L vol. flask, dil. to vol. with H 2 0, and mix thoroly.
(It is not necessary to remove dissolved C0 2 from the H 2 or
to dry salt before weighing.) Prep, fresh every 2 months.
(b) Potassium hydrogen tartrate buffer soln. — Satd soln at
25°, 0.034A/. Add excess (ca 100%) of KHC 4 H 4 6 (NIST SRM
188) to H 2 in g-s bottle or flask, and shake vigorously; few
min shaking is enough for satn (100 mL H 2 at 25° dissolves
ca 0.7 g KHC 4 H 4 6 ). Adjust to 25°, let solid settle, and decant
clear soln, or filter if necessary. Discard when mold appears.
Few crystals of thymol added during prepn will retard mold
growth, and will alter pH by <0.01 unit. For accuracy of ±0.01
pH unit, temp, of soln at satn must be between 20 and 30°.
(c) Acid potassium phthalate buffer soln. — 0.0496M; 0.05m.
Dissolve 10.12 g dried (2 hr at 1 10°) KHC 8 H 4 4 (NIST SRM
185) in H 2 and dil. to 1 L. (Elaborate precautions for exclu-
sion of atm. C0 2 are unnecessary, altho soln should be pro-
640
AOAC Official Methods of Analysis (1990)
Standard Solutions
641
Table 942.25 Temperature Corrections for Volume of Aqueous Solutions
Correction in Milliliters at —
Vol. Std Soln
6°
8°
10°
12°
14°
16°
18°
20°
22°
24°
26°
28°
30°
mL
10
0.01
0.01
0.01
0.01
0.01
0.01
0.00
0.00
0.00
0.00
-0.01
-0.02
-0.02
20
0.03
0.03
0.03
0.02
0.02
0.01
0.01
0.00
-0.01
-0.02
-0.03
-0.03
-0.03
25
0.04
0.03
0.03
0.03
0.02
0.02
0.01
0.00
-0.01
-0.02
-0.03
-0.04
-0.05
30
0.04
0.04
0.04
0.03
0.03
0.02
0.01
0.00
-0.01
-0.02
-0.04
-0.05
-0.07
40
0.06
0.06
0.05
0.04
0.04
0.03
0.01
0.00
-0.02
-0.03
-0.05
-0.07
-0.09
50
0.07
0.07
0.06
0.06
0.05
0.03
0.02
0.00
-0.02
-0.04
-0.06
-0.09
-0.12
tected against evapn and contamination with molds. Replace
soln if mold appears.)
(d) Phosphate buffer soln. — 0.0249M; 0.025m. Dissolve
3.387 g KH 2 P0 4 and 3.533 g Na 2 HP0 4 (NIST SRM 186-1 and
II) in H 2 and dil. to I L. (Dry salts 2 hr at 1 10-130° before
use.)
(e) Phosphate buffer soln.— 0.008663M, 0.008695m KH,PQ 4
and 0.03030M, 0.03043m Na 2 HP0 4 . Dissolve 1.179 g KH 2 P0 4
and 4.303 g Na 2 HP0 4 (NIST SRM 186-1 and II) in H 2 and
dil. to 1 L. (Dry salts 2 hr at 110-130° before use.)
(f) Borax buffer soln.— 0.00996M; 0.0 Lm. Dissolve 3.80
g Na 2 B 4 O 7 .10H 2 O (NIST SRM 187) in H 2 and dil. to 1 L.
(Salt must not be dried in oven before use.) To avoid contam-
ination with C0 2 , stopper bottle except when in use or protect
with soda-lime tube. Use buffer soln within 10 min after re-
moval from bottle.
(g) Sodium bicarbonate-carbonate buffer soln. — 0.0249M;
0.025m (each). Transfer 2.092 g NaHC0 3 (NIST SRM 191;
do not heat) and 2.640 g Na 2 C0 3 (NIST SRM 192; dry 2 hr
at 275°) to I L vol. flask. Dissolve and dil. to vol. with C0 2 -
free H 2 0.
(h) Calcium hydroxide buffer soln. — Satd soln at 25°,
0.02025M. Slowly heat finely granular CaC0 3 , low in alka-
lies, to 1000° in Pt dish and maintain at this temp. 45-60 min.
Cool in desiccator, and add to H 2 with stirring. Heat to bp
with continuous stirring. Cool, and filter on medium fritted
glass filter. Dry at 1 10°, cool, and crush to fine, granular pow-
der.
Place crushed CaO in polyethylene bottle, add H 2 0, shake
vigorously, let settle, and record temp. (Keep large excess of
Ca(OH) 2 in bottle.) For use, filter soln thru medium fritted
glass filter. Use at same temp, at which satn took place, and
discard filtered soln if it becomes turbid. When more buffer
soln is needed, add addnl H 2 to suspension, re-sat., and filter
as above.
Refs.: NIST Certificates for Standard Reference Materials 185e,
186c, 187b, 188, 189, 191, and 192. JAOAC 33,
223(1950); 41, 302(1958); 47, 43(1964).
941.17 Standard Buffers and Indicators
for CoSorimetric pH Comparisons
Final Action
A. Preparation of SuifonphthaJein Indicators
X pH
Bromocresol green 14.3 3.8-5.4
Chlorophenol red 23.6 4.8-6.4
Bromothymol blue 16.0 6.0-7.6
Phenol red 28.2 6.8-8.4
X = mL O.OliV NaOH/0. 1 g indicator required to form mono-
Na salt. Dil. to 250 mL for 0.04% reagent.
&. Preparation of Stock Solutions
Use recently boiled and cooled J-) 2 0.
(a) Acid potassium phtha late soln. — 0.2M. Dry to const wt
at 110-115°. Dissolve 40.836 g in H 2 and dil. to J L.
(b) M onop ota s slum phosphate soln. — 0.2M. Dry KH 2 P0 4
to const wt at 110-115°. Dissolve 27.232 g in H 2 and dil.
to 1 L. Soln should be distinctly red with Me red, and dis-
tinctly blue with bromophenol blue.
(c) Boric acid-potassium chloride soln.—0.2M. Dry H 3 B0 3
to const wt in desiccator over CaCl 2 . Dry KC1 2 days in oven
at 115-120°. Dissolve 12.405 g H,BO, and 14.912 g KC1 in
H 2 0, and dil. to 1 L.
(d) Sodium, hydroxide std soln. — 0.2M. Prep, and stdze as
in 936.16; 0.04084 g KHC 8 H 4 4 = 1 mL 0.2M NaOH. It is
preferable to use factor with soln rather than try to adjust to
exactly 0.2M.
Table 964.24 pH Values for Standard Buffer Solutions as Function of Temperature
0.05m
Satd Potassium
0.05m Acid
0.008695m and
0.025m NaHC0 3
Potassium
Hydrogen
Potassium
0.025m
0.03043m
and 0.025m
Satd Calcium
Temperature
Tetroxaiate
Tartrate
Phthalate
Phosphate
Phosphate
0.01m Borax
Na 2 C0 3
Hydroxide
°C
PH
pH
PH
PH
PH
pH
PH
PH
1.666
4.003
6.982
7.534
9.460
10.321
13.423
5
1.668
—
3.998
6.949
7.501
9.392
10.248
13.207
10
1.670
_
3.996
6.921
7.472
9.331
10.181
13.003
15
1.672
—
3.996
6.898
7.449
9.276
10.120
12.810
20
1.675
—
3.999
6.878
7.430
9.227
10.064
12.627
25
1.679
3.557
4.004
6.863
7.415
9.183
10.014
12.454
30
1.683
3.552
4.011
6.851
7.403
9.143
9.968
12.289
35
1.688
3.549
4.020
6.842
7.394
9.107
9.928
12.133
37
1.691
3.548
4.024
6.839
7.392
9.093
-
12.043
40
1.694
3.547
4.030
6.836
7.388
9.074
9.891
1 1 .984
45
1.700
3.547
4.042
6.832
7.385
9.044
9.859
11.841
50
1.707
3.549
4.055
6.831
7.384
9.017
9.831
1 1 .705
55
1.715
3.554
4.070
-
„
-
-
11.574
60
1.723
3.560
4.085
-
~
-
-
1 1 .449
642
Standard Solutions and Certified Reference Materials
AOAC Official Methods of Analysis (1990)
C, Preparation of Buffer Solutions
Prep, std buffer solns from designated amts stock solns,
941.17, and dil. to 200 mL. For use as colori metric std, mix
20 mL buffer soln with 0.5 mL indicator soln, 941. 17 A.
Phthalate-NaOH Mixtures
0.2M
02M
pH
KH Phthalate (mL)
NaOH (mL)
5.0
50
23.65
5.2
50
29.75
5.4
50
35.25
5.6
50
39.70
5.8
50
43.10
6.0
50
45.40
6.2
50
KH 2 P0 4 ~NaOH Mixtures
47.00
0.2M
0.2M
pH
KH 2 PQ 4 (mL)
NaOH (mL)
5.8
50
3.66
6.0
50
5.64
6.2
50
8.55
6.4
50
12.60
6.6
50
17.74
6.8
50
23.60
7.0
50
29.54
7.2
50
34.90
7.4
50
39.34
7.6
50
42.74
7.8
50
45.17
8.0
50
H,BO r KCl-NaOH Mixtures
46.85
0.2M
0.2M
pH
H3BO3, KCI (mL)
NaOH (mL)
7.8
50
2.65
8.0
50
4.00
8.2
50
5.90
8.4
50
8.55
8.6
50
12.00
Refs.: JAOAC 24, 583(1941). Clark, "Determination of Hy-
drogen-ions/ 1 3rd Ed., pp. 91, 94, 192-202.
936.15
Standard Solution
of Hydrochloric Acid
Final Action
A. Preparation of Standard Solutions
Following table gives approx. vols of 36.5-38% HCl re-
quired to make 10 L std solns:
Approx. normality
mL HCl to be dild to 10 L
0.01
8.6
0.02
17.2
0.10
86.0
0.50
430.1
1.0
860.1
B. Standard Sodium Hydroxide Method
Titr. 40 mL against std alkali soln, 936.16C-E, of ca same
concn as acid being stdzd in 300 mL flask that has been swept
free from C0 2 , using C0 2 -free H 2 and 3 drops phthln.
Normality - (mL std alkali X normality of alkali)/mL HCl
If more coned than desired, dil. soln to required normality
value by following formula:
V } = V 2 X N 2 /N f
where N 2 and V 2 represent normality and vol. of stock soln,
resp., and V t = vol. to which stock soln should be dild to
obtain desired normality, /V,.
Check exact concn of final soln by titrn as above. Normality
will be exact only if same indicator is used in detn as in stdzn.
Restdze if indicators other than phthln are used.
Refs.: JAOAC 19, 107, 194(1936). 49, 250(1966). Kolthoff
& Stenger, "Volumetric Analysis," II, 52(1947).
C. Constant Boiling Method
Dil. 822 mL HCl (36.5-38% HCl) with 750 mL H 2 0. Check
sp gr with spindle and adjust to 1.10. Place 1 .5 L in 2 L flat-
bottom distg flask, add ca 10 SiC grains (ca "20 mesh"), and
connect to long, straight inner- tube condenser. Heat on elec.
hot plate and distil at 5-10 mL/min, keeping end of condenser
open to air. When 1125 mL has distd, change receivers and
catch next 225 mL, which is const boiling HCl, in erlenmeyer
with end of condenser inserted into flask, but above surface
of liq. Read barometer to nearest mm at beginning and end of
collection of 225 mL portion and note barometer temp. Av-
erage readings.
Calc. air wt in g (G) of this const boiling HCl required to
give one equiv. wt of HCl from one of following equations:
For P = 540-669 mm Hg:
G - 162.255 + 0.02415 P
For P = 670-780 mm Hg:
G = 164.673 + 0.02039 P
where P = barometric pressure in mm Hg corrected to 0°C
for expansion of Hg and of barometer scale. For brass scale
barometer, following correction is accurate enough:
P = P t (l - 0.000162/),
where t = barometer temp, in °C
Weigh required amt of const boiling HCl in tared, stoppered
flask to at least 1 part in 10,000. Dil. immediately, and finally
dil. to vol. with C0 2 -free H 2 at desired temp.
Refs.: JAOAC 25, 653(1942); 36, 96, 354(1953); 37, 122,
462(1954).
Standard Borax Method
D. Reagents
(a) Methyl red indicator. — Dissolve 100 mg Me red in 60
mL alcohol and dil. with H 2 to 100 mL.
(b) Reference soln. — Prep. ref. soln of H3BO3, NaCl, and
indicator corresponding to composition and vol. of soln at
equivalence point. For use in detn of end point of titrn with
O.IN HCl, ref. soln should be 0.1M in H3BO3 and 0.05M in
NaCJ.
(c) Std borax.— Sat. 300 mL H 2 at 55° (not higher) with
Na 2 B 4 O 7 .10H 2 O (ACS) (ca 45 g). Filter at this temp, thru folded
paper into 500 mL erlenmeyer. Cool filtrate to ca 10°, with
continuous agitation during crystn. Decant supernate, rinse ppt
once with 25 mL cold H 2 0, and dissolve crystals in just enough
H 2 at 55° to ensure complete soln (ca 200 mL). Recrystallize
by cooling to ca 10°, agitating flask during crystn.
Filter crystals onto small buchner with suction, wash ppt
once with 25 mL ice-cold H 2 0, and dry crystals by washing
with two 20 mL portions alcohol, drying after each washing
with suction. Follow with two 20 mL portions ether. (Just be-
fore use, free alcohol and ether from any possible reacting acids
by vigorously shaking each with 2-3 g of the pure, dry
Na2B4O 7 .10H 2 O and then filtering.) Spread crystals on watch
glass, immediately place dried Na2B 4 O7.10H 2 O in closed con-
AOAC Official Methods of Analysis (1990)
Standard Solutions
643
tainer over soln satd with respect to both sucrose and NaCI,
and let it remain ^24 hr before using. Then transfer the pure
Na 2 B 4 7 . 10H 2 O to g-s container and store in closed container
over soln satd with respect to both sucrose and NaCl (stable
under these conditions 1 year).
E. Standardization
Accurately weigh enough std Na 2 B 4 O 7 .10H 2 O to titr. ca 40
mL and transfer to 300 mL flask. Add 40 mL C0 2 -free H 2 0,
936.16B(a), and stopper flask. Swirl gently until sample dis-
solves. Add 4 drops Me red and titr. with soln that is being
stdzd to equivalence point as indicated by ref. soln.
Normality - g Na 2 B 4 7 . 10H 2 O x 1000/mL acid x 190.69
Ref.: JAOAC 22, 102, 563(1939).
Standard Sodium Carbonate Method
F. Reagents
(a) Methyl orange indicator. — 0.1% in H 2 0.
(b) Reference soln. — 80 mL C0 2 -free H 2 contg 3 or 4 drops
Me orange.
(c) Anhydrous sodium carbonate. — Heat 250 mL H 2 to
80° and add NaHC0 3 (ACS), stirring until no more dissolves.
Filter soln thru folded paper (use of hot H 2 funnel is desir-
able) into erlenmeyer. Cool filtrate to ca 10°, swirling con-
stantly during crystn. Fine crystals of trona that sep. out have
approx. composition: Na2CO3.NaHCO3.2H2O. Decant super-
nate, drain crystals by suction, and wash once with cold H 2 0.
Transfer ppt, being careful not to include any paper fibers,
to large flat-bottom Pt dish. Heat 1 hr at 290° in elec. oven or
furnace with pyrometer control. Stir contents occasionally with
Pt wire. Cool in desiccator. Place the anhyd. Na 2 C0 3 in g-s
container and store in desiccator contg efficient desiccant. Dry
at 120° and cool just before weighing.
Refs.: Kolthoff & Stenger, "Volumetric Analysis," II,
80(1947). Ind. Eng. Chem., Anal. Ed. 9, 141(1937).
JAOAC 22, 563(1939).
G. Standardization
Accurately weigh enough anhyd. Na 2 C0 3 , (c), to titr. ca 40
mL, transfer to 300 mL erlenmeyer, and dissolve in 40 mL
H 2 0. Add 3 drops Me orange and titr. until color begins to
deviate from H 2 tint (ref. soln). (Equivalence point has not
been reached.) Boil soln gently 2 min, and cool. Titr. until
color is barely different from H 2 tint of indicator.
Normality = g Na 2 C0 3 x 1000/mL acid x 52.994
Ref.: JAOAC 22, 102, 563(1939).
939.13 Standard Solution of Eodine
Final Action
A. Preparation of Standard Soiution
Dissolve weighed amts of I (12.7 g/L for 0.17V soln) and
KI, in proportion of 20 g KI to 13 g I, in 50 mL H 2 0. When
I dissolves, transfer soln to g-s vol. flask. Dil. to vol. with
H 2 and mix thoroly. Store in dark brown, g-s bottle away
from light and restdze as frequently as necessary.
B. Standardization
Transfer accurately measured portion of std As 2 3 soln,
939.12B (40-50 mL ca 0.1/V soln for 0AN soln), to erlen-
meyer. Acidify slightly with H 2 S0 4 (1 + 10), neutze with solid
NaHC0 3 , and add ca 2 g excess. Titr. with I soln, using ca
0.2% starch soln (5 mL/100 mL) as indicator. Sat. soln with
C0 2 at end of titrn by adding 1 mL H 2 S0 4 (1 + 10) just before
end point is reached.
Normality = mL As 2 3 x normality As 2 3 /mL I
Refs.: JAOAC 22, 568(1939); 24, 100, 639(1941).
947.13 Standard Solution
of Potassium Bromide-Bromate
Final Action
A. Preparation of Standard Solution
Dissolve ca 2.8 g K'Br0 3 and 12 g KBr in boiled H 2 and
dil. to 1 L with boiled H 2 for ca 0.1JV soln.
B. Standardization
Measure 40 mL std As 2 3 soln, 939. 12B, from buret into
300 mL erlenmeyer. Add 10 mL HCI and 3 drops Me orange,
936.15F(a). Titr. with KBr-KBr0 3 soln until <1 drop causes
color of Me orange to fade completely. Swirl soln constantly
and add last mL dropwise, swirling between drops.
Normality = mL As 2 3 x normality As 2 3 /mL KBr-KBr0 3
Refs.: JAOAC 30, 502(1947); 31, 1 19, 572(1948).
949.13 Standard Solution
of Potassium Dichromate
Final Action
A. Reagent
Starch soln.— Max ca 1 g arrowroot starch with 10 mL H 2
and pour slowly, with const stirring, into 200 mL boiling H 2 0.
Boil until thin, translucent fluid is obtained. Let settle and use
clear supernate. Preserve with Hg.
B. Assay of Stock Potassium Dichromate
If K 2 Cr 2 7 is in small crystals, mix by shaking thoroly in
large, clean jar; if it is in lumps, grind representative sample
to pass No. 60 sieve, and then mix by shaking. Dry portion
for weighings 2 hr at 100°.
Weigh, into each of 3 g-s erlenmeyers, enough K 2 Cr 2 7 (NIST
SRM 136) to give titer of 100.5-102.0 mL 0.UV Na 2 S 2 3 ,
942.27A (0.4928-0.5001 g for 0AN soln). Completely dis-
solve in 100 mL H 2 0, add 4.0 g KI, and swirl mixt. until
dissolved. With buret, add 4.0 mL HCI, stopper flask, mix by
swirling, and let stand in dark 10 min. Cool flask ca 1 min in
ice-H 2 0.
While swirling flask, pipet in 100 mL Na 2 S 2 3 soln. Add 5
mL starch soln and complete titrn with Na 2 S 2 3 soln added
from 10 mL microburet (graduated in 0.05 mL). End point is
from bluish green to clear green; change takes place within
0.01 mL. Record vol. to nearest 0.01 mL. Calc. apparent nor-
mality of Na 2 S 2 3 soln for each of the 3 titrns, and average.
Designate this av. as N mST .
Similarly titr. 3 portions of stock K 2 Cr 2 7 and calc. the 3
apparent normalities. Designate each of these results as Af stock .
Calc. % purity of stock K 2 Cr 2 7 = (N NlST X 100)/A/ slock .
Take av. of the 3 results as % purity of stock K 2 Cr 2 7 .
C. Preparation of Standard Solution
Dissolve theoretical wt K 2 Cr 2 O v (NIST SRM 136) (4.9032
g for 0. IN soln), or wt stock K 2 Cr 2 7 , 949.13B, found to have
oxidimetric value 99.95-100.05% of NIST SRM, in enough
H 2 to make I L. (Dry K 2 Cr 2 7 2 hr at 100° before using.)
Refs.: JAOAC 32, 587(1949); 33, 225(1950).
644
Standard Solutions and Certified Reference Materials
AOAC Official Methods of Analysis (1990)
940.35 Standard Solution
of Potassium Permanganate
Final Action
A. Preparation of Standard Solution
Dissolve slightly more than desired equiv. wt (3.2 g for 0. IN)
of KMn0 4 in 1 L H 2 0. Boil soln 1 hr. Protect from dust and
let stand overnight. Thoroly clean 15 cm glass funnel, per-
forated porcelain plate from Caldwell crucible, and g-s bottle
(preferably of brown glass) with warm chromic acid cleaning
soln. Digest asbestos for use in gooches on steam bath 1 hr
with ca 0.1 Af KMn0 4 that has been acidified with few drops
H 2 S0 4 (1 + 3). Let settle, decant, and replace with H 2 0. To
prep, glass funnel, place porcelain plate in apex, make pad of
asbestos ca 3 mm thick on plate, and wash acid -free. (Pad
should not be too tightly packed and only moderate suction
should be applied.) Insert stem of funnel into neck of bottle
and filter KMn0 4 soln directly into bottle without aid of suc-
tion.
B. Standardization
For OAN soln, transfer 0.3 g dried (1 hr at 105°) N a oxalate
(NIST SRM 40) to 600 ml beaker. Add 250 mL H 2 S0 4 (5 +
95), previously boiled 10-15 min and then cooled to 27 ± 3°.
Stir until Na 2 C 2 4 dissolves. Add 39-40 mL KMn0 4 soln
at rate of 25-35 mL/min, stirring slowly. Let stand until pink
disappears (ca 45 sec). If pink persists because KMn0 4 soln
is too coned, discard and begin again, adding few mL less of
KMn0 4 soln. Heat to 55-60°, and complete titrn by adding
KMn0 4 soln until faint pink persists 30 sec. Add last 0.5-1
mL dropwise, letting each drop decolorize before adding next.
Det. excess of KMn0 4 soln required to turn soln pink by
matching with color obtained by adding KMn0 4 soln to same
vol. of boiled and cooled dil. H 2 S0 4 at 55-60°. This correction
is usually 0.03-0.05 mL. From net vol. KMn0 4 , calc. nor-
mality:
Normality - g Na 2 C 2 4 x 1000/mL KMn0 4 x 66.999
Refs.: JAOAC 23, 543(1940); 31, 568(1948). J. Research NBS
15, 493(1935), Research Paper No. 843.
941.18
Standard Solution
of Silver Nitrate
Final Action
A. Preparation of Standard Solution
Dissolve slightly more than theoretical wt of AgN0 3 (equiv.
wt, 169.87) in halogen-free H 2 and dil. to vol. Thoroly clean
glassware, avoid contact with dust, and keep prepd soln in
amber g-s bottles away from light.
Mohr Method
B. Reagents
(a) Potassium chloride. — Recrystallize KC1 3 times from
H 2 0, dry at 1 10°, and then heat at ca 500° to const wt. Equiv.
wt KC1 = 74.555. Or, preferably, use NIST SRM 999.
(b) Potassium chromate soln. — 5% soln of K 2 Cr0 4 in H 2 0.
C. Standardization
Accurately weigh enough KC1 to yield titrn of ca 40 mL (ca
0.3 g for OAN soln), and transfer to 250 mL g-s erlenmeyer
with 40 mL H 2 0. Add 1 mL K 2 Cr0 4 soln and titr. with AgN0 3
soln until first perceptible pale red -brown appears. From titrn
vol., subtract mL of the AgN0 3 soln required to produce end
point color in 75 mL H 2 contg 1 mL K 2 Cr0 4 soln. From net
vol. AgN0 3 , calc. normality:
Normality = g KC1 x 1000/mL AgN0 3 x 74.555
Volhard Method
D. Reagents
(a) Ferric alum indicator soln. — Satd soln of
FeNH 4 (S0 4 )2.12H 2 in H 2 0.
(b) Potassium or ammonium thiocyanate std soln. — Prep.
ca OAN soln, 942. 26B. Det. working titer by accurately mea-
suring 40-50 mL std AgN0 3 soln, adding 2 mL Fe alum soln
and 5 mL HN0 3 (1 + 1), and titrg with the thiocyanate soln
until soln appears pale rose after vigorous shaking.
E. Standardization
Accurately weigh enough KC1, 941.18B(a), to yield titrn of
ca 40 mL (ca 0.3 g for 0.1/V soln) and transfer to 250 mL
g-s erlenmeyer with 40 mL H 2 0. Add 5 mL HN0 3 (1 + 1)
and excess AgN0 3 soln. Mix, and let stand few min protected
from light. Filter thru gooch prepd with medium pad of as-
bestos previously rinsed with 2% HN0 3 . Wash flask and ppt
with several small portions of 2% HN0 3 , passing washings
thru crucible until filtrate and washings measure ca 150 mL.
Add 2 mL Fe alum soln and titr. residual AgN0 3 with thio-
cyanate soln. From titrn, together with ratio of the 2 solns,
calc. net vol. AgN0 3 soln. (Errors of blank are compensating
and may be disregarded.) From net vol. AgN0 3 , calc. nor-
mality as in 941. 18C.
Ref.: JAOAC 24, 100, 631(1941).
936.16 Standard Solution
of Sodium Hydroxide
Final Action
Standard Potassium Hydrogen Phthalate Method
A. Apparatus
Use buret and pipet calibrated by NIST or by analyst. Pro-
tect exits to air of automatic burets from C0 2 contamination
by suitable guard tubes contg soda-lime. Use containers of al-
kali-resistant glass,
B. Reagents
(a) Carbon dioxide-free water. — Prep, by one of following
methods: (J) Boil H 2 20 min and cool with soda-lime pro-
tection; (2) bubble air, freed from C0 2 by passing thru tower
of soda-lime, thru H 2 12 hr.
(b) Sodium hydroxide soln. — (1 + 1). To 1 part NaOH (re-
agent quality contg <5% Na 2 C0 3 ) in flask add 1 part H 2 and
swirl until soln is complete. Close with rubber stopper. Set
aside until Na 2 C0 3 has settled, leaving perfectly clear liq. (ca
10 days).
(c) Acid potassium phthalate. — NIST SRM for Acidimetry
84. Crush to pass No. 100 sieve. Dry 2 hr at 120°. Cool in
desiccator contg H 2 S0 4 .
C. Preparation of Standard Solution
Following table gives approx. vols of NaOH soln (1 + 1)
to make 10 L of std solns:
Approx.
mL NaOH to I
normality
dild to 10 L
0.01
5.4
0.02
10.8
0.10
54.0
0.50
270.0
1.0
540.0
AOAC Official Methods of Analysis (1990)
Sodium Hydroxide
645
Add required vol. of NaOH soln (1 . + 1) to 10 L C0 2 -free
H 2 0. Check normality, which should be slightly high, as in
936. 16D, and adjust to desired concn by following formula:
V } - V 2 x N 2 /N t
where N 2 and V 2 represent normality and vol. stock soln, resp.,
and V], vol. to which stock soln should be dild to obtain de-
sired normality, N { . Stdze final soln as in 936. 16D or E.
D. Standardization
Accurately weigh enough dried KHC 8 H 4 4 to titr. ca 40 mL
and transfer to 300 mL flask that has been swept free from
C0 2 . Add 50 mL cool C0 2 -free H 2 0. Stopper flask and swirl
gently until sample dissolves. Titr. to pH 8.6 with soln being
stdzd, taking precautions to exclude C0 2 and using as indi-
cator either glass-electrode pH meter or 3 drops phthln. In lat-
ter case, det. end point by comparison with pH 8.6 buffer soln,
941. 17C, contg 3 drops phthln. Det. vol. NaOH required to
produce end point of blank by matching color in another flask
contg 3 drops phthln and same vol. C0 2 -free H 2 0. Subtract
vol. required from that used in first titrn and calc. normality.
Normality - g KHQH 4 4 x 1000/mL NaOH x 204.229
Refs.: JAOAC 19, 107, 194(1936). NIST Certificate for Stan-
dard Reference Material 84.
Constant Boiling Hydrochloric Acid Method
E. Standardization
Accurately weigh from weighing buret enough const boiling
HC3, 936. 15C, to titr. ca 40 mL, into erlenmeyer previously
swept free from C0 2 . Add ca 40 mL C0 2 -free H 2 0, then
3-5 drops desired indicator, and titr. with soln being stdzd.
Normality = g HC1 X 1000/mL titer x G
where G has value given in 936. 15C.
Refs.: JAOAC 25, 653(1942); 36, 96, 354(1953); 37, 122,462
(1954).
890.01
Standard Solutions
of Sulfuric Acid
Final Action
942.27
Standard Solutions
of Sodium Thiosulfate
Final Action
A. Preparation of Standard Solution
Dissolve ca 25 g Na 2 S 2 3 .5H 2 in 1 L H 2 0. Boil gently 5
min and transfer while hot to storage bottle previously cleaned
with hot chromic acid cleaning soln and rinsed with warm boiled
H 2 0. (Temper bottle, if not heat-resistant, before adding hot
soln.) Store soln in dark, cool place; do not return unused por-
tions to stock bottle. If solns less coned than 0. IN are desired,
prep, by diln with boiled H 2 0. (More dil. solns are less stable
and should be prepd just before use.)
B. Standardization
Accurately weigh 0.20-0.23 g K 2 Cr 2 7 (NIST SRM 136
dried 2 hr at 100°) and place in g-s I flask (or g-s II ask). Dis-
solve in 80 mL Cl-free H 2 contg 2 g Kl. Add, with swirling,
20 mL ca IN HO and immediately place in dark 10 min. Titr.
with Na 2 S 2 3 soln, 942.27A, adding starch soln after most of
I has been consumed.
Normality = g K 2 Cr 2 7 x 1000/mL Na 2 S 2 3 x 49.032
Refs.: JAOAC 25, 659(1942); 27, 557(1944); 28, 594(1945);
38, 382(1955); 47, 43, 46(1964); 48, 103(1965).
A. Preparation of Standard Solution
Following table gives approx. vols of 95-98% H 2 S0 4 nec-
essary to make 10 L std solns:
Approx.
normality
0.01
0.02
0.10
0.50
1.0
mL H 2 S0 4 to be
dild to JO L
2.8
5.6
27.7
138.1
276.1
0. Standard Borax Method Standardization
See 936.15E.
C. Specific Gravity Method
Dil. H 2 S0 4 with enough H 2 to make convenient vol. of ca
70% H 2 S0 4 by wt. Det. sp gr in air at convenient temp.
(0-40°) as in 945. 06C (or sp gr may be detd with Sprengel
pyenometer), protecting soln from contact with air. Calc. ex-
act % H 2 S0 4 by wt,
P = 5(85.87 + 0.057 - 0.0004 r 2 ) - 69.82
where S = sp gr (in air) at 7°, compared with H 2 at t°.
Weigh exactly W g prepd acid contg P% H2SO4 and dil. to
n L to make required soln contg G g H 2 S0 4 /L. Calc. W from
equation:
W = nG x 100/P
Refs.: J. Chem. Soc. Trans. 57, 64(1890). J. Soc. Chem. Ind.
(1899), 109L JAOAC 24, 636(1941).
948.28 Standard Solutions
of Titanium Trichloride
Final Action
A. Preparation of Standard Solution
To 200 mL com. 15% TiCl 3 soln add 150 mL HC1 and dil.
to 2 L. Make soln ca 0.W, place in container with H atm.
provision (e.g., JAOAC 5, 207(1921)), and let stand 2 days
for absorption of residual O.
B. Standardization
Weigh 3 g FeS0 4 (NH 4 ) 2 S0 4 .6H 2 and transfer to 500 mL
flask. Introduce stream of C0 2 , and add 50 mL recently boiled
H 2 and 25 mL 40% (by wt) H 2 S0 4 . Then, without inter-
rupting current of C0 2 , rapidly add 40 mL 0.LV K 2 Cr 2 7 ,
949. 13C. Add TiCl 3 soln until near calcd end point. Then
quickly add 5 g NH 4 SCN, and complete titrn. Det. blank on
3 g FeS0 4 (NH 4 ) 2 S0 4 .6H 2 0, using same vols of H 2 0, H 2 S0 4;
and NH 4 SCN, and current of C0 2 . From net vol. TiCl 3 , calc.
normality:
Normality = mL K 2 Cr 2 7 X normality K 2 Cr 2 7 / mL TiCl 3
Refs.: JAOAC 31, 573(1948); 32, 589(1949).
982.35
Certified Reference Wlateriais
A. Definitions
Reference Material {RM). — Material or substance one or more
properties of which are sufficiently well established to be used
for calibrating app., or assessing measurement method, or as-
signing values to materials.
646
Standard Solutions and Certified Reference Materials
AOAC Official Methods of Analysis (1990)
Certified Reference Material (CRM). — Ref. material one or
more of whose property values are certified by valid proce-
dure, or accompanied by or traceable to certificate or other
documentation which is issued by certifying body.
B. General Information
Following tables list selected CRMs, certification, and code
of issuing organization. (Tables 982.35A-F). Table 982.35A
shows source codes together with names and addresses of these
organizations.
Particular CRMs were selected on basis of present and po-
tential applicability to AOAC activities. Listing of CRMs from
organization does not imply either AOAC endorsement of CRMs
or AOAC recommendation of organization supplying them.
Organizations listed are only representative of those supply-
ing CRMs. No claim is made as to completeness of infor-
mation supplied. More complete listing of organizations sup-
plying CRMs is available from the International Organization
for Standardization. Catalogs or literature describing CRMs are
available from individual organizations. However, because in-
ventories of CRMs are continually changing, inquiries should
be made of the organizations concerning current availability
of specific CRMs.
Table 982.35A
Source Code
Sources of Certified Reference Materials-
Organizations and Addresses
Name and Address
BCR Community Bureau of Reference — BCR
Directorate General XII
Commission of the European Communities
200, rue de la Loi
B-1049 Brussels, Belgium
IAEA International Atomic Energy Agency
Analytical Quality Control Services
Laboratory Seibersdorf
PO Box 100
A-1400 Vienna, Austria
NIST National Institute of Standards and Technology
Office of Standard Reference Materials
Room B311, Chemistry Building
Washington, DC 20234, USA
NIES National Institute for Environmental Studies
Yatabe-Machi, Tsukuba, Ibaraki 305, Japan
LGC National Physical Laboratory
Laboratory of the Government Chemist
Office of Reference Materials
Teddington, Middlesex TWtt OLY, UK
NRCC National Research Council
Marine Analytical Chemistry Standards
Montreal Rd
Ottawa, Ontario, Canada K1A 0R9
USP U.S. Pharmacopeial Convention, Inc.
USP-NF Reference Standards
12601 Twinbrook Parkway
Rockville, MD 20852, USA
WHO World Health Organization
Collaborating Center for Chemical Reference Substances
Apotekens Centrallaboratorium
Box 3045
T-17103 Solna, Sweden
Refs.: ISO Directory of Certified Reference Materials (CRM),
International Organization for Standardization, 1982.
Available from (J) ISO Central Secretariat, Case post-
ale 56, CH-1211, Geneva, Switzerland; (2) American
National Standards Institute, 1430 Broadway, New York,
NY 10018.
Table 982.35B Certified Reference Materials for Animal Tis-
sues, Plant Tissues, Foods, Alcoholic Bever-
ages, Animal Feedstuffs
Designation
Certification
Source Code
1566a
Oyster Tissue
Note a
NIST
1577a
Bovine Liver
Note a
NIST
H-4
Muscle
Note a
IAEA
1572
Citrus Leaves
Note a
NIST
1573
Tomato Leaves
Note a
NIST
1575
Pine Needles
Note a
NIST
No. 1
Pepperbush
Note a
NtES
60
Aquatic Plant
{Lagarosiphon m Major)
Note a
BCR
61
Aquatic Plant
( Platihypnidium riparioides)
Note a
BCR
62
Olive Leaves
{Olea europaea)
Note a
BCR
1567a
Wheat Flour
Note a
NIST
1568
Rice Flour
Note a
NIST
1549
Nonfat Milk Powder
Note a
NIST
1569
Brewers Yeast
Cr
NIST
17c
Sucrose
Note b
NIST
41c
Dextrose
Note c
NIST
1590
Wine
EtOH
NIST
A15-01
DL-5"Vinyloxazolidin-2-thione
(VOT)
Purity
LGC
063
Skim Milk Powder, natural
Note a
BCR
150
Skim Milk Powder, spiked
Note a
BCR
151
Skim Milk Powder, spiked
Note a
BCR
162
Soya-maize Oil Blend
Note d
BCR
163
Beef-pig Fat Blend
Note d
BCR
184
Bovine Muscle
Note a
BCR
185
Bovine Liver
Note a
BCR
186
Pig Kidney
Note a
BCR
189
Wholemeal Flour
Note a
BCR
191
Brown Bread
Note a
BCR
273
Single Cell Protein
Matrix
BCR
274
Single Cell Protein
Tr. Elem.
BCR
279
Sea Lettuce
Note a
BCR
282
Aflatoxin M-, in Whole Milk
BCR
283
Powder
Very low level
284
Aflatoxin M, in Whole Milk
285
Powder
Aflatoxin Mt in Whole Milk
Low level
BCR
Powder
Medium level
BCR
Aflatoxin M, in Whole Milk
Powder
High level
BCR
1563
Cholesterol & Fat-Sol.
Vitamins in Coconut Oil
Fortified levels
NIST
1588
Organics in Cod Liver Oil
Note e
NIST
TORT-1
Lobster Hepatopancreas
Note a
NRCC
DORM-1
Dogfish Muscle
Note a
NRCC
DOLT-1
Dogfish Liver
Note a
NRCC
3 Elemental composition.
b Optical rotation, index of refraction, and density.
5 Purity and specific rotation.
AOAC Official Methods of Analysis (1990)
Certified Reference Materials
647
Table 982.35C Certified Reference Materials for Biochemi-
cals, Ctinicals, Drugs, Industrial Hygiene,
Pharmaceuticals
Table 982.35E Certified Reference Materials for Pesticides
Designation
Certification
Designation
Certification
Source
Code
900
Antiepilepsy Drug Level
Note a
NIST
1599
Anticonvulsant Drug Level
Note b
NIST
909
Human Serum
Selected electrolytes, orgs
NIST
2671a
Freeze-dried Urine
F- (0.55 and 5.7 mg/L)
NIST
2672a
Freeze-dried Urine
Hg (2 cones)
NIST
2670
Freeze-dried Urine
Selected elements
Purity, other properties
NIST
(Note c)
NIST
—
Clinical CRMs
Purity, other properties
(Note d)
NIST
—
Drugs, clinicals . . .
USP
—
Pharmaceuticals
Purity, other properties
WHO
147
Lyophilized thromboplastin
Human plain
Note e
BCR
148
Lyophilized thromboplastin
Bovine
Note e
BCR
149
Lyophilized thromboplastin
Rabbit
Note e
BCR
194
Lyophilized bovine blood
(low conc'n)
Pb, Cd
BCR
195
Lyophilized bovine blood
(medium conc'n)
Lyophilized bovine blood
Pb, Cd
BCR
196
(high conc'n)
Pb, Cd
BCR
303
Calcium in Human Blood 1
Ca
BCR
304
Calcium in Human Blood II
Ca
BCR
192
Lyophilized Human Serum
(low conc'n)
Cortisol
BCR
193
Lyophilized Human Serum
(high conc'n)
Cortisol
BCR
319
Lyophilized CGT from pig
kidney
Note f
BCR
8430
AST (E.C.2.6.1.1)— Human
Erythrocyte
Note f
NIST
1951
Cholesterol in Human
Serum (Frozen)
Choi.
NIST
1952
Cholesterol in Human
Serum (Freeze-Dried)
Choi.
NIST
1507
Tetrahydrocannabinol (THC)
in Urine
THC
NIST
1598
Inorganic Constituents in
Bovine Serum
Tr. Elem.
NIST
5
Human Hair
Tr. Elem.
NIES
A-13
Freeze dried animal blood
Tr. Elem.
IAEA
a Phenytoin, ethosuximide, phenobarbital, and primidone at 3 concn
levels plus serum blank.
b Carbamazepine and valproic acid at 3 concn levels plus serum blank.
c Approximately 20 clinical calibration materials.
Drugs of abuse, veterinary drugs, enzymes, food chemicals clinicals,
vitamins ...
e Calibrated against Inter. Ref. Preparation of WHO (IRP 67/40).
f Based on I FCC method.
Table 982.35D Certified Reference Materials for Fertilizers and
Related Materials
Designation
Certification
Source Code
120c Phosphate Rock (Florida)
Note a
NIST
694 Phosphate Rock (Western)
Note a
NIST
32 Phosphate Rock (Moroccan)
Note a
BCR
33 Superphosphate
Note a
BCR
193 Potassium Nitrate
N and K concn
NIST
194 Ammonium Dihydrogen
N and P concn
NIST
Phosphate
200 Potassium Dihydrogen
P and K concn
NIST
Phosphate
P1V01 to P11-31
Chlorinated compds
P12-04 to P12-07
Organophosphorus compds
P13-01 to P13-12
Phenoxy-acids and related compds
P14-01 to P14-05
Substituted urea compds
P16-01 to P16-34
Heterocyclic and miscellaneous
compds
P17-01 to P17-02
Pyrethroids
1583
Chlorinated compds in isooctane
1492
Chlorinated Pesticides in Hexane
1491
Aromatic Hydrocarbons in Hexane/
Toluene
1579
Powdered Pb-base Paint
1582
Petroleum Crude Oil
1584
Priority Pollutant Phenols in Methanol
1585
Chlorinated Biphenyis in Isooctane
1586
Isotopically Labeled & Unlabeled
Priority Pollutants in Methanol
1587
Nitrated Polycyclic Aromatic
Hydrocarbons in Methanol
1589
Polychlorinated Biphenyis (As Aroclor
1260) in Human Serum
1596
Dinitropyrene Isomers & 1 -Nitropyrene
in Methylene Chloride
1597
Complex Mixture of Polycyclic Aromatic
Hydrocarbons from Coal Tar
1614
Dioxin (2,3,7,8 TCDD) in Isooctane
Dioxin
1618
V & Ni in Residual Fuel Oil
1636
Pb in Reference Fuel
1639
Halocarbons (in CH 3 OH) for H 2 Anal.
1650
Diesel Particulate Matter
2694
Simulated Rainwater
Source
Code
High purity
LGC
High purity
LGC
High purity
LGC
High purity
LGC
High purity
LGC
Note a
LGC
Note b
NIST
Note c
NIST
23 org. compds
NIST
Pb
NIST
6 org. compds.
NIST
10 org. compds.
NIST
8 org. compds.
NIST
10 org. compds.
NIST
6 org. compds.
NIST
Aroclor 1260
4 org. compds.
NIST
12 PAHs
NIST
NIST
V, Ni
NIST
Pb
NIST
7 org. compds.
NIST
6 org. compds.
NIST
Note c
NIST
a No purity figure is given but full anal, data supplied.
b Concns at i^g/mL levels.
c Concns approx. 200 ng/g
3 Chem. composition.
648
Standard Solutions and Certified Reference Materials
AOAC Official Methods of Analysis (1990)
Table 982.35F Certified Reference Materials for Water, Sediments, Gases, Particulates, Fuels
Designation
Certification
Source Code
1641b
Mercury in Water
fxg/mL level
NiST
1643b
Trace Elements in Water
ng/mL level
NIST
1644
Generator Columns
Note a
NIST
1647a
Priority Pollutants (in CH 3 CN)
Note b
NIST
NASS-2
Seawater
Trace elements
NRCC
CRM 046-097
Polynuclear Aromatic Hydrocarbons
Purity
BCR
2
Pond Sediment
Note c
NIES
SL-1
Lake Sediment
Note c
IAEA
2704
Buffalo River Sediment
Note c
NIST
1646
Estuarine Sediment
Note c
NIST
BCSS-1
Marine Sediment I
Note c
NRCC
MESS-1
Marine Sediment II
Note c
NRCC
Soil-5
Soil
Note c
IAEA
1658a-1659a
Methane in Air
1-10 (xmol/mol
NIST
1661/1696
Sulfur Dioxide in N 2
50-3500 ^mol/mol
NIST
1665b-1669b
Propane in Air
3-500 txmol/mol
NIST
2643-2648
Propane in N 2
100-5000 jj-mol/mo!
NIST
2649-2650
Propane in N 2
1.0-2.0 mol %
NIST
2651-2652
Propane/Oxygen in N 2
2 (5.0-10.0 mol %) C 3 H 8 (0.01 mol %)
NIST
1683/2631
Nitric Oxide in N 2
5-3000 ixmol/mol
NIST
1677C/2638
Carbon Monoxide in N 2
25-5000 ixmol/mol)
NIST
2639-2642
Carbon Monoxide in N 2
1.0-8.0 mol %
NIST
2612a-2614a
Carbon Monoxide in Air
9.9-43 mol %
NIST
2632-2634
Carbon Dioxide in N 2
300-800 ixmol/mol
NIST
261 9a- 1675b
Carbon Dioxide in N 2
0.5-15 mol %
NIST
2657-2659
Oxygen in N 2
2.0-21 mol %
NIST
1805-1806
Benzene in N 2
0.25-10 (xmol/mol
NIST
1625-1627
Sulfur Dioxide Permeation Tube
2.8-0.56 M-g/min (Note d)
NIST
1629a
Nitrogen Dioxide Permeation Tube
0.5-1.5 jxg/min
NIST
1911
Benzene Permeation Tube
0.3-0.5 jxg/min (Note d)
NIST
1648
Urban Particulate, Inorganic
As, Cd, Pb + 11 others
NIST
1649
Urban Dust, Organics
Polynuclear aromatic hydrocarbons
NIST
2676b
Toxic Metals on Filters
Cd, Mn, Pb, Zn at |xg levels
NIST
Air-3/1
Trace Elements on Filters
13 elements at jxg levels
IAEA
1619-1624a
Sulfur in Fuel Oil
0.15-5 wt %
NIST
1634a
Trace Elements in Fuel Oil
Pb, V, + 6 others
NIST
1581
Polychlorinated Biphenyls in Oil
100 ng/g
NIST
1580
Shale Oil
9 org. compds
NIST
1636a- 1638a
Lead in Reference Fuel
11.2-764 jig/g
NIST
2682-2685
Sulfur in Coal
S(0.5-4.6 wt %); plus ash and calorific values
NIST
1632a
Trace Elements in Coal (Bituminous)
As, Cd, Pb + 15 others
NIST
1635
Trace Elements in Coal (Subbituminous)
As, Cd, Pb + 11 others
NIST
1630
Trace Mercury in Coal
0.13 ^g/g
NIST
1633a
Trace Elements in Coal Fly Ash
As, Cd, Pb + 17 others
NIST
a Generates certified concns of anthracene, benz(a)anthracene, and benzo(a)pyrene in H 2 0.
b Certified concns of 16 polynuclear aromatic hydrocarbons.
c Certified for chem. composition,
d Permeation rate is certified.
Appendix: Laboratory Safety
Eugene C. Cole, Associate Chapter Editor
University of North Carolina
introduction
This chapter is not intended to be an exhaustive treatise on
laboratory safety. These precautionary notes serve only as a
reminder of possible hazards involved in the use of particular
operations or substances. Refer to recommended texts at end
of chapter for fuller treatment of subject. Follow safety re-
quirements of your organization and state, provincial, or fed-
eral government. Consult guidelines issued by professional as-
sociations and government agencies.
Cautionary Statements
Nature and amt of each chemical and its prescribed use were
criteria used in detg if cautionary statement for method was
indicated.
Safety hazard was considered to exist when nature, amt, and
use of chemical or equipment specified in method appeared
likely to produce any of following:
(a) Concn of vapors from flammable liq. exceeding 25% of
lower flammability limit of that liq. described by National Fire
Protection Association, Boston, MA.
(b) Contact between analyst and amts of material highly ac-
tive physiologically or toxic to humans in excess of Threshold
Limit Values published by American Conference of Govern-
mental Industrial Hygienists, P.O. Box 1937, Cincinnati, OH
45201.
(c) Contact between analyst and amts of highly corrosive
material sufficient to produce serious injury.
(d) Contact between analyst and radiations which could be
harmful.
(e) Explosion or violent reaction.
(f ) Injury to analyst by hazards in equipment or processes
which are not readily detectable by analyst.
When in doubt about possible hazards not covered in this
chapter, consult refs at end of chapter and other sources of
information such as hazard warnings on labels and manufac-
turers' material safety data sheets.
Potential Hazards of Equipment
Refrigerators
Should be explosion proof or explosion resistant when used
for storage of ether and other highly volatile, flammable liqs.
Ordinary refrigerator can be made explosion resistant by re-
moval of light switch, receptacle, and associated wiring and
placing thermoregulation controls on outside of refrigerator.
Glass
Dispose of chipped or broken glassware in special con-
tainers; minor chips may be fire-polished and glassware re-
tained. If glassware is to be repaired, mark defective area plainly
and store in special location until repairs are completed.
Use heat-resistant glassware for prepn of solns that generate
heat (e.g., not bottles or graduates).
Fire Extinguishers
Class B and C dry chemical fire extinguishers (for flam-
mable liq. and elec. fires) should be conveniently available to
each laboratory room. Carbon dioxide fire extinguishers should
be used on fires in electronic equipment.
Become familiar with their location and methods for effec-
tive use.
Blenders
Motor on high-speed blenders used to mix flammable solv.
with other materials should be explosion proof. Blend toxic or
flammable liqs in effective fume removal device.
Centrifuges
Adjust all tubes to equal wt before loading them into centrf.
Make certain that stoppers of tubes placed in pivot- type head
will clear center when tubes swing to horizontal. Do not open
centrf. cover until machine stops completely. Before removing
tubes, turn elec. switch to "off." Do not rely on zero-set rheo-
stat. Use only tubes specially designed for centrfg. Do not ex-
ceed safe speed for various tube materials (glass, cellulose ni-
trate, polyethylene, etc.) recommended by tube manufacturer.
Cellulose nitrate tubes may explode if autoclaved. Heating cel-
lulose nitrate tubes >60° may cause them to produce harmful
nitrogen oxide fumes.
Atomic Absorption Spectrophotometer
Follow all manufacturer's instructions for installation, op-
eration, safety, and maintenance. Use only hose/tubing to
conduct gases approved by manufacturer and supplier. Use ef-
fective fume removal device to remove gaseous effluents from
burner. Use only C 2 H 2 which is dissolved in solvent recom-
mended by manufacturer. Open C 2 H 2 tank stem valve only l / 4
turn. Change tank when C 2 H 2 pressure shows 75-100 lb. If
instrument has a drain trap, ensure that it is filled with H 2
before igniting burner. Following repair to C 2 H 2 supply line,
check for gas tightness at all connections with soap solution
or combustible gas detection system. Whenever solutions are
aspirated which contain high concentrations of Cu, Ag, or Hg,
spray chamber should be rinsed with 50-100 mL H 2 before
shutting down to clean these metals from chamber. See safety
notes on compressed gas cylinders.
Flame Photometer
Use effective fume removal device to remove gaseous burner
effluents.
Photofluorometer
Considerable amts of 3 are formed by UV light radiated
by quartz lamp. Ozone is toxic even in low concns; remove
thru effective fume removal device placed near quartz lamp.
Monitoring Equipment
Monitor unattended operations with equipment that will au-
tomatically shut down process if unsafe condition develops.
Ref.: N. V. Steere, "Handbook of Laboratory Safety" (1971);
CRC Press, Inc., 2255 Palm Beach Lakes Blvd, West
Palm Beach, FL 33409.
649
650
Laboratory Safety
AOAC Official Methods of Analysis (1990)
Compressed Gas Cylinders
Identify by name(s) of gas(es) contents of compressed gas
cylinders on attached decal, stencil, or tag, instead of by color
codes. Move cylinders (with protective cap) upright secured
to cart. Secure cylinders in upright position by means of strap,
chain, or non-tip base. Let contents of C 2 H 2 cylinders settle
and let all cylinders come to room temp, prior to opening. Use
only correct pressure gages, pressure regulator, flow regulator,
and hose/tubing, for each size of gas cylinder and type of gas
as specified by supplier. Use soap solution or combustible gas
detection system to check all connections, especially when
system is pressurized and gas is not flowing, to check for slow
leak. Use special heater on N 2 gas line. Close gas tank valve
and diaphragm on regulator (turn counter-clockwise) when gas
not in use. Service regulator at least yearly. Use toxic gases
only in effective fume removal device. When burning gas, use
flashback prevention device in gas line on output side of reg-
ulator to prevent flame being sucked into cylinder.
Ref.: Handbook of Compressed Gases (1981) Compressed Gas
Assoc, Van Nostrand Reinhold Co., New York, NY.
Distillation, Extraction, and Evaporations
(a) Flammable liquids. — Perform operations behind safety
barrier with hot H 2 0, steam, or elec. mantle heating. Use ef-
fective fume removal device to remove flammable vapors as
produced. Set up app. on firm supports and secure all con-
nections. Leave ample headroom in flask and add boiling chips
before heating is begun. All controls, unless vapor sealed, should
be located outside vapor area. Dispose of waste flammable
solvs by evapn as above unless other provisions for safe dis-
posal are available.
(b) Toxic liquids. — Use effective fume removal device to
remove toxic vapors as produced. Avoid contact with skin. Set
up app. on firm supports and secure all connections. Dispose
of waste toxic solvs by evapn, using effective fume removal
device unless other provisions for safe disposal are available.
Electrical Equipment
Accidents involving elec. equipment may result in mech.
injury, e.g., fingers being caught in chopping mill knives; elec.
shock, which may be due to lack of or improper grounding,
defective equipment, exposed wiring, or inadequate mainte-
nance; and fire thru ignition of flammable vapors by electri-
cally produced spark. Ground all elec. equipment to avoid ac-
cidental shock. Installation, maintenance, and repair operations
should be performed by qualified electricians.
Parr Bomb
Follow manufacturer's directions closely to avoid explosion.
Pressure
Do not conduct pressure operations with std glassware. In
certain circumstances, glassware specifically designed to with-
stand pressure may be used. Observe manufacturer's recom-
mended safeguards when using pressure app. such as calorim-
eter bomb, hydrogenator, etc.
Vacuum
Tape or shield with safety barrier containers and app. to be
used under vac. to minimize effects of possible implosion. Vac.
pump drive belts must have effective guards.
Hazardous Radiations
UV radiation is encountered in A A spectrophotometry, fluo-
rometry, UV spectrophotometry, germicidal lamps, and both
long- and shortwave UV lamps used to monitor chromatgc
sepns. Never expose unprotected eyes to UV light from any
source either direct or reflected (e.g., flames in flame photom-
eter, lamps, elec. arcs, etc.). Always wear appropriate eye
protection such as goggles having uranium oxide lenses, weld-
er's goggles, etc., when such radiations are present and un-
shielded. Keep skin exposure to UV radiations to min.
Safety Technics and Practices
Spraying Chromatograms
When strong corrosive and toxic reagents are sprayed on
chromatograms, use gloves, face shield, respiratory protec-
tion, and appropriate fume removal device to protect skin, eyes,
and respiratory tract against mists or fumes generated by spraying
device.
Pipets
Do not pi pet hazardous liqs by using mouth suction to fill
pipet. Use pipet fillers or rubber tubing connected thru trap to
vac. line for this purpose.
Wet Oxidation
This technic is among most hazardous uses of acids but can
be performed safely. Observe precautions in this chapter for
particular acids used and rigorously follow directions given in
specific method being used.
Hazardous or After Hours Work
Anyone working alone after hours or on hazardous proce-
dures should arrange to be contacted periodically as safety
measure.
Glass Tubing
Protect hands with heavy towel or gloves when inserting
glass tubing into cork or rubber stopper. Fire polish all raw
glass cuts.
Open ampules in fume removal device over tray large enough
to hold contents if ampule should break. If contents are vol-
atile, cool before opening.
Safe Handling of Acids
Use effective acid-resistant fume removal device whenever
heating acids or performing reactions which liberate acid fumes.
In dilg, always add acid to H 2 unless otherwise directed in
method. Keep acids off skin and protect eyes from spattering.
If acids are spilled on skin, wash immediately with large amts
of H 2 0.
Acetic Acid and Acetic Anhydride
React vigorously or explosively with Cr0 3 and other strong
oxidizers. Wear face shield and heavy rubber gloves when us-
ing.
CAS- 108-24-7 (acetic anhydride)
Chromic and Perch romic Acids
Can react explosively with Ac 2 0, HO Ac, EtOAc, isoamyl
alcohol, and benzaldehyde. Less hazardous with ethylene gly-
col, furfural, glycerol, and MeOH. Conduct reactions behind
safety barrier. Wear face shield and heavy rubber gloves.
Formic and Performic Acids
Strong reducing agents; react vigorously or explosively with
oxidizing agents. Irritating to skin, forming blisters. Performic
acid (formyl hydroperoxide) has detonated for no apparent rea-
AOAC Official Methods of Analysis (1990)
Acids
651
son while being poured. Wear face shield and heavy rubber
gloves when using.
CAS-64-18-6 (formic acid)
Hydrofluoric Acid
Very hazardous with NH 3 . It can cause painful sores on skin
and is extremely irritating to eyes. Use effective removal de-
vice. Wear goggles and acid-resistant gloves.
CAS-7664-39-3 (hydrofluoric acid)
Nitric Acid
Reacts vigorously or explosively with aniline, H 2 S, flam-
mable solvs, hydrazine, and metal powders (especially Zn, Al,
and Mg). Gaseous nitrogen oxides from HN0 3 can cause se-
vere lung damage. Copious fumes are evolved when coned
HNO3 and coned HC1 are mixed. Avoid premixing. Use ef-
fective fume removal device when fumes are generated. Han-
dle with disposable polyvinyl chloride, not rubber, gloves.
Oxalic Acid
Forms explosive compd with Ag and Hg. Oxalates are toxic.
Avoid skin contact and ingestion.
CAS- 144-62-7 (oxalic acid)
Perchioric Acid
Contact with oxidizable or combustible materials or with de-
hydrating or reducing agents may result in fire or explosion.
Persons using this acid should be thoroly familiar with its haz-
ards. Safety practices should include following:
(a) Remove spilled HCIO4 by immediate and thoro washing
with large amts of H 2 0.
(b) Hoods, ducts, and other devices for removing HC10 4
vapor should be made of chem. inert materials and so designed
that they can be thoroly washed with H 2 0. Exhaust systems
should discharge in safe location and fan should be accessible
for cleaning.
(c) Avoid use of org. chems in hoods or other fume removal
devices used for HCIO4 digestions.
(d) Use goggles, barrier shields, and other devices as nec-
essary for personal protection; use polyvinyl chloride, not rub-
ber, gloves.
(e) In wet combustions with HC10 4 , treat sample first with
HNO3 to destroy easily oxidizable org. matter unless otherwise
specified. Do not evap. to dryness.
(f ) Contact of HCIO4 soln with strong dehydrating agents
such as P 2 ? or coned H 2 S0 4 may result in formation of an-
hyd. HCJO4 which reacts explosively with org. matter and with
reducing agents. Exercise special care in performing analyses
requiring use of HC10 4 with such agents. Extremely sensitive
to shock and heat when concn is >72%.
(g) Also observe precautions outlined in (1) "Perchloric Acid
Solution," Chemical Safety Data Sheet SD-11 (1965), Man-
ufacturing Chemists Association of the US, 1825 Connecticut
Ave, NW, Washington, DC 20009; (2) "Applied Inorganic
Analysis/' W. F. Hillebrand, G. E. F. Lundell, H. A. Bright,
and J. I. Hoffman, 2nd ed. (1953), pp. 39-40, John Wiley
and Sons, Inc., New York, NY; (3) "Notes on Perchloric Acid
and Its Handling in Analytical Work," Analyst 84, 214-
216(1959); (4) "Perchlorates," ACS Monograph No. 146, J.
C. Schumacher, ed., Reinhold (1960). See also refs at end of
this chapter.
Picric Acid
Highly sensitive to shock when in dry state. In contact with
metals and NH3, it produces picrates which are more sensitive
to shock than picric acid. Readily absorbed thru skin and ir-
ritating to eyes. Wear heavy rubber gloves and eye protection.
CAS-88-89-1 (picric acid)
Sulfuric Acid
Always add H 2 S0 4 to H 2 0. Wear face shield and heavy rub-
ber gloves to protect against splashes.
CAS-7664-93-9 (sulfuric acid)
Fuming Acids
Prep, and use with effective fume removal device. Wear
acid-resistant gloves and eye protection.
Safe Handling of Alkalies
Alkalies can burn skin, eyes, and respiratory tract severely.
Wear heavy rubber gloves and face shield to protect against
coned alkali liqs. Use effective fume removal device or gas
mask to protect respiratory tract against alkali dusts or vapors.
Ammonia
Extremely caustic liq. and gas. Wear skin, eye, and respi-
ratory protection when handling in anhyd. liq. or gaseous state.
NH 3 vapors are flammable. Reacts violently with strong oxi-
dizing agents, halogens, and strong acids.
Ammonium Hydroxide
Caustic liq. Forms explosive compds with many heavy met-
als such as Ag, Pb, Zn, and their salts, especially halide salts.
CAS- 1336-2 1-6 (ammonium hydroxide)
Sodium, Potassium, Lithium, and Calcium Metals
Violently reactive with H 2 or moisture, C0 2 , halogens,
strong acids, and chlorinated hydrocarbons. Emit corrosive
fumes when burned. Can cause severe burns. Wear skin and
eye protection when handling. Use only dry alcohol when pre-
paring Na alcoholate and add metal directly to alcohol, one
small piece at a time. Avoid adding metallic Na to reaction
thru condenser.
CAS-7440-23-5 (sodium)
Sodium Peroxide
Less caustic than Na and K hydroxides but reacts violently
with H 2 0, org. matter, charcoal, glycerol, Et 2 0, or P. Wear
skin, eye, and respiratory protection when handling multigram
amts.
CAS- 13 13-60-6 (sodium peroxide)
Calcium Oxide (Burnt Lime)
Strongly caustic! Reacts violently with H 2 0. Protect skin,
eyes, and respiratory tract against contact with dust.
CAS- 1305-78-8 (calcium oxide)
Sodium and Potassium Hydroxides
Extremely caustic. Can cause severe burns. Protect skin and
eyes when working with these alkalies as solids or coned solns.
Add pellets to H 2 0, not vice versa.
CAS-1310-58-3 (potassium hydroxide)
CAS- 13 10-73-2 (sodium hydroxide)
Sodium Biphenyl, Sodium Methylate, and Sodium Ethylate
Less caustic than NaOH but can be injurious. React vigor-
ously with H 2 0. Protect skin and eyes when handling.
652 Laboratory Safety
AOAC Official Methods of Analysis (1990)
Safe Handling of Organic Solvents
(Do not mix waste solvs.)
Flammable Solvents
Do not let vapors cone, to flammable level in work area,
since it is nearly impossible to eliminate all chance of sparks
from static electricity even tho elec. equipment is grounded.
Use effective fume removal device to remove these vapors when
released .
Toxic Solvents
Vapors from some volatile solvs are highly toxic. Several
of these solvs are readily absorbed thru skin. Use effective
fume removal device to remove vapors of these solvs as they
are liberated.
Refs.: Gosselin, Smith, and Hodge, "Clinical Toxicology of
Commercial Products (Home and Farm)," 5th ed. (1976);
The Williams & Wilkins Co., 428 E Preston St, Bal-
timore, MD 21202.
American Conference of Governmental Industrial Hy-
gienists, "Threshold Limit Values"; PO Box 1937,
Cincinnati, OH 45201.
Sax, "Dangerous Properties of Industrial Materials,"
7th ed. (1988); Van Nostrand Reinhold Publishing Corp.,
New York, NY 10022.
N. V. Steere, "Handbook of Laboratory Safety" (1971);
CRC Press, Inc., 2255 Palm Beach Lakes Blvd, West
Palm Beach, FL 33409.
Journal of the American Society of Safety Engineers 7,
Feb. 1964.
See also references at end of chapter.
Safe Handling of Special Chemical Hazards
Pesticides
Many pesticide chemicals are extremely toxic by various
routes of exposure, especially in coned form. These chemicals
include org. CI, carbamate, and org. P insecticides, mercuri-
als, arsenic als, nicotine, and other chemicals. As an example,
org. P family of pesticides is consistently highly toxic, not
only by oral ingestion, but dermally and by inhalation as well.
Observe following min. precautions at all times. Consult safety
data sheets or labels for addnl information.
(a) Do all laboratory sampling, mixing, weighing, etc., un-
der effective fume removal device in area having good forced
ventilation of nonrecirculated air, or wear gas mask of proper
type. If mask is used, replace cartridges as recommended, since
using contaminated mask may be worse than no mask.
(b) Keep off skin. Wear clean protective clothing and non-
permeable gloves (such as polyethylene gloves) as necessary.
Wash thoroly with soap and water to avoid contaminating food
and smoking materials.
(c) Label all sample containers with name and approx. con-
tent of all pesticides.
(d) Have readily available and study information on symp-
toms of poisoning and first aid treatment for each type of pes-
ticide being handled.
(e) Consult physician about preventive measures and anti-
dotes for use in emergencies when pesticide poisoning is sus-
pected.
(f ) Follow your organization's procedures when disposing
of waste pesticides. The manufacturer can be contacted for
advice on disposal problems.
(g) Do not enter pesticide residue or other laboratories after
handling pesticide formulations until protective clothing and
gloves have been removed and face and hands thoroly washed
with soap and water.
U.S. Environmental Protection Agency operates "hotline"
staffed to handle pesticide questions, called National Pesticide
Telecommunications Network (NPTN). To reach this hotline,
dial: 800-858-7378.
Refs.: Gosselin, Smith, and Hodge, "Clinical Toxicology of
Commercial Products (Home and Farm)," 5th ed.
(1984), The Williams and Wilkens Co., Baltimore, MD
21202.
Farm Chemicals Handbook, 75th ed. (1989), Meister
Publishing Company, 37841 Euclid Ave, Willoughby,
OH 44094.
Morgan, D.P., Recognition and Management of Pes-
ticide Poisonings, 4th ed. (1989), U.S. Environmental
Protection Agency, Office of Pesticide Programs,
Washington, DC.
Citizens Guide to Pesticides (1987), U.S. Environmen-
tal Protection Agency, Office of Pesticides and Toxic
Substances, Washington, DC.
Aniline
Toxic. Avoid contact with skin and eyes. Use effective fume
removal device. Highly toxic when heated to decomposition.
Flammable. May react vigorously with oxidizing agents. Ig-
nites in presence of fuming HN0 3 . May react violently with
3 .
CAS-62-53-3 (aniline)
Acetonitrile
Toxic. Avoid contact with skin and eyes. Use effective fume
removal device.
Ammoniacal Silver Nitrate
Use soon after prepn and do not allow to stand for long
periods of time.
Benzene
Toxic. Highly flammable. Avoid contact with skin. Do not
breathe vapors. Use effective fume removal device. Decom-
poses violently in presence of strong oxidizing agents. Reacts
violently with CI. Considered to be tumor producing agent.
CAS-71-43-0 (benzene)
Acetone
Highly flammable. Forms explosive peroxides with oxidiz-
ing agents. Use effective fume removal device. Do not mix
with CHC1 3 .
CAS-67-64-1 (acetone)
Bromine and Chlorine
Hazardous with NH 3 , H, petroleum gases, turpentine, ben-
zene, and metal powders. Extremely corrosive. Use effective
fume removal device. Protect skin against exposure.
Carbon Disulfide
Extremely flammable with low ignition temp. Toxic. Use
effective fume removal device. Can react vigorously to vio-
lently with strong oxidizing agents, azides, and Zn. Avoid static
electricity.
CAS-75-15-0 (carbon disulfide)
AOAC Official Methods of Analysis (1990)
Chemical Hazards
653
Carbon Tetrachloride
Reacts violently with alkali metals. Toxic. Fumes may de-
compose to phosgene when heated strongly. Use effective fume
removal device.
CAS-56-23-5 (carbon tetrachloride)
Cyanides
React with acids to form highly toxic and rapid acting HCN
gas. Use only in effective fume removal device. Destroy res-
idues with alk. NaOCl soln.
Cyclohexane
Highly flammable. Use effective fume removal device. Can
react vigorously with strong oxidizing agents.
CAS- 110-82-7 (cyclohexane)
Di- and Triethylamine
Flammable. Toxic. Corrosive to skin and eyes. Use effec-
tive fume removal device. Can react vigorously with oxidizing
materials.
Dimethylformamide
Toxic. Flammable. Avoid contact with skin and eyes. Use
effective fume removal device. Can react vigorously with ox-
idizing agents, halogenated hydrocarbons, and inorg. nitrates.
Diethyl Ether
Store protected from light. Extremely flammable. Unstable
peroxides can form upon long standing or exposure to sunlight
in bottles. Can react explosively when in contact with CI, 3 ,
LiAlH 4 , or strong oxidizing agents. Use effective fume re-
moval device. Avoid static electricity. See also safety notes
on peroxides.
CAS-60-29-7 (ether)
Ethanot
Flammable. Use effective fume removal device when heat-
ing or evapg.
Chloroform
Can be harmful if inhaled. Forms phosgene when heated to
decomposition. Use effective fume removal device. Can react
explosively with Al, Li, Mg, Na, K, disilane, N 2 4 , and NaOH
plus MeOH. Considered to be tumor producing agent.
CAS-67-66-3 (chloroform)
Ethyl Acetate
Flammable, especially when being evapd. Irritating to eyes
and respiratory tract. Use effective fume removal device.
CAS- 141 -78-6 (ethyl acetate)
Formaldehyde
A suspect human carcinogen. Exposure to high concns may
cause skin irritation and inflammation of mucous membranes,
eyes, and respiratory tract. Use skin protection and effective
fume removal device.
CAS-50-00-0 (formaldehyde)
Hydrogen Sulfide
Hazardous with oxidizing gases, fuming HN0 3 , and Na 2 2 .
Forms explosive mixts with air. Toxic. Use effective fume re-
moval device.
CAS-7783-06-4 (hydrogen sulfide)
Hypophosphorus Acid
Reacts violently with oxidizing agents. On decomposition,
emits highly toxic fumes (phosphine) and may explode. Use
effective fume removal device.
Hexane
Highly flammable. Use effective fume removal device.
CAS- 110-54-3 (hexane)
Isooctane
Highly flammable. Use effective fume removal device.
CAS-26635-64-3 (isooctane)
Magnesium
When finely divided, liberates H in contact with H 2 0. Burns
in air when exposed to flame. Can be explosive in contact with
CHC1 3 or CH3CI.
Magnesium Perchlorate
Explodes on contact with acids and reducing materials. Use
as drying agent on inorg. gases and materials only.
CAS- 10034-8 1-8 (magnesium perchlorate)
Mercury
Hazardous in contact with NH 3 , halogens, and alkali. Va-
pors are extremely toxic and cumulative. Regard spills on hot
surfaces as extremely hazardous and clean up promptly. Powd
S sprinkled over spilled Hg can assist in cleaning up spills.
High degree of personal cleanliness is necessary for persons
who use Hg. Handle only in locations where any spill can be
readily and thoroly cleaned up. When Hg evapn is necessary,
use effective fume removal device.
To avoid environmental contamination, dil. liq. remaining
in Kjeldahl distn flask to ca 300 mL with H 2 0, cool to room
temp., and add 50 mL 30% H 2 2 . (If Raney powder method
is used, 6 mL is enough.) Warm gently to initiate reaction, let
reaction go to completion in warm flask, and sep. pptd HgS.
Reserve ppt in closed labeled container for recovery of Hg or
disposal appropriate for Hg.
See also safety notes on mercury salts.
Methanol
Flammable. Toxic. Avoid contact with eyes. Avoid breath-
ing vapors. Use effective fume removal device. Can react vig-
orously with NaOH plus CHC1 3 , and KOH plus CHC1 3 or
HC10 4 .
CAS-67-56-1 (methanol)
Methyl Cellosolve
Vapors can be harmful. Use effective fume removal device.
CAS-109-86-4 (methyl cellosolve)
Nitrobenzene and Other Nitroaromatics
Readily absorbed thru skin. Symptoms of intoxication are
sense of well-being and bluish tint on tongue, lips, and fin-
gernails. Wear resistant rubber gloves when handling. Heat or
evap. in effective fume removal device.
CAS-98-95-3 (nitrobenzene)
Oxidizers
(Perchlorates, peroxides, permanganates, persulfates, perbor-
ates, nitrates, chlorates, chlorites, bromates, iodates, coned
H 2 S0 4 , coned HN0 3 , Cr0 3 )
654
Laboratory Safety
AOAC Official Methods of Analysis (1990)
Can react violently with most metal powders, NH 3 , and NH 4
salts, P, many finely divided org. compds, flammable liqs,
acids, and S. Use exactly as specified in method. Handle in
effective fume removal device from behind explosion-resistant
barrier. Use face shield.
Peroxides
(a) Hydrogen peroxide. — 30% strength is hazardous; can
cause severe burns. Drying H 2 2 on org. material such as pa-
per or cloth can lead to spontaneous combustion. Cu, Fe, Cr,
other metals, and their salts cause rapid catalytic decomposi-
tion of H 2 2 . Hazardous with flammable liqs, aniline, and ni-
trobenzene. Since it slowly decomposes with evolution of O,
provide stored H 2 2 with vent caps. Wear gloves and eye pro-
tection when handling.
(b) Ether peroxides. — These peroxides form in Et 2 0, diox-
ane, and other ethers during storage. They are explosive and
must be destroyed chem. before distn or evapn. Exposure to
light influences peroxide formation in ethers. Filtration thru
activated alumina is reported to be effective in removing per-
oxides. Store over Na ribbon to retard peroxide formation.
CAS-7722-84-1 (hydrogen peroxide)
Phosphotungstic Acid
Emits highly toxic fumes when heated to decomposition or
in strong alkali.
Pyridine
Toxic. Flammable. Use effective fume removal device. Re-
leases toxic cyanides when heated to decomposition.
CAS-1 10-86-1 (pyridine)
Petroleum Ether
Extremely flammable. Use effective fume removal device.
Avoid static electricity.
CAS-8030-30-6 (petroleum ether)
Pentane
Extremely flammable. Use effective fume removal device.
Avoid static electricity.
CAS-109-66-0 (pentane)
Radioactive Chemicals
Consult NBS Handbook No. 92, "Safe Handling of Radio-
active Materials" (available as NCRP Report No. 30 from Na-
tional Council on Radiation Protection, Publications Dept., 4201
Conn. Ave. NW, Washington, DC 20008) and NCRP Report
No. 39 "Basic Radiation Protection Criteria," before handling
these materials.
Silver Nitrate
Powerful oxidizing agent; strongly corrosive. Dust or solid
form is hazardous to eyes. Handle as noted for oxidizers.
Silver lodate
Powerful oxidizing agent. Can initiate combustion in contact
with org. material (e.g., paper or cloth). Can react vigorously
with reducing agents. Handle as noted for oxidizers.
Arsenic Trioxide
Toxic. Forms toxic volatile halides in contact with halide
acids. Forms volatile, highly toxic arsine when reduced in acid
soln. Protect skin and respiratory tract when handling. Use
effective fume removal device when arsine or arsenic trihalide
is formed.
CAS- 1327-53-3 (arsenic trioxide)
Mercury Salts
Mercuric salts are quite toxic and mostly H 2 0-sol. Use skin
and respiratory protection when dry mercuric salts are to be
used. Use skin protection when coned aq. solns of mercuric
salts are used. Mercurous salts are generally less toxic than
mercuric salts. Use of personal protection is advisable when
handling these salts and their coned solns.
See also safety notes on mercury.
Permanganates
Moderately toxic. Readily sol. in H 2 0. Strong oxidizing agent.
May form explosive mixt. with H 2 S0 4 or HC10 4 . When using
with strong acids to destroy org. matter, perform reaction be-
hind safety barrier.
Sulfur Dioxide
Toxic gas. Forms H 2 S0 3 in contact with moisture. Use ef-
fective fume removal device to remove S0 2 vapors released
by reaction or from gas cylinder. Avoid contact with skin, eyes,
and respiratory tract.
CAS-7446-09-5 (sulfur dioxide)
Di- and Trichloroacetic and Trifluoroacetic Acids
Protein precipitants. Can cause severe burns to skin and res-
piratory tract. Use rubber gloves, eye protection, and effective
fume removal device to remove vapors generated.
Uranyl Acetate
Highly toxic. Avoid skin contact and breathing dusts.
Toxic Dusts
Use gloves and goggles to avoid contact with skin and eyes.
Use effective fume removal device or other respiratory pro-
tection.
Carcinogens
Regulations of U.S. Department of Labor require special
precautions to avoid exposure of persons to carcinogenic chems.
Consult 29CFR1910.93c (U.S. Government Printing Office,
Washington, DC 20402) and Guidelines for the Laboratory Use
of Chemical Substances Posing a Potential Occupational Car-
cinogenic Risk, USDHEW, 1978.
Asbestos
Dry asbestos fibers are hazardous when inhaled. Wet fibers
form a mat which does not constitute a hazard. Transfer dry
fibers in hood to container of distd H 2 and store under H 2
until needed, e.g., for prepn of mats in Gooch crucibles. Do
not dry asbestos in forced draft oven, only in convection oven.
Open oven doors slowly to avoid developing convection cur-
rents that will make fibers airborne. Reuse of filtering mats is
often possible by washing, drying, and ignition, as appropri-
ate.
CAS-80 12-0 1-9 (asbestos)
SPECIAL REFERENCES
Chemical Safety
Safe Storage of Laboratory Chemicals, D.A. Pipitone (ed.)
(1984), John Wiley & Sons, Inc., New York, NY 10158.
Dangerous Properties of Industrial Materials, 7th ed., N.I. Sax
(1988), Van Nostrand Reinhold, New York, NY 10001.
Prudent Practices for Disposal of Chemicals from Laborato-
ries, National Academy of Sciences (1983), National Acad-
emy Press, Washington, DC.
AOAC Official Methods of Analysis (1990)
Safety References
655
Flammable and Combustible Liquids Code Handbook, 2nd ed.
(1984), National Fire Protection Association, Boston, MA
02110.
Guidelines for Selection of Chemical Protective Clothing, A.D.
Schwope, P.P. Costas, J.O. Jackson, and D.J. Weitzman
(1983), American Conference of Governmental Industrial
Hygienists, Inc., Cincinnati, OH.
Health and Safety for Toxicity Testing, D.B. Walters and C.W.
Jameson (1984), Butterworth Publishers, Stoneham, MA.
Compendium of Safety Data Sheets for Research and Indus-
trial Chemicals, Parts 1, 2, and 3, L.H. Keith and D.B.
Walters (1985), VCH Publishers, Inc., Deerfield Beach, FL.
Health Risks to Female Workers in Occupational Exposure to
Chemical Agents, R.L. Zielhuis, A. Stijkel, M.M. Verberk,
and M. Vande Poel-Bot (1984), Springer- Verlag, Berlin,
FRG.
Guide to Safe Practices in Chemical Laboratories (1986), Royal
Society of Chemistry, Letchworth, Herts SG6 1HN, UK.
Hazards in the Chemical Laboratory, L. Bretherick (1986),
Royal Society of Chemistry, Letchworth, Herts SG6 IHN,
UK.
Solvents in Common Use: Health Risks to Workers (1986),
Royal Society of Chemistry, Letchworth, Herts SG6 IHN,
UK.
Sichere Arbeit im Chemischen Laboratorium; Ein Lernpro-
gram (1977), 2 Volumes, Berufsgenossenschaft der Chem-
ischen Industrie, Heidelberg, FRG.
Onderzoek Kwaliteit Zuurkasten, G. Bolkesteijn (1985),
Technical University Twente, Enschede, The Netherlands.
Handboek Bedrijfsveiligheid (1986), 4 Volumes, Kluwer,
De venter, The Netherlands.
La Securite dans les Laboratoires Utilisant des Substances Chi-
miques: Guide Practique (1983), p. 305, Centre National de
Prevention et de Protection (CPP), Paris, France.
Zuurkasten In Laboratoria: Hoe zijn ze, Hoe blijven ze en hoe
moeten we er mee werken, A.G. Kroes (1982), pp. 68, Cur-
sus Hogere Veiligheidskunde, Royal Shell Laboratory Safety,
Environment and Security, Amsterdam, The Netherlands.
Biological Safety
Laboratory Biosafety Manual, World Heath Organization
(1983), WHO Publications Center USA, 49 Sheridan Ave,
Albany, NY 12210.
Biohazards Reference Manual, AIHA Biohazards Committee
(1985), American Industrial Hygiene Association, Akron,
OH 44331-1087.
Biosafety in Microbiological and Biomedical Laboratories, 2nd
ed., Centers for Disease Control and National Institutes of
Health (1988), HHS No. (NIH) 88-8395, Superintendent of
Documents, U.S. Government Printing Office, Washington,
DC 20402.
EPA Guide for Infectious Waste Management (1986), Stock
No. PB86- 199130, National Technical Information Service,
Springfield, VA 22161.
Laboratory Safety
CRC Handbook of Laboratory Safety, 2nd ed., N.V. Steere
(1971), CRC Press, Inc., Boca Raton, FL 33431.
Prudent Practices for Handling Hazardous Chemicals in Lab-
oratories, National Academy of Sciences (1981), National
Academy Press, Washington, DC.
Handbook of Laboratory Waste Disposal, M.J. Pitt and E. Pitt
(1985), Ellis Horwood Ltd, Halstead Press-John Wiley &
Sons, New York, NY.
Handling Radioactivity — A Practical Approach for Scientists
and Engineers, D.C. Stewart (1981), John Wiley & Sons,
New York, NY.
Radionucleotiden- Laboratoria; Richtlijnen voor Indenting van
en Werken in Radio-Nucleotiden-Laboratoria (1983), Min-
isterie van Volkshuisvesting, Ruimtelijke Ordening, en
Milieubeheer, 44 pp., Publication 83-02, The Hague, The
Netherlands.
Appendix: Reference Tables
PAGE
977.38 Table of atomic weights, 1977 657
935.70 Various strength solutions of the common acids, alkalies, and alcohol 658
955.57 Optical crystallographic properties of some crystalline drugs 659
955.58 Table of rafractive indices for drugs, arranged according to ascending value of the lowest
index 668
963.37 Nomograph relating absorbance, concentration, and absorptivity (1 cm cell) 672
SURPLUS TABLES
10th ed.
Section
Progressive accumulation of radium emanation 43.029
Element for which known variations in isotopic composition in normal terrestrial material prevent a more precise atomic weight being
given; A r {E) values should be applicable to any "normal" material.
Element for which geologic specimens are known in which the element has an anomalous isotopic composition, such that the dif-
ference between the atomic weight of the element in such specimens and that given in the Table may exceed considerably the
implied uncertainty.
Element for which substantial variations in A r from the value given can occur in commercially available material because of inadvertent
or undisclosed change of isotopic composition.
Element for which the value of A r is that of the radioisotope of longest half-life.
656
AOAC Official Methods of Analysis (1990)
Reference Tables 657
977.38 Table of atomic weights (1981) from Commission of Atomic Weights, International Union of Pure and
Applied Chemistry
Scaled to the relative atomic mass, A,{ ,2 C) = 12
The atomic weights of many elements are not invariant but depend on the origin and treatment of the material.
The footnotes to this Table elaborate the types of variation to be expected for individual elements. The values of
A r (E) given here apply to elements as they exist naturally on earth and to certain artificial elements. When used
with due regard to the footnotes, they are considered reliable to =bl in the last digit or ±3 when followed by an
asterisk*. Values in parentheses are used for certain radioactive elements whose atomic weights cannot be quoted
precisely without knowledge of the origin of the elements; the value given is the atomic mass number of the iso-
tope of that element of longest known half life.
Sym-
Atomic
Atomic
Foot-
Sym-
Atomic
Atomic
Foot-
Name
bol
Numbe
r Weight
notes
Name
bol
Number
Weight
notes
Actinium
Ac
89
227.0278
z
Mercury
Hg
80
200.59*
Aluminum
Al
13
26.98154
Molybdenum
Mo
42
95.94
X
Americium
Am
95
(243)
Neodymium
Nd
60
144.24+
X
Antimony
Sb
51
121.75*
Neon
Ne
10
20.179+
x.y
Argon
Ar
18
39.948*
w,x
Neptunium
Np
93
237.0482
2
Arsenic
As
33
74.9216
Nickel
Ni
28
58.69
Astatine
At
85
(210)
Niobium
Nb
41
92.9064
Barium
Ba
56
137.33
x
Nitrogen
N
7
14.0067
Berkelium
Bk
97
(247)
Nobelium
No
102
(259)
Beryllium
Be
4
9.01218
Osmium
Os
76
190.2
X
Bismuth
Bi
83
208.9804
Oxygen
O
8
15.9994+
w,x
Boron
B
5
10.81
w ( y
Palladium
Pd
46
106.42
x
Bromine
Br
35
79.904
Phosphorus
P
15
30.97376
Cadmium
Cd
48
112.41
x
Platinum
Pt
78
195.08*
Calcium
Ca
20
40.08
X
Plutonium
Pu
94
(244)
Californium
Cf
98
(251)
Polonium
Po
84
(209)
Carbon
C
6
12.011
w
Potassium
K
19
39.0983*
Cerium
Ce
58
140.12
X
Praseodymium
Pr
59
140.9077
Cesium
Cs
55
132.9054
Promethium
Prn
61
(145)
Chlorine
CI
17
35.453
Protactinium
Pa
91
231.0359
2
Chromium
Cr
24
51.996
Radium
Ra
88
226.0254
x,z
Cobalt
Co
27
58.9332
Radon
Rn
86
(222)
Copper
Cu
29
63.546*
w
Rhenium
Re
75
186.207
Curium
Cm
96
(247)
Rhodium
Rh
45
102.9055
Dysprosium
Dy
66
162.50*
Rubidium
Rb
37
85.4678+
X
Einsteinium
Es
99
(252)
Ruthenium
Ru
44
101.07+
X
Erbium
Er
68
167.26*
Samarium
Sm
62
150.36*
X
Europium
Eu
63
151.96
X
Scandium
Sc
21
44.9559
Fermium
Fm
100
(257)
Selenium
Se
34
78.96+
Fluorine
F
9
18.998403
Silicon
Si
14
28.0855+
Francium
Fr
87
(223)
Silver
Ag
47
107.8682+
X
Gadolinium
Gd
64
157.25*
X
Sodium
Na
11
22.98977
Gallium
Ga
31
69.72
Strontium
Sr
38
87.62
X
Germanium
Ge
32
72.59*
Sulfur
S
16
32.06
w
Gold
Au
79
196.9665
Tantalum
Ta
73
180.9479*
Hafnium
Hf
72
178.49*
Technetium
Tc
43
(98)
Helium
He
2
4.00260
X
Tellurium
Te
52
127.60*
X
Holmium
Ho
67
164.9304
Terbium
Tb
65
158.9254
Hydrogen
H
1
1.00794
w,x,y
Thallium
Tl
81
204.383
Indium
In
49
114.82
X
Thorium
Th
90
232.0381
x,z
Iodine
1
53
126.9045
Thulium
Tm
69
168.9342
Iridium
Ir
77
192.22*
Tin
Sn
50
118.69*
Iron
Fe
26
55.847*
Titanium
Ti
22
47.88*
Krypton
Kr
36
83.80
x,y
Tungsten
Lanthanum
La
57
138.9055*
X
(Wolfram)
W
74
183.85*
Lawrencium
Lr
103
(260)
U ra n i u m
u
92
238.0289
x,y
Lead
Pb
82
207.2
w,x
Vanadium
V
23
50.9415*
Lithium
Li
3
6.941*
w,x,y
Xenon
Xe
54
131.29*
x,y
Lutetium
Lu
71
174.967*
Ytterbium
Yb
70
173.04*
Magnesium
Mg
12
24.305
X
Yttrium
Y
39
88.9059
Manganese
Mn
25
54.9380
Zinc
Zn
30
65.38
Mendelevium
Md
101
(258)
Zirconium
Zr
40
91.22
X
(See preceeding page for footnotes.)
658
Reference Tables
AOAC Official Methods of Analysis (1990)
935.70 Various strength solutions of the common acids, alkalies, and alcohol 3
(a) Ammonia sofns: Specification requires >28-<30%
NH 3 by wt. Sp gr of 28.0% NH 3 soln = 0.9 at 15°. Mix and
dil. tol L.
NH 3 Strength
Desired
g/L
Reagent NH3 Required
ml
5
10
15
20
25
50
75
100
150
200
17.86
35.71
53.57
71.43
89.29
178.57
267.86
357.14
535.71
714.29
19.8
39.7
59.5
79.4
99.2
198.4
297.6
396.8
595.2
793.7
(c) Hydrochloric acid sofns: Specification requires
> 36.5- < 38.0% HCI by wt. Sp gr of 37.2% HCI soln - 1.19
at 15°. Mix with H 2 and dil. to 1 L.
HCI Strength
Desired
HCI Required
g/L
g
ml
5
13.44
11.29
10
26.88
22.59
15
40.32
33.88
20
53.77
45.18
36.46
98.01
82.36
IN soln
50
134.41
112.95
100
268.82
225.90
150
403.23
338.85
200
537.63
451.79
222.6
598.39
502.85
Constant boiling
278.4
748.39
628.90
Sp gr 1.125
300
806.45
677.69
(e) Sulfuric acid sofns: Specification requires >95.0-
<98.0% H2SO4 by wt. Sp gr of 96.0% soln = 1.84 at 15°.
Pour acid into excess of H2O and dil. to 1 L.
H 2 S0 4
Strength
Desired
g/L
H2SO4 Required
g
ml
5
12.5
20
30
40
49
100
150
250
300
400
5.21
13.02
20.83
31.25
41.67
51.04
104.17
156.25
260.42
312.50
416.67
2.8
7.1
11.3
17.0
22.6
27.7
56.6
84.9
141.5
169.8
226.5
For crude fiber
IN soln
(b) Sodium hydroxide sofns: Specification requires
>97% NaOH in sticks or pellets of caustic soda. Dis-
solve and dil. to 1 L.
NaOH Strength
Desired
NaOH Required
g/L
g
12.5
30
40
50
75
100
150
200
250
300
12.89
30.93
41.24
51.55
77.32
103.09
154.64
206.19
257.73
309.28
For crude fiber
IN soln
(d) Nitric acid sofns: Specification requires >69.0-
<71.0% HNO3 by wt. Sp gr of 70.4% HNO3 soln = 1.42 at
15°. 1 ml coned HNO3 contains ca 1.00 g HNO3. Mix with
H 2 Oand dil. to 1 L.
HNO3
Strength
Desired
HNO3 Required
g/L
g
ml
5
10
20
30
40
50
63
70
100
150
200
300
7.10
14.20
28.41
42.61
56.82
71.02
89.49
99.43
142.05
213.07
284.09
426.14
5.0
10.0
20.0
30.0
40.0
50.0
63.0
70,0
100.0
150.0
200.1
300.1
IN soln
(ff) Afcohofic sofns : b Specification requires 95%
C2H5OH by vol. Sp gr = 0.810 at 25°. Mix and dil. to 1 L.
Alcohol
Strength
Desired
Alcohol Required
ml/L
g
ml
50
100
150
200
250
300
400
500
700
42.63
85.26
127.89
170.52
213.16
255.78
341.04
426.32 (proof)
596.84
52.6
105.3
157.9
210.5
263.2
315.9
421.1
526.3
736.8
Prepd by G. C. Spencer and H. J. Fisher, 1935 and updated by W. D. Hubbard, 1970.
6 Alcohol of any desired strength may be obtained by taking number of ml 95% alcohol equiv. to desired strength
and dilg soln to 95 ml ; e.g., to obtain soln of 70% alcohol, take 70 ml 95% alcohol and dil. to 95 ml.
AOAC Official Methods of Analysis (1990)
Reference Tables
659
D.
O
co
QO C
c o
£ '£
LU
c c
~.2
UJ
.2 c
♦^ 00
a--
O w
oa
■a
c
3
O
a
E
o
O
cd
c
CD .
■- &0
X
a) to
o o
c »-
o -o
to
CD
>
V
8.8
IS
Cl to
co o
■M «
C ^ V
* — ^
CD — c
^E*
to o 2
o ° "Hi
2 >-
CD qz
E -
-a
o
C
0)" c
S: o
4- X -o
CD
o
k.
■o
c
CD
to d a —
iO v. 0-
„ « «J . lu
CD CO fli —
w M • 5 o *
-fj c a "C to .2
o o o co _. c
DC UJ 2 CO D
£ 5
X)
8<*
0)
to
3
XI
S S
O
« £
11
P c
-Q
CD
O
^ *>
+ + + + +
I I
+ -H +1
a. a a a .- a a a
+ I + +
+ I +
+ + l I I I + I +
l l 4-
I +4- +
m r-% .-4 m in o
r*» <r> o in an o oo
w w mo m u> in
Tfro comiftooHOOTttDHoo
roroinooHiN^owiflHOOiviD cr»
OOSf«1tf)Cfl<DCn'S-HCMOOOOo
mNHmfOinHmooHWrto
lOUjtoiotomioiNiniciciooo
AAA
OJ OO O 00
n m O) oo
m ^o ur> in
tniOin«efrinh-lOtOOC\l <m
OHOWOOOHO)fOOO^ cm
mmioioixjiom^omio <x>
fl- m h en
r*- en oo in
in in in <x>
o en oo it) o in o
10 en ro in io in io
in ^j- «x> in in in in
OOlDOOOONffJONNininoo
lOinNOPOfOHiniDN^^iD
minm«)inininiflinwiniftin
o o
X I
a> cd CD
.£,Ei
"O jC *
a) o a ®
•;aor
~ CD C O
O Q. g CD
o
sz
3? E
ii
C C £
op c
*3 £ «
CD
O
<<<<<< CD
CD
o
I
CD
CD
*♦-
3
"E
'■5
o>
C
"5
CD
c
CD
c
X
CD
C
O
X
O
X
•2
"5
CD
c
E
C
C
CD
<D
a
>
c
c
c
c
CD
CD
CD
CD
CD
X)
CD
C
C
o
sz
Cl
o
C)
o
x:
o
x:
c
CD
C
"CD
c
'3
C
Q.
i.
w.
D
3
■M
CJ
U
u
U
w
CD
(D
k.
k.
CD
O
O
o
o
CD
0QCDDQ0QOOOOOOUOOQ
£.E£
CD O - 1 -.
c *- —
JJ <U CD
S.E.E
> j= x:
x a a
O k- k-
a o o
SEE
Q. > >,
o -*- 1 *-
t. CD CD
CD « .2
O Q O
CD CD
c
1 5 " £
Sx «^
o
<u X
c -
— >» £ a ^ +3 CD 'm
E
CD
o
X}
^ <->
CD
t- x
C
CD O •
E
c . c
CO
(D CD O
o
v. T3 3
o
= CD «
a a.£
u
(A
x:
511
CD
£
° c
I*
a
O
'—
<D
CD
O
O
>
k.
II
*-*
a)
E
>
F
■o
to
CD
II
11
to
k.
■*
>
CD
*J
~
X
CO
01)
CD
to
a
II
II
660
Reference Tables
AOAC Official Methods of Analysis (1990)
CO
E
co
QO C
c o
o -^
u
•5 .2
UJ
M c
O ^
c
3
O
a
E
o
O
5
DO
C
00 <*-
£ c
O (0
£ «
0) oo
E I
o S
1/5 O
- .c
♦3 «
S « 8 ~
•- £ to a>
a o a £
v CO
£>« Ss
o ca a> 5
UJ ±
o ti
C « '- ,
E M > J
O C -ft
c B aS
a
o
<u
E ^
2 « a
k. O "O
3 TO w
00 - 00
00
5 E
Of)
>N J2 VO
>
-Hill
+ + I
+
+ I
I I
I I I
+ l +
+ l
+ + 4- +
in (^
in in cm © <r> p~» ^r
«ef <v> m Cft r*1 cm CT>
io <jo id «) i^ yD in
A A
CO <T>
i£> «
O O O O
CM en r~* ro
<D VD VO UD
w o oo <o »
cm er> m ** ao en
US UO lO (O Ii3 *o
in o o o m o
cm en cm o> ro en
ID lO IO«Mf>
vo r-
in r»* in
O CO
moifioo m
i-< rH
UD O CM
*H O
00 lO M fO tO H
in tx>
ID lO ID
id m
in <x> to in in in
00 VD
m cm
in m
o o m m in m r-.
00 V q- N in N N
in in in vo in in m
oo as o o in o
O rrt o oo io cm
in in to in m vo
o o
N N O Ol 00
<T> r^
ID H tH OJ V S
in in
in <d in in in in
O
X
a>
c
o
■o
CM
O x
a?
c
(0
<r> *- —
o
CO —
O
o
* fO O
a 5 ^
X
X
T I co a) a)
© aJ
c c
.C CO
>>
c
JJ Q) t U
c c ™ "C
o o
N N
£5
5 O
Q.
O
5
a a Q. «j «j
sssJJ
TO CO
c c
a) a>
0- CL
o
I
c £
3 ±1
c « w
V 5 .E £ a)
c c c c c
v a) .- .- .-
£ JC 3 3 3
CL CL COO
o
X
X -o
• a)
E a
c «
.- o
3 ro
OK
o
X
CO
X
0)
c
X c
a? cy
c c
O
X
a a> oj
& c c
tn) — —
C -O A
■5 E E
o > ^ E 2 '£
o +-*-<>. o o
E
to c c
-H I -H
a a a d
+ I
(V) Ch CTi O
h in io ro
in (fl ifl n
r-l i-t I r-l
*r m r**« id
N M n m
E
CO
a
o
X
0)
c
O
o
o
o
c
c
o
QJ
0}
>.
-C
£.
V
u
a
a
(0
>1
E
o
E E
CO CO
JQ
o
o
o
CO
JO
x:
n
O O O O
AOAC Official Methods of Analysis (1990)
Reference Tables
661
o
E
(Z
(0
DO C
c o
o -^
UJ
c c
•^ o
X '■£
o v
a
E
o
O
TO
GO
c
Q)
\-
00
F
ao
00
o
(0
r-t
u
•a
II
t/i
a
.5?
jc
c
TO
<D
(0
•6
to
(0
o
X
a
^
tr
UJ
O
c
.^ ab
a
o
5 >
,9 A
■D
Q)
m <a- o 2 2
k_
U
T
*•"
*
fc
c
o
E
E
o
o
M
* X
o
"ai
E
o
u
C
TO
t»
03
TO w
o
a> <y
(/)
t/)
c
C
T> —
c —
9?
c
op
0)
o o
E E
C TO
X
*o
o
TO
DO
c
fc
E
o w
>, (0
.£ E
to
a
c
TO
TO
X
o
o
O
TO «J
. o
X o
h c/>
O
c
OQ
UJ
C
c
m o
0)
—
_
F
o
bo
TO
n
.2
<d
tt)
d a>
l -
E
E
bo
60
tiO 00
>
>
^
T)
»-
k- L.
to
V)
tt)
to
TO
TO TO
a)
id
<D
E
>
>
>
+ I + I ++ I + -H
+ + I
+ -H + -H +
aaaaaaaaaaa
a a a a
aaaaaaaaaa
I + + I + + I + + + +
+ +
+ I + I +
I +
Oto«3tnor*.ooocMir>rooro «s- en cm o o
^(OlOHHlONlfifflHlOOON in CvJ CO O 0O
^rjininmmminminminr^m to io id n n
m o oa
r-t ^H ^r
ID U3 UD
kOiDUJiOiDtOiftSNif)
CO CO CO US m (D ro M N n m
•trmooor-i ro cm m © r^ m
in w * in in in in t in in in
o en in in id
S O 00 CO rr
in tn in 10 in
MMMfOiDOlCOOfVJfl-OOOOHHOOinMtlO
OONinoOOlO^OMHOOChrl^^OlDHm^
minin^rTrrrininm^r^rininLntommirjin
n in m
uDr-iOtnOT— tinrocooo
mmiomLnLnmmmm
E
o
a) <d
0) o
<" ■£;
°£
>l
TO w
TO
=. °- E
40 a) t: o
C -I-' TO 3
o — — —
> 2 * ?
E «55
o & d> <v
a c c c
cu o y o
.fc >% >» >»
S e E E
2 o o o
_ i_ i_ »_
^ -C -C £
>> -M *- *-
£ >s > >,
5 UJ UJ UJ
O
I
3 —
c c
O «
-E X .
c c
O O L> O O
> >
E E
o o
E E
o o
c .r
j= j= x x:
4-» +w +J ^J
X > > >
L- W *_ k_
UJ UJ UJ UJ
E
o
J2
uj u. O
5 E
fc TO
TO
. r "D QJ <D
O O C C
3
TO tf) ~ ™
i/i
C
.E . >> >»
o o ww
(J
o o c: TO TO
E
of 5 22
u
>>«>,>,
<u
O O >% X X
z
Z Z Z O O
0J
c
xi
CD
a)
£ I
P* a) OJ
.■= a; tt)
■d -^. -i
CD CD CD
c c c
.= TO
-c J= a
CD CD CD CD CD
c c c c c
o fc
II
O TO
JZ O I
o a •
oo ?
a o o u o
tt)
CL
a) cu (D
0. Q. CL
C C C C C
tt) tt) <D <D 4)
0. CL CL CL 0.
2 2 TO
c c £,
a) a) <u
CL CL H
oo a
0) >
662
Reference Tables
AOAC Official Methods of Analysis (1990)
a
o
a.
o
jE
a.
ea
k.
at
o
Q.
O
E
cr
TO
00 C
c o
£ ~
LU
6
c c
'■3.2
X -M
UJ
£ c
Tt
£*>
Q)
O to
3
C
■*■•
c
o
o
I
c-
w
CO
O)
3
k.
■o
a>
c
—
Qa
(0
+->
CO
£
o
a>
E
o
(0
8
4-
o
(0
o
a
E
o
U
CD
E
00
is
x a
£: a .
"5 ^J «*
<u cd .-
fc « E £
— T3 00
e^ O (0 o3
c a
to o
w to
.5? 3
*■ oo
* c
TO -M
. O
« E
0)
E
bo
3
00
CD
TJ
O
W
TJ
O
TO S^
_ w a
« £.E o
t fl)££
CO > I- CO
c °8 «
o « E
E a> oo
E 5 2
° a «
u <D
. «> >
B E
a £
£ 5
00 CD .=
. — </>
x x .5?
k.
a
o3
CD
2 8
TO
k- +-
CD (0
cr
to a
V)
Etj
°8
£tj
V)
13
C
3
a
o
TO *- 3 .
tj cr
CD <D ,
TJ £
'? «
ID CO
TJ ^
™ TO TO
c 00 .
w .2 c a
o « o o
CT LU I
c
00
(0
3
00
CD
CD
■a
r
o
Si c
oo
c
o
CD
00
(rt h-
o
TO .fc
E
w
"k_
a
X»
X)
3
» E
?a.
5 v
_ ra
« a
.2 *- x o ™
_TO y
3 '■£
oo to
2! E
>— 'Z
3 « ^
w o £
CC CO
TJ
O
cr
to °8 o
*"' « 0(
J5 -5 5
a 3 .
„ oo x
T3 O
O <U
cr cr
CD
S> So
CO
E E
<1>
00
I +
+ + I
4- +
I I +
+ + +
s q- in in m •<$■
h ro yj id *r rr>
va r>- to kd ix> r^
O OO OO PO LX">^CM *t m M
r^O 1 *^) (T> m no s roiurn
U> m id r-% io s to r^ixjr^.
no r-.
co o t—t m
fO rn
in oo a> o
r-> r*-
ID ID lO N
A V
m t o o ro m
co in h id o c\J
m ID ID ID ID ID
m m o
N ID O
id m vo
r-i in ** in i-H
h r^. m
in t*** o o ro ro
o ^h C7^ .-i oo m
in id in id in in
co in in
o cm oo
in in in
oo rs. r*.
N W N
r«. O
ID O CM O
0O V lO
r-t <T>
ID CD H 00
in in id
ID «3
in in id in
RJ
a)
V
(a
CD
E
to
<u
rr — X cm
O o . .
■ - 1 -. c c
® c N *N
a)
(0 —
o -^
c c
fc
(D
C
E
S
'E
O.E
X E
"O CD
c u - =
0) CO
a a
— -M >^ >^
£ JC
— o o o
W r l i.
£ c o
o o
a
t.
k. o
•m o
a x
C l££
-C .c
JZ
>% a>
<mou
a o
O
O Q
© 3
U +■*
ra w
w .ii
'^ -c
g 5i
3 OO -
^ O 0)
? « I
E &a
b 5
o
i
0) c
I E
.E to
o c
(J c
3 0)
Q> a
.E r
i2 o «
$£ £
o u. cu
a o. 5
T3
x:
o
I
c
cu
x: >>r; -m
o
u
X
cu
ra
CD T5 x cu
* c -=
U
C
CD
^ Jo
a fe l.
x: 5r
co J2 X
f E a;
E ® c
S-- ' S
2 E *
CD TO +j
XI ,h <D
_ *
O - TO
x o a;
cJ X g
C CD t
M C CD
TJ
oo o
^o^
a)
CD
5
CM
; cd co g
1 r x: c
0. CL £L
fl N C TO
£ » c " -
r +- TO
g !i
a. o. a.
TO 3
£J3
*- o
o ^
CD rr
1 1 1
In
- > D
* N Tx
c c fj;
cd o a
x: x: r
AOAC Official Methods of Analysis (1990)
Reference Tables 663
Q.
o
E
cr
<o
DO C
C O
° ~
UJ
u
c c
•^ 2
x '+*
UJ
O <"
oa
T3
C
3
O
a
E
o
o
a)
c
(0
to
•o
o
c
o
c
o
E
E
o
o
tfj
a a
O O
4- "D
O o =
ft o ~
c "»
(0 £
"D U)
O O
a: oc
IS lo
■i|
m 4>
°I
<■> C O o« ™
03 e . -.
ui poo
*«- o ° x x
DC CD CO
a3 ™
c
a>
o
£
■a
a;
H
<a
o
c
u
°3
>^
i-
to CO
<u
•D "O
>
O O
oa
DC QC
E
3
0)
E
I s !
TS
i +
+ + +
-H I
+ I
+ + +
a a
§
I + I
I + I
I + I
I I -f
in in o o o in cm
cm q- <o o oo rH m
<£> m in m in <n in
^h in
ai ^t h w
m r-.
tt m m in ^t
CM CM
^r ro tn l£S
cm to
CM C\J IT) CM ro
ir> in
IT) ID in in
in id
lO ID IT) Lf) O
cm oo m i-i oo cm en
r^. r^. <r> oo ^r m cm
in in in in in m in
10 in en oo o
in in in in in
mow rn *t
in in in u> m
iDrtMnowrjin
i-Hmi£>ocM^r»Htn
m in «* in in - -^
m o
in o ro in *»■ in
H N N rr CI ID
in id ^r in in rj-
NOOOCn H
"3- ir> n- «s- in
'o
3
o
x:
a
C
0)
a
o
B 1
— w
co
E
3
El
D (fl
E
TJ
o
CO
«? s~ «.
E
3
O
« « iS 5 -2 ? .S3
-gxrooofixiw
= Tt CQI0 103
<<<<<C0C0CQ
S o —
.* - (0
£ = - c
"5 CO <u
+rf -M ■*-"
3 3 3
OQ CD CO
■*= .hf io £* -Q
a « 3 £ ■ w
flj «J .t
3 ■= "2 ro =
; x> -a
CO CO
(0 /-s
O TJ
cow
-S5
JQ ■" "T <0 « ■
■M CO
XI QLX1 ^ J3 -g
o o 5 x x o. c
o o x x 5 a.
CO CO
XI XI
o o
c c
a>
co xi
X3 O
o o
Q. </)
-o o ^ a co
O E 3 O X3
S .5? - Z .E
664
Reference Tables
AOAC Official Methods of Analysis (1990)
Q>
■c
0)
Q.
O
k_
a
o
a.
O
TO
DO C
C O
u
2 c
x '-P
UJ
O <"
c
O
a
E
o
CO
O
E
TO
x:
T7
•+-<
c
£
TO
-
00
tu
"to
*■
V)
r
m
%
o
X
c
to
m
o
E
F
V)
o
u
o
c
X
X3
r
c
o
E
o
r-
a»
ro
u
to
00
aJ
to
0)
TO
u
v.
DO
>
Csi
E
o
o
5
_TO
<D
**-
—
;*•
13
O
x:
"So
c
LL
E
E
.2
'x
TO
£
V)
C
T3
C
Q)
o
>> -
(13
X
c
CQ
«
CD
D
C
a)
CO
rr>
00
o o
O ID
|
JS
0O LO
\_
O
L0
a
10
"'
to
c
CJ
oo
o
+
1
1
1
1 +
c
a
j
(0
<£>
r\j
OO
rr»
rn
•«r
«*■ O^J
I
CTi
<\j
lo
in r^.
UO
LD
LO
LO
KD LO
to
cm
CO
CM
o
00 <3-
a>
CM
OO
rvj
i-H LO
uo
UO
LD
to
to
tD UO
H
i-H
O
OO
■*r
r-^
<M
CM I--
*J-
t— 1
LO
CO
LO
UO
LO
IT)
uo
uo un
CD
en
o
c
<D
I
o
-Q
_
ro
CD
a>
( )
TO
c
to
o
X
3
«
E
0)
(D
O
A3
ra
r
c
E
C
E
c
E
TJ
a
to
ra
ro
E
E
(0
0)
<D
x:
0)
x:
to
©
a
F
a
F
a
F
^
o
>
TO
m
ro
0J
a)
o
Q
X
o
a?
X
o
X
o
CO
u
b
ro
*->
X
X
■a
u
r
u
u
(D
■n
O
T
<u
r>
a>
<D
tt>
0)
to
IT)
0)
£>
>.X
c
0)
(U
c
c
u
<\J
>-
TO
>
a
u
."2 c
00 f\i
a> 6
0) <S)
x:
1
<
x: •
11
o
c
0)
v-/
*>
\s
^i-
«* w
*T \s
r.
x; to
13
*t
no
OO
rr>
0-
0- Q_
a
■a
c
o
w 8
is?
- § s
> UJ C
E
<D
s|
^ W n ^
> TO ? »
Axt <2
^ ca o cl
CD
^
o
M
TO
uo
CD
<r
TO
*
II
X
>
A
C\J
UJ
w
i5 A
(0 (^ +j T3 —
*j W W O u
E E
■- P '
E E
= E
J5 ^
« op ^ E "-5
1 0)
oo tv> >
5 E
+ +-H -H
+ + -f I + + + I + l i
I I + + I + 4- +
r-LOuo,— trstooo inrocoMNNm^oONOH
uouoc\iojr-~m^r(r)Of ir )Oicr>o , >"T^TOror\jouD
tOLOUOtOLOUOUOI^r-^UOLOUOUDt£)tjDr^UDtOr-«UO
CNJCVJfSJfVJOMHNffl
mrsir«TirsHNO
uouououoinuovououo
PvJHMinMOOi^
^rr\jfvjr\juoLOOor-.
touououounuoioLO
tOfV(MO^^c^iD«)OH<triorvjHmr\j^
LOUOUO UnLOlOLOLOLOUOLOir>tOLOU->UOUOUOUOLO
2 £
E
£ o
0) x:
CQ O
0) o
0) o
CCO
o
o o o
O 5 (D
o £^
U 0) w
E >
x: = re
« >C m 3^
<D CD .- CT V)
OUOOOOUJUJ
.—
no
CD
TO
o a
O
fl)
Q)
CD
C
o
<u
<D
o o
co to
k.
(0
tf>
to
a
TO
TO
TO
o
o
O
C C
r
x:
r
QJ
n
trt
tn
T3 13
n
n
n
c
a> gj
o
CD
CD
<i>
aJ
aJ
E
O
a a
^
c
c
c
o
n
n
> >
TO
o
o
o
V)
x: x:
l.
k.
u
Tt
■n
to
UJ
to
UJ
to
UJ
11
2
U-
X
X
0) 0>
AOAC Official Methods of Analysis (1990)
Reference Tables
665
■o
CO
DO C
C O
o -^
O
C c
■J .2
x +*
W
o w
oa
a
e
o
o
F 3
£ 3
00 CO
2 « «
JO O >»
a. o
Eg
I i
+ 4- + + l l
ID O IT) CM
rv rv oi <J>
■o id W in
id in r-.
m OO ID N
ro «3- «*■ .— i
ir> Ln in in
-C
CO
0)
o
a
■4-J
0)
to
-Q
(0
a)
QJ
CD
n>
c
CD
C
C
o
c
o
c
o
o
o
o
<D
aj
c
(/J
U)
(/)
n
u
c
<D
03
E
o
QO
ft)
CD
O
O
to
a>
CL
a
Q_
h-
K
o
(J
c
(0
3
a
<d
o
o a
a o
bb
o
W (0
o x
CC CO
no o
, »- c
w x£ a
— W (0 3 v
oo ce o h-
<D
c
E
■o
a)
h
a
o
(0
u
JC
<^
V)
o3
T3
O
<0 (\j
E ^
-H -H -H
I I
+ 4-
+ +
4- I
ONO<rtHomNinoo(v)
r-*r^oor^.r^r^cor^,ioiDf^r^
o en m
in <y> ro ,_(
W ID N OO
O ro o ro i-t r-.
O m oo no rg ai
n rs rs fN to io
<D <3" in rr> m CTi O
r^H to r-. id tD T (T>
<D m to to to id in
co tn cr> id
n in m n
<U ID N N
w iJi u) in id id fO cor-.^.— i
n in cri h o oo i-i io co oo to
in tf) in id io in in in m in «)
in o o o
If) t OO N
m in * id
o t co in io m
Ch r-» CT> <X> (71 O
in io in id n id
(0
+
o>
-g
E
0)
TJ
F
-C
m
<.;
C
^
CO
; co
* 0)
O
I
TJ "D
£ CD
£2 <d •%
iE.E.E ~ -~ o
W TO N N S m r
c -o "o bo bo = :
(j (0 (0 CO CO (0 CO .
3 3 3 3 3 3 3
co oo oo co oo co oo
O
0)® .
C CD
C C N O
(0 N
sz
%-*
E E
CD
rt
a a
O
X
■a
o
(D <D
iE E
'3 3
00 00
-*- CO CD
•£? T> —
CD CD
^ C C
O -£ .
E
a a
(0 (0 CO CO
O <D (d"~"<D CD 0>
■ £ 3 o o o 3
TJ N Nl N N N
■J3 co to co to to
£ 3 j= 3 i o
CO CO .CO co .—
00 00
D 3 3 3
3 3 D D Z> 3
00 CO CO 00 00 00
0} CD
3 »-
= ■2
o (o
•^ o
a
a
E
■o
c
3
O
3
£
S
CO
CO
CO
•a
CD
o
(0
CD
T)
JC
0)
E
o
<D
c
o
r
10
CO
c
CD
c
CD
in
CD
O
a
C
o
b
CD
E
CO
CJ
o
o
^1
r
u
CD
(0
CD
CD
c
(/)
TD
CD
CO
k.
^
CD
CD
Q,
k_
O
CD
a
fc
W)
3
O
c
o
u
E
r
a
o
o
o
a
"o
V)
3'
o
a
n
CD
■o
JO
3
c
j-
O
o
i-
<D
CJ
co
O
E
CD
c
CO
CD
+J
3
1-
c
CT
h ai u
666
Reference Tables
AOAC Official Methods of Analysis (1990)
to
a
a.
o
o.
(0
h.
O)
o
75
(0
u
15
o
■s.
o
to
E
a>
tr
TO
00 c
c o
J2 ~
UJ
S .2
a ■-
O w
■o
c
O
a
E
o
o
w w
■o
O
« c
TO O
ti E
£E
o o
■o °
£ «
J2 °
■o TO
5 a
ro
o E
c
o
-C
<7)
i
TO
c/)
u
<1)
u
TO
; <n E °3
! 5.2 vi
> to *- a>
-_ a -m
a ^ «
a; -m _
op O *yj
lO UJ to I
(1) w
£ X
T XI
<D .-
"D «
_TO 0>
o
E
E
o
o w
. a)
w *-
oo ffl
'ȣ a
n *°
°S
w X3 ■
^ lO
|£°
to »- -a
£ .to <d
l. 3 ■=
(0 M O
— c -Q
5s E
0) u o
± ex: en
c
o
E
E
o
o
w
bo
x
CD
eg
l'£
0J TO
00 °
. JC
X '—
ro -6
d£
o ^
■* o
i3 >
c
E
DO
£T 00
O U
DO
a
o
■d
0)
a
to
«
C
o
E
c
o
£
E
o
c ■ »
r*= X
E a
no -a
to a)
J2 03
o
E
E
o
E
oc
E
J: co
a to
.SP2 o
H^ W TO
"O
o
«*-
QJ
jrt
<l)
C
to <y
3
o
O
o w
k.
C/)
X o
m
E
TO o
F
CO
CL
CO
a
o
E
00
£: m « L
IO >>
« TO
a.
3
cr
Q) • w
*- 2 *
**" ro Q.
«i Q °
00 O T3
^ _ a>
. TO C
O . TO C
F x a-
£ XI - ••
fc .. tn «
" ^ 5 «
■ * W £
x N5
" TO O W
-J DC t£>
jo
a
S " »
2 *- vi
£ «*-
s « ™
= «> E E
00 C v. 3 (o o^ M .5
> > E E
■i: S .h 5 £
TO TO —
00 00 00
I -H -H I I
■H +
I I
a « a a a
4- +
+ + I +
I + I + I + +
+ + +
H W <J
f-H «3 ^1
vr> <X) v£)
T PI TO NIT) ^J-OOinOO^OlDO
»x)0(r>oorn fr>tO"rrr-.ir)Crtr-4fM
ud u3 id in fN p*-tr>^)(i>u5ir)iO<i>
**■ om w o
i-t in o m try
^f CT> CO
to 00 o
(M Ol r-l
oo oo ro
in in id
m in m in is o^r<\ioor--.ir)r-*o
oo ^r o (och oooinKroo oom^
m m id in m to^mtoin inmin
in o *r
o <m o
U3 iO (O
OO w o
o rr oj
in in in
(Or-ioooo ON^cocvi oom
LnLomintn ninmifiifi inmin
rn r-H ro o
io r- «r r>>
in m in in
OMO to
^t H00
in in in
■9
■*•_ a «
CD <D 0)
C C C
E E E
TO TO TO
aj a; a>
.c x: jc
a a a js
E E E 2
T3 TD "O O
TO
i - =
x t aw
aj a> <u
c c c
a>
a
E
TO TO O —
% <D X w
aa* S
E E »
TO TO -Q "D
o aj a>
x x a a .3
o) <u uj ui a
a
c
w .E
CO c 0)
d'i « ,E
•c ? a O
E,£c 3
ro ^ £ - w
o?r^ g
aa ? • E
X X iw ^J
$* o o a<+=
2^^ 3 E
o
X
a a
P 4)E
TO
o
gx : •
3 fli-
rt c E
S E S
E«i
S^TO
c E 5
r- w t:
at 5
S5=5
.5
o
N
TO
a
TO
z
o
X
T3
c
0)
JZ
Q.
O
X .
6
c
E
TO
O
C
TO
a
o
E
•5 >,
c c
a> <d
x: x:
a cl
Is
£ a) O
ci
Ji -o »
^ • .E
a.c l.
O a T3
o. ® »
3oi
>tj a
c s a>
Q) » w
x: « to
a. cl cc
a>
O
TO
- +
o w —
0)
c
©
X 0)0
TO
C
■ C I
a
0) m z *
0)
x:
c J= £
JC
a
•c a.E
o
0)
x: <n ■=
c
c
nep
Syn
laze
£
CO
TO
«i
>N^ O
3
T3
CO "0 h-
y~
AOAC Official Methods of Analysis (1990)
Reference Tables
667
v>
v.
<0
£
a:
>
CM
W> c
c o
o •£
uj
g c
5= .2
UJ
■8
3
.£2 c
o ^
a
i
(0
en
3
?-
■a
c
B
oa
U
4>
E
o
o
B
0)
0)
a.
o
k.
Q.
O
!E
Q.
O)
o
75
o
75
o
a.
O
id
uj
ui
■o
c
3
O
a
E
o
O
o
o
c
a>
E
GO
(0
a
c
<c
•o
E
r
o
<♦-
E
T3
VI
N
a
(0
n
(A
a>
>
TJ
k.
O
w
<D
«o
<T
•a w>
(0 w
« '"
p c
"' "S »
'55 .a
0)
c
c a>
.2E
> a
5 jo
to u. —
3 a)
0) _w
o ft
+ +
a a
+ + + + I
«r o in >o o <j-
r-> oo en *t a> ■**
vo r^ r-. ir> tr> io
A A
rv o uj tn
<■ i-i co oo
to n mm
oo *r «» o cm to
CO CO 00 CO P"* «-H
uj to m ir> in in
X<3
x * .E <u
1 IPJ
§ ° o .£ 5 S.
< O O uj O 5
a
X
CM
(0
a
o
k.
a.
o
668
Reference Tables
AOAC Official Methods of Analysis (1990)
955.58 Table of refractive indices for drugs, arranged according to ascending value of the lowest index 9
a /3 7 Compound
Alkaloids and Related Amines
1.490
1.701
>1.734
1.499
1.572
1.597
1.500
1.535
>1.733
1.508
1.516
1.628
1.509
1.566
1.613
1.512
1.595
1.688
1.513
_
1.619
1.522
1.574
1.612
1.526
1.597
1.639
1.529
1.538
1.646
1.530
1.603
1.638
1.536
1.617
>1.673
1.537
__
1.556
1.540
1.590
1.635
1.543
1.636
1.684
1.545
1.581
1.618
1.545
1.595
1.653
1.545
1.617
1.661
1.545
1.620
1.632
1.548
1.563
1.688
1.550
1.583
1.595
1.550
1.734
>1.734
1.555
__
1.60
1.555
1.590
1.655
1.555
nyl.733
>1.733
1.556
1.607
1.627
1.558
1.581
1.585
1.559
1.580
1.676
1.560
—
1.575
1.560
1.565
1.589
1.561
1.642
1.661
1.562
__
1.581
1.562
_
>1.65
1.562
1.604
1.660
1.565
1.607
1.670
1.567
1.585
1.623
1.568
1.636
1.638
1.568
1.659
si >1.800
1.57
1.61
1.69
1.570
1.596
1.618
1.570
1.608
1.630
1.570
1.685
1.690
1,575
1.590
1.627
1.577
_
1.594
1.578*,
1.613«
1.580
1.615
1.617
1.580
1.625
1.645
1.580
1.665
1.690
1.586
1.622
1.698
1.590
1.610
1.669
1.600
—
1.620
1.600
__
1.635
1.610
1.625
1.675
1.610
1.626
1.668
1.612
1.637
1.644
1.617
1.660
>1.690
1.620
1.625
1.630
1.625
1.690
>1,690
1.638
1.658
1.701
1.474
1.484
1.513
1.483
1.488
1.515
1.484
1.492
1.516
1.490
1.515
1.567
1.496
1.506
1.510
1.498
1.507
1.563
Berberine
Alphaprodine.HCI
Berberine. HCI. 2H 2
Phenmetrazine. HCI
Cetylpyridinium chloride
Brucine sulfate
Ethylhydrocupreine. HCI
Isobucaine. HCI
6 -Monoacetylmorphine. HCI
Syrosingopine
/-Ephedrine.HCI
Phensuximide
Ethoheptazine citrate
Morphine. HCI. H 2
Codeine
Meperidine. HCI
Levallorphan tartrate
Cinchonine.HCI.2H 2
Morphine sulfate. 5H2O
Yohimbine
Atropine
Hydra stine
Atropine sulfate
Arecoline.HBr
Papaverine. HCI
Diacetylmorphine
Methylphenidate.HCI
Codeine. HCI
Aconitine
Benzethonium chloride
Codeine sulfate
Hyoscyamine
Brucine
Cinchonidine sulfate
Quinidine sulfate
Scopolamine. HBr
Dextropropoxyphene napsylate
Methaqualone.HCl
Yohimbine. HCI
Cocaine. HCI
Racephedrine.HCI
Cinchonine
Pentazocine
Pentazocine. HCI
Diacetylmorphine. HCI . H2O
Methscopolamine bromide
Morphine. H2O
Quinidine
Desipramine. HCI
Quinine. HCI
Phenylbutazone
Diphenylhydantoin
Cinchonidine
Strychnine. HCi.2H 2
Morphine, HCI, anhyd.
Strychnine
Quinine
Papaverine
Apomorphine.HCI
Antibiotics
Carbomycin
Erythromycin estolate
Erythromycin oxalate. 2H 2
Erythromycin ethylsuccinate
Erythromycin ethylcarbonate
Erythromycin stearate
See 52.023 for symbols.
(Continued)
AOAC Official Methods of Analysis (1990) Reference Tables 669
955.58 Table of refractive indices for drugs, arranged according to ascending value of the lowest index 3
— Continued.
a
7
Compound
Antibiotics — Continued
1.506
—
1.528
Erythromycin gluceptate
1.512
1.523
1.532
Erythromycin . 2H2O
1.512
1.583
1.682
Nystatin
1.518
ca 1.572
>1.780
Fumagillin
1.522
1.548
1.566
Dihydrostreptomycin . 3HCI
1.523
1.608
1.659
Chloramphenicol
1.523
1.622
1.630
Penicillin G benzathine
1.527
__
1.569
Chloramphenicol palmitate
1.528
1.536
1.550
Erythromycin . HI . H2O
1 538
1.646
s! >1.787
Tetracycline. 3H2O
n 1.541
Neomycin sulfate
1.541
ca 1.553
1.573
Gramicidin
1.541
1.585
1.656
Penicillin O chloroprocaine
1.545
_
1.593
Penicillin O potassium
1.545
1.570
1.685
Penicillin G procaine
1.546
1.635
1.730
Oxytetracycline. HCI
1.550
—
1.603
Penicillin G potassium
1.550
1.609
1.620
Penicillin G sodium
1.552
1.558
1.566
Dihydrostreptomycin sulfate
1.553
—
1.584
Tyrocidine.HCI
1.556
ca 1.590
1.619
Penicillin G hydrabamine
1.565
—
1.629
Novobiocin sodium
1.567
__
1.613
Penicillin G dibenzylamine
1.575
—
1.610
Penicillin G /-ephedrine
1.583
—
1.630
Cycloserine
1.583
1.590
1.648
Penicillin G /-ephenamine
1.601
1.608
1.632
Penicillin G.HI diethylaminoethyl ester
1.603
1.685
1.714
Tetracycline. HCI
1.608
1.638
1.654
Novobiocin acid, form 2
1.634
1.646
>1.700
Oxytetracycline. 2H2O
1.635
1.706
1.730
Chlortetracycline.HCI
Antihistamines
1.505
1.585
1.617
Anthallan®.HCI
1.509
1.564
1.683
Dexchlorpheniramine maleate
ca 1.525
1.563
1.598
Doxylamine succinate
1.533
nil. 668
si <1.734
Chlorpheniramine maleate
1.548
1.574
1.665
Pheniramine maleate
1.553
1.625
>1.734
Chlorothen.HCI
1.566
1.614
1.653
Pyrrobutamine phosphate
1.577
1.631
1.672
2(4-Morpholinyl) ethyl benzhydryl ether.
HCI
1.580
1.655
1.705
Tripelennamine. HCI
1.583
1.603
1.645
Chtorothen citrate
1.585
1.600
1.668
p-Fluorotripelennamine. HCI
1.587
1.635
1.734
Phenbenzamine.HCI
1.588
1.654
>1.695-
<1.734 Methapyrilene.HCI
1.590
—
1.680
Thenyl diamine. HCI
1.590
1.610
1.665
ChlorcycNzine.HCI
1.602
1.625
1.630
Diphenhydramine. HCI
1.604
1.675
1.733
M etna phenilene. HCI
1.610
1.660
1.665
Chlorcyclizine.2HCI
1.612
1.679
1.691
Thonzylamine. HCI
1.617
1.654
1.734
Bromothen.HCI
1.617
1.691
1.733
Promethazine. HCI
1.620
1.647
1.738
Cyproheptadine. HCI
1.667
1.675
>1.733
Promethazine. HBr
1.690
—
1.737
Pyrathiazine.HCI
Barbiturates
1.445
1.548
1.580
Barbital
1.454
1.518
1.556
Butethal
1.465
—
1.565
Pentobarbital
1.465
1.529
1.532
Butabarbital sodium
1.467
1.533
1.560
Amobarbital
1.473
1.519
1.549
Hexethal
(Continued)
670 Reference Tables AOAC Official Methods of Analysis (1990)
955.58 Table of refractive indices for drugs, arranged according to ascending value of the lowest index 3
—Continued.
a y Compound
Barbiturates — Continued
1.477 — 1.523 Pentobarbital sodium
1.477 1.573 1.624 Probarbital
1.487 1.557 1.563 Secobarbital
1.490 nil. 500 1.525 Secobarbital sodium
n 1.505 Amobarbital sodium
1.506 1.544 1.672 Vinbarbital
1.508 n;l- 521 1.577 Butalbital
1.512 1.532 1.615 Barbital sodium
1.515 1.546 1.621 Cyclobarbital
1.516 1.572 1.625 Allobarbital
1.519 1.583 1.634 Sigmodal®
1.520 1.575 1.626 Cyclopal®
1.520 1.581 1.600 Aprobarbital
1.524 1.577 1.603 Butallylonal
1.532 — 1.629 Probarbital sodium
1.534 1.634 — Thiopental
1.546 1.608 1.634 Hexobarbital
1.551 1.578 1.645 Alphenal
1.557 1.620 1.667 Phenobarbital
1.594 1.610 1.651 Mephobarbital
Hallucinogens
1.517 1.612 1.679 3,4-Methylenedioxyamphetamine. HCI
1.518 1.622 1.632 4-Methyl-2,5-dimethoxyamphetamine. HCI
1.527 1.554 1.672 Psilocybin
1.537 — 1.629 3,4-Methylenedioxyamphetamine sulfate
1.540 1.596 1.676 d-Lysergic acid diethylamide tartrate
1.564 1.586 1.598 3,4-Methylenedioxyamphetamine carbonate
1,572 1.618 1.654 Phencyclidine. HCI
1.572 1.620 1.654 Phencyclidine. HBr
Steroids
1.506 — 1,598 Estradiol dipropionate
1.510 1.542 1.572 Dehydrocholic acid
1.511 1.621 1.697 Estrone, phase 2
1.512 1.552 1.621 Cortisone acetate
1.517 1.567 1.592 Triamcinolone diacetate
1.520 1.532 1.566 Cholesterol
1.520 1.642 1.692 Estrone, phase 1
1.529 1.550 1.630 Desoxycorticosterone acetate
1.529 1.575 1.676 Progesterone, beta
1.531 1.550 1.638 Hydrocortisone
1.534 1.677 1,705 Equilin
1.542 1.554 1.663 Progesterone, alpha
1.543 1.589 1.627 Hydrocortisone acetate
1.546 e 1.595^ Triamcinolone acetonide
1.548 1,565 1.670 Testosterone
1.552 — 1.561 Methylprednisolone sodium succinate
1.552 1.572 1.625 Cortisone
1.553 1.572 1.648 Dexamethasone
1.554 — 1.667 Betamethasone
1.555 ca 1.565 1.620 Methyltestosterone
1.562 1.575 1.700 Methylprednisolone acetate
1.562 1.568 1.704 Fluorometholone
1.576 1.625 1.645 Ethisterone
1.586 1.603 1.633 Estradiol benzoate
1.587 1.590 1.651 Prednisone
1.594 1.611 1.73 Diethylstilbestrol, trans
1.594 1.628 1.647 Estrone, phase 3
Sulfonamides
1.513 1.590 >1.690-<1.733 Sulfallantoin®
1.540 1.655 1.690 Sulfanilamide, HCI
1,555 1.672 1.85 Sulfanilamide, phase B (anhyd.)
1.559 1.564 1.727 Sulfacetamide
(Continued}
AOAC Official Methods of Analysis (1990) Reference Tables 671
955.58 Table of refractive indices for drugs, arranged according to ascending value of the lowest index 3
—Concluded.
a. /? 7 Compound
Sulfonamides — Continued
1.568 1.657 1.687 Sulfamerazine
1,578 1.676 1.710 Succinylsulfathiazole
1.584 1.623 >1.778 Sulfamethazine
1.586 1.649 1.731 Sulfaguanidine.H 2
1.587 — 1.675 Sulfamerazine 6
1.590 — 1.700 Sulfapyridine sodium. H 2
1.596 — 1.621 Sulfathiazole sodium. lVa^O
1.596 1.675 1.830 Sulfadiazine
1.598 1.741 1.780 Sulfathiazole, phase II
1.605 1.642 1,697 Sulfisoxazole
1.606 1.663 1.734 Sulfaguanidine
1.615 1.663 >1.734 Sulfadiazine 6
1.661 1.678 >1.733 Sulfamidazole®
1.670 1.736 1.813 Sulfapyridine, phase I
1.674 1.685 >1.733 Sulfathiazole, phase I
1.680 1.733 >1.733 Sulfapyridine 6
1.695 n t 1.733 >1.733 Sulfathiazole 6
Sympathomimetic Amines
1.458 w 1.468 e Tuaminoheptane sulfate
1.501 1.545 1.603 Dextroamphetamine sulfate
1.507 1.604 1.668 p-Hydroxyephedrine.HCI
1.508 1.582 1.611 d/-Amphetamine.HCI
1.508 1.648 1.670 Isoxsuprine.HCI
1.516 1.552 1.645 p-Hydroxymethamphetamine sulfate
1.516 n t 1.620 1.689 d/-Synephrine (+) tartrate (neutral salt)
1.520 1.531 1.614 d/-Amphetamine sulfate
1.530 1.537 1.615 Methamphetamine.HCI
1.530 1.585 1.596 Mephentermine sulfate
1.530 1.603 1.638 /-Ephedrine. HCI
1.535 1.540 1.620 d/-Methamphetamine. HCI
1.535 1.602 1.607 Phendimetrazine. HCI
1.540 1.565 1.587 /-Ephedrine sulfate
1.543 — 1.632 Pseudoephedrine.HCI
1.544 1.594 1.614 Phendimetrazine tartrate
1.546 1.583 1.664 Dextroamphetamine phosphate, dibasic
1.546 1.604 ca 1.725 d/-Synephrine base
1.548 1.597 >1.735 Epinephrine
1.549 1.589 1.665 d/-Amphetamine phosphate, dibasic
1.549 1.605 1.664 Synephrine. HCI
1.560 1.592 1.622 Dextroamphetamine. HCI
1.560 1.619 >1.740 Naphazoline nitrate
1.560 1.680 1.734 Hydroxyamphetamine. HBr
1.563 1,618 1.650 Phenylpropanolamine. HCI
1.570 1.608 1.630 Racephedrine. HCI
1.572 1.587 1.650 Leva mfeta mine succinate
1.577 — 1.603 Phenylpropylmethylamine.HCI
1.586 1.604 1.703 Tolazoline.-HCI
Tranquilizers
ca 1.515 — ca 1.544 Meprobamate
1.530 1,536 1.546 Ethinamate
1.572 1.585 1.590 Glutethimide
1.584 — >1.735 Chlorpromazine.HCI
1.589 1.609 1.700 Thiopropazate.2HCI
1.634 ca 1.710 >1.780 Chlordiazepoxide. HCI
1.638 1.647 1.674 Azacyclonol . HCI
6 The second set of optical properties in each case represents intermediate data which are commonly found in
some commercial samples. They probably represent an anhydrous form or merely a different common orientation
of the crystal.
^672
Reference Tables
AOAC Official Methods of Analysis (1990)
963.37 Nomograph relating absorbance, concentration, and absorptivity (1 cm cell)
(A straight edge placed at known values on two appropriate axes (i.e., absorbance and absorptivity) will intersect
the corresponding value on the third axis (i.e., concentration).)
1.0
| 3
2 5
2.0
—
1.0
0.8
20
0.6
" — '
30
,
4.0
~~
0.4
. 1.5
6.0
0.2
-^
8.0
0.1
—
10.0
— .
0.08
i l.o
0.06
200
i 09
—
0.04
0.8
-
30.0
-
0.02
40.0
07
••x
— *
— - 6
cr _
UJ __
0.01
0.008
^ —
600
t
0.006
5 -
800
0.5
-i
tc\ • —
0.004
u _a
100
gram:
i 1 i
5 _
O
— • 0.4
002
200
z
<
CD
J
^~*
0.001
> ~"
300
cr
0.3
z _
O __
0.0008
r-
400
0.0006
>
CO
CD
- 0.25
< —
0,0004
H —
0. J,
Ct —
o „r
CO
CD
< -
600
<
0.2
a. -
r-
z —
UJ _
O
z —
0.0002
0001
800
1000
- 0.1
o —
000008
2000
o —
000006
_
_
—
0.00004
—
3000
-
—
4000
— 0.1
0,00002
-
—
6000
I_ 0.09
0.00001
8000
— 08
000008
_JI
10000
0.000006
— 0.07
0.000004
— 0.06
20000
-
0. 000002
-
— 0.05
—
30000
-
0.00000 1
—
40000
— 004
—
60000
-
—
80000
100000
003
Reprinted from The Analyzer 4, No. 2 (1963)
Beckman Instruments, Inc., Fullerton, Calif.
Appendix: Guidelines for Collaborative Study Procedure to
Validate Characteristics of a Method of Analysis
(incorporates symbols, terminology, and recommendations accepted by consensus by the participants at the IUPAC Workshop on
Harmonization of Collaborative Analytical Studies, Geneva, Switzerland, May 4-5, 1987.)
This document has been prepared from the point of view of AOAC administrative arrangements. In AOAC, the Associate Referee
is the individual scientist responsible for choosing the method, conducting intra- and interlaboratory tests, evaluating the results,
and recommending approval of a method of analysis. Other organizations who may utilize this document may wish to substitute
their own terminology. Although the directions were developed for chemical studies, some parts may be applicable to all types
of collaborative studies.
Summary Statement of AOAC Recommendation for the Design of a Collaborative Study:
1. Minimum number of materials: 5 (only when a single level specification is involved for a single matrix may this minimum
be reduced to 3).
2. Minimum number of laboratories: 8 reporting valid data for each material (only in special cases involving very expensive
equipment or specialized laboratories may the study be conducted with a minimum 5 laboratories, with the resulting expansion in
the confidence interval for the statistical estimates of the method characteristics).
3. Minimum number of replicates: 1, if within-laboratory repeatability parameters are not desired; 2, if these parameters are
required. Replication should ordinarily be attained by blind replicates or split levels (Youden pairs).
Guide to Collaborative Study Procedure
(Section numbers correspond to Outline; not all section numbers are included in guide.)
1 . Preliminary Work
1 . 1. Determine purpose of method
1.2 Choose method
1.3 Optimize method
1.5 Prepare description of method
1 .6 Invite participation
2. Design of Collaborative Study
2.1 General principles
2.2 Laboratories
2.3 Materials
2.4 Replications
3. Preparation of Materials
4. Submission of Materials
5. Obligations of Collaborators
6. Statistical Analysis
6.1 Initial review of the data
6.3 Outliers
6.4 Bias (systematic error)
6.5 Precision (random error)
6.5.1 Reproducibility
6.5.2 Repeatability
6.6 False positive and false negative values
7. Final Report
8. References
1. Preliminary Work (Within One Laboratory)
1.1 Determine Purpose and Scope of the Study and Method
Determine purpose of the study (e.g., to determine attributes of a method, proficiency of analysts, reference values of a
material, or to compare methods), the type of method (empirical, screening, practical, reference, definitive), and the
probable use of the method (enforcement, surveillance, monitoring, acceptance testing, quality control, research). Also,
on the basis of the relative importance of the various method attributes (bias, precision, specificity, limit of determination),
select the design of the interlaboratory study. The directions in this document pertain primarily to determining the precision
characteristics of a method, although many sections are also appropriate for other types of studies.
1.2 Alternatives for Method Selection
1.2.1 Sometimes obvious (only method available)
1.2.2 Critical literature review (reported within-laboratory attributes are often optimistic)
1 .2.3 Survey of laboratories to obtain candidate methods; comparison of within-laboratory attributes of candidate methods
(sometimes choice may still not be objective)
1.2.4 Selection by expert (AOAC-preferred procedure — selection by Associate Referee with concurrence of General Ref-
eree)
1.2.5 Selection by committee (ISO-preferred procedure — often time-consuming)
1.2.6 Development of new method or modification of existing method when an appropriate method is not available.
(Proceed as a research project.) (This alternative is time-consuming and resource-intensive; use only as a last resort).
673
674 Guidelines for Collaborative Study Procedure AOAC Official Methods of Analysis (1990)
13 Optimize Either New or Available Method
1.3.1 Practical principles
(a) Do not conduct collaborative study with an unoptimized method. An unsuccessful study wastes a tremendous amount
of collaborators' time and creates ill will. This applies especially to methods that are formulated by committees and
have not been tried in practice.
(b) Conduct as much experimentation within a single laboratory as possible with respect to optimization, ruggedness,
and interferences. Analysis of the same material on different days provides considerable information on variability
that may be expected in practice.
1.3.2 Alternative approaches to optimization
(a) Conduct formal ruggedness testing for identification and control of critical variables. See Youden and Steiner (8.1,
pp, 33-36, 50-55). The actual procedure is even simpler than it appears. (This is an extremely efficient way for
optimizing a method.)
(b) Use Deming simplex optimization to identify critical steps. See Dols and Armbrecht (8.3).
(c) Conduct trials by changing one variable at a time.
1.4 Develop Within-Laboratory Attributes of Optimized Method
(Some items can be omitted; others can be combined.)
1.4.1 Determine calibration function (response vs concentration in pure or defined solvent) to determine useful mea-
surement range of method. For some techniques, e.g., radioimmunoassay, linearity is not a prerequisite. Indicate
any mathematical transformations needed.
1.4.2 Determine analytical function (response vs concentration in matrix, including blank) to determine applicability to
commodity(ies) of interest.
1.4.3 Test for interferences (specificity):
(a) Test effects of impurities, ubiquitous contaminants, flavors, additives, and other components expected to be present
and at usual concentrations.
(b) Test nonspecific effects of matrices.
(c) Test effects of transformation products, if method is to indicate stability, and metabolic products, if tissue residues
are involved.
1 .4.4 Conduct bias (systematic error) testing by measuring recoveries of analyte added to matrices of interest and to
extracts, digests, or other treated solutions thereof. (Not necessary when method itself defines the property or
component.)
1.4.5 Develop performance specifications for instruments and suitability tests for systems (which utilize columns or
adsorbents) to ensure satisfactory performance of critical steps (columns, instruments, etc.) in method,
1.4.6 Conduct precision testing at the concentration levels of interest, including variation in experimental conditions
expected in routine analysis (ruggedness).
In addition to estimating the "classical" repeatability standard deviation, s r , the initiating laboratory may estimate
the total within-laboratory variability expected by determining the variability at different days and with different
calibration curves, by the same or different analysts within a single laboratory. This total within-laboratory estimate
includes both between-run (between-batch) and within-run (within-batch) variability.
1 .4.7 Delineate the range of applicability to the matrices or commodities of interest.
1.4.8 Compare the results of the application of the method with existing tested methods intended for the same purposes,
if other methods are available.
1.4.9 If any of the preliminary estimates of the relevant performance of these characteristics are unacceptable, revise the
method to improve them, and retest as necessary.
1.4.10 Have method tried by analyst not involved in its development.
1.4.1 1 Revise method to handle questions raised and problems encountered.
7.5 Prepare Description of Method
Note: A collaborative study of a method involves practical testing of the written version of the method, in its specific style
and format, by a number of laboratories on identical materials.
1 .5.1 Prepare method in format and style given in the Handbook for AOAC Members (1989), or other recognized manual,
e.g., ISO Guide 18 (8.4).
1.5.2 Clearly specify requirements for chromatographic materials, enzymes, antibodies, and other performance-related
reagents.
1.5.3 Clearly describe and explain every step in the analytical method so as to discourage deviations. Use imperative
directions; avoid subjunctive and conditional expressions as options as far as possible.
1.5.4 Edit method for completeness, credibility (e.g., buffer pH consistent with specified chemicals, volumes not greater
than capacity of container), continuity, and clarity.
1.5.5 Check for inclusion of performance specifications and systems suitability tests (1.4.5), defined critical points, and
convenient stopping points. Incorporate physical or chemical constants of working standard solutions, e.g., absorp-
tivities, half-scale deflections, recoveries, etc., or properties of operating solutions and chromatographic materials,
e.g., pH, volumes, resolution, etc., and any other indicators (e.g., sum equals 100%) that suggest analysis is pro-
ceeding properly.
1.5.6 If time and resources are available, conduct pilot study involving 3 laboratories.
AOAC Official Methods of Analysis (1990) Instructions for Collaborators 675
1.6 Invite Participation
1.6.1 Selection of candidate laboratories. Laboratories invited to participate should have personnel experienced in the
basic techniques employed; experience with the method itself is not a prerequisite for selection. Lists of possible
participants can be developed through personal contacts, technical societies, trade associations, or literature search,
and advertisements in the AOAC's news publication "The Referee."
1.6.2 Letter of invitation. Address a formal letter to the individual responsible for assignment of laboratory effort. State
reason for selecting that laboratory (e.g., as a volunteer or has responsibility or familiarity with the problem or
method), estimated number of person-hours required for performance, number of materials to be sent, number of
analyses to be required, expected date for material distribution, and target date for completion of the study. Em-
phasize the importance of management support in assigning the necessary time for the project. Enclose a copy of
the method and a return form or card (with postage affixed, if domestic), requiring only a check mark for acceptance
or refusal of the invitation, a signature, and space for address corrections, telephone number, and date.
With large studies, involving several analysts per laboratory, several familiarization materials, receipt of items at
different times, or similar recurrent situations, acceptance of the invitation should be followed by a letter suggesting
that a study coordinator be appointed. The study coordinator should be responsible for receiving and storing the
materials, assigning the work, dispensing materials and information related to the study, seeing that the method is
followed as written, accumulating the data, assuring that the data are correctly reported, and submitting the report
within the deadline.
(A file of letters previously used for these purposes is- available).
1 . 7 Instructions and Report Forms
1.7.1 Carefully design and prepare instructions and forms, and scrutinize them before distribution. A pilot study (1.5.6)
is also useful for uncovering problems in these documents.
1.7.2 Send instructions and report forms immediately on receipt of acceptance, independent of materials, if selection of
laboratories is not to be based on performance in pilot or training studies. The instructions should include in bold
face or capital letters a statement: "THIS IS A STUDY OF THE METHOD, NOT OF THE LABORATORY. THE
METHOD MUST BE FOLLOWED AS CLOSELY AS PRACTICABLE, AND ANY DEVIATIONS FROM THE
METHOD AS DESCRIBED, NO MATTER HOW TRIVIAL THEY MAY SEEM, MUST BE NOTED ON THE
REPORT FORM."
1.7.3 Include instructions on storage and handling, markings, and identifications to be noted, any special preparation for
analysis, and criteria for use of practice or familiarization materials, if included (see 1.8). Precode the form for
each laboratory and provide sufficient space for as much sequential data as may be required for proper evaluation
of the results, including a check of the calculations.
Indicate the number of significant figures to be reported.
When recorder tracing reproductions are required to evaluate method performance, request their submission both in
the instructions and as a check item on the form. Provide instructions with regard to labeling of recorder tracings,
such as identification with respect to item analyzed, axes, date, submitter, experimental conditions, and instrument
settings.
Include in the report form a signature line for the analyst and lines for a printed or typed version of the name and
address for correct acknowledgment. Provide for a review by the laboratory supervisor. An example of a completed
form is helpful. A questionnaire may be included or sent after completion of the analyses in which the questions
can be designed to reveal if modifications have been made at critical steps in the method,
1 .7.4 Request a copy of the calibration curve or other relationship between response and concentration or amount of analyte
so that if discrepancies become apparent after examining all of the data, it can be determined whether the problem
is in the calibration or in the analysis.
1.8 Familiarization or Practice Materials
If deemed necessary, supply as far ahead as practicable, familiarization materials, with instructions, before actual materials
are sent. When familiarization materials have been submitted, supply forms for reporting progress toward satisfactory
performance.
2. Design of the Collaborative Study
2.1 General Principles
2.1.1 The purpose of the collaborative study is to provide a realistic estimate of the attributes of a method, particularly
the systematic and random deviations, to be expected when the method is used in actual practice. A collaborative
study usually provides information on the best performance to be expected.
2.1.2 The design should attempt to identify and to include the possible sources of significant variability that may occur
in actual practice, including between days, between runs, and between calibration curves, if these are significant
factors. (Within-laboratory performance, 1.4.6, particularly on different days and with different calibration curves
may provide a clue with respect to between-laboratory performance and is required information for quality control.)
The best measure of within-laboratory variability is obtained by using blind replicates or split levels (Youden pairs).
The design must take into account how the data will be analyzed statistically.
676 Guidelines for Collaborative Study Procedure AOAC Official Methods of Analysis (1990)
2.1.3 Present materials sent for analysis as unknowns (blind) and coded in a random pattern. If necessary to conserve
analyst time, an indication of the potential range of concentration or amount of analyte may be provided. If spiking
solutions are used, provide one coded solution for each material. All spiking solutions should be identical in ap-
pearance and volume. Do not provide a single solution from which aJiquots are to be removed for spiking. Any
information with regard to concentration (e.g., utilizing factorial aliquots or serial dilutions of the same spiking
solutions) or known replication is likely to lead to an underestimate of the variability.
2.1.4 The study must be extensive enough to assure sufficient data surviving in the face of possible loss of materials
during shipment, inability of collaborators to participate after acceptance, and a maximum outlier rate of 2/9 (2
laboratory failures in a 9-laboratory study, when a minimum design is used).
2.1.5 Improper preparation of reference standards and standard solutions can cause a significant portion of the analytical
error. A decision must be made whether such error is to be considered separately or as part of the method, i.e.,
will the analysts procure their own standards and prepare their own standard solutions or will standards be provided
by the Associate Referee. The decision depends primarily on the availability of the standard. If the standard is
readily available, the analysts should prepare their own. If the standard is not readily available, the standard may
be supplied, but physical constants, e.g., absorptivity, of working standard solutions should be incorporated into
the description as a check on proper preparation of the solution.
2.1.6 Obtain the necessary administrative and operational approvals. Review by potential users of the method is also
desirable.
2.2 Laboratories
Laboratories must realize the importance of the study. A large investment is being made in testing the method and this
probably will be the only collaborative study of the method that will be performed. Therefore, it is important to have a
fair and thorough evaluation of the method.
2.2.1 Type. The most appropriate laboratory is one with a responsibility related to the analytical problem. Laboratory
types may be representative (selection of laboratories that will be using the method in practice), reference (assumed
to be "best"), or the entire population of laboratories (usually certified or accredited) that will be using the method.
Final selection of participants should be based on a review with the General Referee and other Associate Referees
of each laboratory's capabilities and past performance in collaborative studies, followed up, if possible, by telephone
conversations or by personal visits. Selection may also be based on performance with familiarization materials.
Sometimes only laboratories with dedicated or very specialized instruments must be used. If the study is intended
for international consideration, laboratories from different countries should be invited to participate.
2.2.2 Number of laboratories: Minimum of 8 laboratories submitting valid data (to avoid unduly large confidence bands
about the estimated parameters). Only in special cases of very expensive equipment or specialized laboratories may
the study be conducted with a minimum of 5 laboratories. Fewer laboratories widen the confidence limits of the
mean and of the variance components (see design considerations 2.4.1 and 2.4.2). The optimum number of labo-
ratories, balancing logistics and costs against information obtained, often is 8-10. However, larger studies are not
discouraged.*
2.2.3 Analysts: Most designs require only 1 analyst per laboratory. If analyst- within- laboratory variability is a desired
variance component, multiple analysts should be requested from all participating laboratories. Ordinarily 2 analysts
from the same laboratory cannot be substituted for different laboratories, unless standard solutions, reagents, chro-
matographic columns and/or materials, instrument calibrations, standard curves, etc., are prepared independently,
and no consultation is permitted during the work. Different laboratories from the same organization may be used
as separate laboratories if they operate independently with their own instruments, standards, reagents, and super-
vision.
2.3 Test Materials
2.3.1 Materials must be homogeneous. This is critical. Establish homogeneity by testing a representative number of lab-
oratory samples taken at random before shipment. (A collaborator who reports an outlying value will frequently
claim receipt of a defective laboratory sample.) The penalty for inhomogeneity is an increased variance in the
analytical results that is not due to the intrinsic method variability.
2.3.2 Code laboratory samples at random so that there is no preselection from order of presentation.
2.3.3 Concentration range: Choose analyte levels to cover concentration range of interest. If concentration range of interest
is a tolerance limit or a specification level, bracket it and include it with materials of appropriate concentration. If
design includes the determination of absence of analyte, include blank (not detectable) materials as part of range of
interest.
2.3.4 Number of materials: Minimum number of materials is 5. However, when a single-level specification is involved,
this may be reduced to an absolute minimum of 3.
2.3.5 Nature of materials: Materials should be representative of commodities usually analyzed, with customary and ex-
treme values for the analyte.
2.3.6 Size of laboratory samples: Furnish only enough material to provide the number of test portions specified in the
instructions. If additional test portions are required, the collaborator must request them, with an explanation.
* In some cases Associate Referees are unable to obtain more than 5 participants. In such cases a study may be conducted with 5 laboratories, but it must be
realized that the reliability of the resulting estimates of performance parameters is reduced considerably. Furthermore, in such cases the number of test materials in
the study should be increased to provide some additional assurance, but the analysis of more test materials is not a substitute for fewer laboratories. Methods adopted
on the basis of such a design may not be acceptable to other organizations that have adopted the IUPAC-87 recommendations.
AOAC Official Methods of Analysis (1990) Design Considerations 677
2.3.7 Interferences: If pertinent, some materials, but not all, should contain contaminants and interferences in concentra-
tions likely to be encountered, unless they have been shown to be unimportant through within- laboratory testing
(see 1.4.4). The success of the method in handling interference on an intralaboratory basis will be demonstrated by
passing systems suitability tests.
2.3.8 Familiarization materials: With new, complex, or unfamiliar techniques, provide material(s) of stated composition
for practice, on different days, if possible (see 1.8). The valuable collaborative materials should not be used until
the analyst can reproduce the stated value of the practice samples within a given range. However, it should be
pointed out that one of the assumptions of analysis of variance is that the underlying distribution of results is in-
dependent of time (i.e., there is no drift). The Associate Referee must be satisfied that this assumption is met.
2.4 Replication
When within-laboratory variability is also of interest, as is usually the case, independent replication can be ensured by
applying at least one of the following procedures (listed in suggested order of desirability; the nature of the design should
not be announced beforehand):
2.4.1 Split levels (Youden pairs): A pair of materials of slightly different composition obtained either naturally or by
diluting (or by fortifying) one portion of the material with a small amount of diluent (or of analyte). Both portions
are supplied to the participating laboratories under a random code number and each portion should be analyzed only
once; replication defeats the purpose of the design.
2.4.2 Split levels for some materials and blind duplicates for other materials in the same study (obtain only single values
from each portion supplied).
2.4.3 Blind duplicate laboratory samples, randomly coded. Note: Triplicate and higher replication are relatively inefficient
when compared with duplicate test samples because replication provides additional information only on individual
within-laboratory variability, which is usually the less important component of error. It is more effective to utilize
resources for the analysis of more levels and/or materials rather than for increasing the number of replicates for the
individual materials.
PRACTICAL PRINCIPLE: With respect to replication, the greatest net marginal gain is always obtained in going
from 2 to 3 as compared to going from 3 to 4, 4 to 5, etc.
2.4.4 Independent materials. (Note: Unrelated independent materials may be used as a split level in the calculations of
the precision parameters or for plotting, but the more they differ, particularly with respect to concentration, the less
reliable the information they provide on within-laboratory variability.)
2.4.5 Use of known replicates is a common practice. It is much preferable to use the same resources on blind replicates
or split levels. (See Note of 2.4.3.)
2.4.6 Instead of obtaining repeatability parameters through the collaborative study, use of quality control materials in each
laboratory individually, for its own use, independent of the collaborative study, for a separate calculation of s r ,
using 2 (or more) replicates from each quality control test, according to the pattern developed for each product.
2.5 Other Design Considerations
2.5.1 The design can be reduced in the direction of less work and less cost, but at the sacrifice of reduced confidence in
the reliability of the developed information.
2.5.2 More work (values) is required if more confidence is needed, e.g., greater confidence is required to enforce a
tolerance at 1 .00 ppm than at 1.0 ppm. (The distinction is a precision requirement of the order of 1% rather than
10%.)
2.5.3 The estimate of the standard deviation and of the corresponding relative standard deviation is a random variable.
For example, 30 data points from a single population will permit estimation of the standard deviation of an individual
reading only to within ±25% with about 95% confidence; 200 data points are required to estimate that standard
deviation to within about 10%, with about 95% confidence. The distribution of standard deviations is actually asym-
metrical for small numbers of values, e.g., the relative distribution about 1.00 for 30 values (about 95% confidence
limits) is 0.78-1.30; for 200, 0.91-1.10.
2.5.4 The validity of extrapolating the use of a method beyond concentrations and components tested can be estimated
only on the basis of the slope of the calibration curve (sensitivity) observed as a function of the nature and con-
centration of the matrix and contaminant components. If the signal is more or less independent of these variables,
a reasonable amount of extrapolation may be utilized. The extrapolator assumes the burden of proof as to what is
reasonable .
3. Preparation of Materials for Collaborative Studies
3.1 General Principles
3. 1.1 Heterogeneity between portions from a single test material must be negligible compared to analytical variability, as
measured within the Associate Referee's laboratory. See also 2.3.1.
3.1.2 The containers must not contribute extraneous analytes to the contents, and they must not adsorb or absorb analytes
or other components from the matrix, e.g., water.
3.1.3 If necessary, the materials may be stabilized, preferably by physical means (freezing, dehydrating), or by chemical
means (preservatives, antioxidants) which do not affect the performance of the method.
3.1.4 Compositional changes must be avoided, where necessary, by the use of vapor-tight containers, refrigeration, flush-
ing with an inert gas, or other protective packaging.
678 Guidelines for Collaborative Study Procedure AOAC Official Methods of Analysis (1990)
3.2 Materials Suitable for Collaborative Studies
3.2.1 A single batch of homogeneous, stable product such as milk powder, peanut butter, vegetable oil, starch, etc., is
the best type of material.
3.2.2 Reference materials supplied by standards organizations such as the National Bureau of Standards are excellent,
unless they have easily recognizable characteristics (e.g., odor and color of NBS Orchard Leaves). However, they
are of limited availability, composition, and analyte level. If available, they are expensive. Sometimes the certifi-
cation organization may be interested in making reference materials available for the analyte under study, in which
case it may assist in providing the material for the study.
3.2.3 Synthetic materials may be especially formulated with known amounts of analytes by actual preparation for the
study. This procedure is best used for macroconstituents such as drugs or pesticide formulations.
3.2.4 Spiked materials consisting of normal or blank materials to which a known amount of analyte has been added may
be used. The amount of analyte added should not be excessive in relation to the amount present (e.g., about 2x),
and the analyte added should be in the same chemical form as present in the commodities to be analyzed subse-
quently.
In drug and pesticide residue-type problems, it is often necessary to use spiked materials in order to assess recovery.
However, because incurred residues are likely to present different problems from those of spiked residues, collab-
orative studies should include some incurred test samples to ensure that the method is applicable under these con-
ditions as well.
(a) Preparation in bulk: This requires thorough and uniform incorporation of the analyte, often by serial dilution of
solids. The danger of segregation due to differences in densities always exists. Fluid materials susceptible to seg-
regation should be prepared under constant agitation. Uniformity should be checked by direct analysis, with an
internal standard, or by a marker compound (dye or radioactive label).
(b) Laboratory samples, individually prepared: A known amount of analyte is either weighed directly or added as an
aliquot of a prepared solution to premeasured portions of the matrix in individual containers. The collaborator is
instructed to use each entire portion for the analysis, transferring the contents of the container quantitatively or a
substantial weighed fraction of the portion. (This is the preferred alternative to spiked solid materials at trace [ppm]
levels, at the expense of considerably more work.)
(c) Concentrated unknown solutions for direct addition by collaborators to their own commodities: Should be used only
as a last resort when instability of the analyte precludes distribution from a central point. To preclude direct analysis
of the spiking solution, supply individual coded solutions to be added in their entirety to portions of the matrix for
single analyses by each laboratory. All solutions should have the same volume and appearance. This type of material
is analogous to that of 3.2.4(b) except for the source of matrix. This case should be used only for perishable
commodities that are altered by all available preservation techniques.
3.2.5 Materials analyzed by another, presumably accurate, method, if available, in the Associate Referee's laboratory or
by some or all the collaborators.
3.2.6 Only as an absolutely last resort {usually with unstable materials and preparation of material studies) should the
collaborators be permitted to prepare their own materials for analysis. Since it is impossible to avoid the personal
bias introduced by knowledge of the composition of the material, the materials should be prepared in each laboratory
by an individual who will not be involved in the analyses.
3.3 Blanks
When the absence of a component is as important as its presence, when determinations must be corrected for the amount
of the component or the presence of background in the matrix, or when recovery data are required, provision must be
made for the inclusion of blank materials containing "none" (not detected) of the analyte. It is also important to know the
variability of the blank and the tendency of the method to produce false positives. There are 2 types of blanks; matrix
blanks and reagent blanks. Since laboratories often will utilize reagents from different sources, each laboratory should
perform reagent blanks. Matrix blanks, when required, are an intrinsic part of the method, and the number of blanks
needed depends on the combined variance of the material (s M ) and of the blank (s B ). The total variability of a blank corrected
value will be s = (s M 2 + s B 2 ) 1/2 .
3 A Limit of Determination
If the limit of determination is important, it is necessary to provide a design which gives special attention to the number
of blanks, and to the necessity for interpreting false positives and false negatives. In all cases, the definition of limit of
determination used in the study must be given by the Associate Referee.
3.5 Controls
When separation from interferences is critical to the analysis, appropriate materials incorporating these interferences must
be included.
3.6 Practical Principle
Always allow for contingencies and prepare more sets (e.g., 25% more) of laboratory samples than there are collaborators.
Some packages may never arrive, some materials may spoil, and some may be lost or the container broken. New labo-
AOAC Official Methods of Analysis (1990) Obligations of Collaborators 679
ratories may have to be substituted for those which are unable to complete the promised work. Some sets may have to be
analyzed at a later time for different purposes, such as to verify stability on storage.
4. Submission of Laboratory Samples
4. 1 Label laboratory samples legibly and without ambiguity.
4.2 Pack shipping cartons well and label properly to avoid transportation delays. If the containers are breakable, pack well
to minimize possibility of breakage. If material is perishable, ship frozen with solid C0 2 , sufficient to last several days
longer than anticipated travel time. Notify collaborators of shipping arrangements, including waybill numbers, arrival
time, and required storage conditions. Use special transportation services, if necessary. For international delivery, mark
as "Laboratory samples — no commercial value" or other designation as required by customs regulations of the country
to which the package is being sent. Hazardous materials must be packed and labeled as required by transportation reg-
ulations. Animal and plant products sent across international borders may require special certification from health au-
thorities.
4.3 Include a return slip, to confirm safe receipt, with each package. If not sent previously, include copy of method, in-
structions, and report forms.
4.4 Provide instructions for proper storage of laboratory samples between unpacking and analysis. Do not use thawed or
decomposed test samples without consulting the Associate Referee.
4.5 When it is important to have instruments calibrated with the same reference material, supply reference material to col-
laborators. Provision for supplying reference standards is particularly important when commercial sources of standards
have not yet been developed. The inclusion of a working standard solution as an unknown is useful to establish a consensus
value for standardization of quality control parameters, such as absorptivity, retention time, and sensitivity (change in
signal intensity divided by the change in concentration).
5. Obligations of Collaborators
5.1 Analyze materials at times indicated, according to submitted protocol. With unstable materials (e.g., with microbial or
decomposition problems) analyses must be started at specified times.
5.2 FOLLOW METHOD EXACTLY (this is critical). Any deviation, such as the necessity to substitute reagents, columns,
apparatus, or instruments, must be recorded at the time and reported. If the collaborator has no intention of following
the submitted method, he or she should not participate in the study. If the collaborator wishes to check another method
on the same materials, additional laboratory samples should be requested for that purpose, to be analyzed separately.
5.3 Conduct exactly the number of determinations stated in the instructions. Any other number complicates the statistical
analysis. Too few determinations may require discarding the results from that laboratory for that material or inserting
"missing values"; too many values may require discarding the contribution of that laboratory or at least some of the
values. If a laboratory cannot follow instructions as to number of analyses to perform, it raises a question as to its ability
to follow the method.
5.4 Report individual values, including blanks. Do not average or do other data manipulations unless required by the instruc-
tions. Undisclosed averaging distorts statistical measures. If blank is larger than determination, report the negative value;
do not equate negative values to zero. Follow or request instructions with regard to reporting "traces" or "less than."
Descriptive (i.e., nonquantitative) terms are not amenable to statistical analysis and should be avoided. When results are
below the limit of determination, report actual calculated result, regardless of its value.
5.5 Supply raw data, graphs, recorder tracings, photographs, or other documentation as requested in the instructions.
5.6 If analytical results appear unreasonable, investigate possible cause immediately, first by checking for transcription and
calculation mistakes, and then by reanalysis, if permitted by the protocol. Call Associate Referee to discuss suspicious
values. If Associate Referee indicates a value may be an outlier, review the determination promptly to the extent possible,
by recalculation, reanalysis, or preparation of new standards. If time and materials are available, obtain new laboratory
samples for repeat analysis.
Since collaborators may have no basis forjudging whether a value is an outlier, the results should be communicated to
the Associate Referee as soon as the protocol is complete and before time and equipment are reassigned, so that repeat
assays may be performed at once, if necessary.
Note: The sooner an apparent outlier is investigated, the greater the likelihood of finding a reason for its occurrence. The
most frequent causes of correctable outliers are:
5.6.1 Incorrect calculations and arithmetic errors.
5.6.2 Errors in reporting, such as transposition of numbers, misplacement of the decimal point, or use of the wrong
units.
5.6.3 Incorrect standards due to weighing or volumetric errors (check physical constants or compare against freshly
prepared standard solutions).
5.6.4 Contamination of reagents, equipment, or test materials.
680 Guidelines for Collaborative Study Procedure AOAC Official Methods of Analysis (1990)
6. Statistical Analysis
6.1 Initial Review of Data (Data Audit)
The Associate Referee should first plot the collaborative data material by material (or one value against the other for a
split level [Youden pair]), values vs laboratory, preferably in ascending or descending order of reported average con-
centration. Usually major discrepancies will be apparent: displaced means, unduly spread replicates, outlying values,
differences between methods, consistently high or low laboratory rankings, etc.
Only valid data should be included in the statistical analysis. Valid data are values that the collaborator has no reason to
suspect as being wrong. Invalid data may result when: (a) the method is not followed; (b) a nonlinear calibration curve
is found although a linear curve is expected; (c) system suitability specifications were not met; (d) resolution is inadequate;
(e) distorted absorption curves arise; (f) unexpected reactions occur; or (g) other atypical phenomena materialize. Other
potential causes of invalid data are included in 5.6.1-5.6.4.
62 Statistical Approach
Perform calculations on each material individually or as split levels, as appropriate. Only if the variances are not sig-
nificantly different from each other should the results across materials be pooled for analysis of variance. If the relative
standard deviations of the results from different materials are not significantly different, it may be convenient to average
them over the range tested and to thereby report just a single relative standard deviation. Consultation with a statistician
is always desirable.
6.3 Outliers
Collaborative studies seem to have an inherent level of outliers, the number depending on the definition of outliers and
the basis for calculation (analytes, materials, laboratories, or determinations). Rejection of more than 2/9 of the data
from each material in a study, without an explanation (e.g., failure to follow the method), is ordinarily considered ex-
cessive. This corresponds to rejection of more than 1 laboratory from a 5-6 laboratory study or 2 from a 9 laboratory
study. For larger studies, a smaller acceptable percentage of rejections may be more appropriate. Determine the probability
that the apparent aberrant value(s) is part of the main group of values considered as a normal population by applying the
following tests in order:
6.3.1 Cochran test for removal of laboratories (or indirectly for removal of extreme individual values from a set of
laboratory values) showing significantly greater variability among replicate (within-laboratory) analyses than the
other laboratories for a given material. Apply as a 1 -tail test at a probability value of 0.01.
To calculate the Cochran test statistic: Compute the within-laboratory variance for each laboratory and divide the
largest of these by the sum of all of these variances. The resulting quotient is the Cochran statistic which indicates
the presence of a removable outlier if this quotient exceeds the critical value listed in the Cochran table for P =
0.01 (1-tail) and L (number of laboratories), Appendix A-l.
6.3.2 Grubbs tests for removal of laboratories with extreme averages. Apply in the following order: single value test (2-
tail; P = 0.01); then if no outlier is found, apply pair value test (2 values at the highest end, 2 values at the lowest
end, and 2 values, one at each end, at an overall P = 0.01).
To calculate the single Grubbs test statistic: Compute the average for each laboratory and then calculate the standard
deviation (SD) of these L averages (designate as the original s). Calculate the SD of the set of averages with the
highest average removed (s H ); calculate the SD of the set of averages with the lowest average removed (s L ). Then
calculate the percentage decrease in SD as follows:
100 x [l - (s L /s)] and 100 x [1 - (s H /s)]
The higher of these 2 percentage decreases is the single Grubbs statistic, which signals the presence of an outlier
to be omitted if it exceeds the critical value listed in the single Grubbs tables at the P = 0.01 level, 2-tail, for L
laboratories, Appendix A-2.
To calculate the Grubbs pair statistic, proceed in an analogous fashion, except calculate the standard deviations
S2L, s 2H , and s HL , following removal of the 2 lowest, the 2 highest, and the highest and the lowest averages,
respectively, from the original set of averages. Take the smallest of these 3 SD values and calculate the corre-
sponding percentage decrease in SD from the original s. A Grubbs outlier pair is present if the selected value for
the percentage decrease from the original s exceeds the critical value listed in the Grubbs pair value table at the
P = 0.01 level, for L laboratories, Appendix A-2.
6.3.3 If the single value Grubbs test signals the need for outlier removal, remove the single Grubbs outlier and recycle
back to the Cochran test as shown in the flowchart, Appendix A-3.
If the single value Grubbs test is negative, check for masking by performing the pair value Grubbs test. If this
second test is positive, remove the 2 values responsible for activating the test and recyle back to the Cochran test
as shown in the flowchart, Appendix A-3, and repeat the sequence of Cochran, single value Grubbs, and pair value
Grubbs. Note, however, that no outliers should be removed if such removal results in an overall reduction of more
than 2/9 in the number of laboratories whose values are removed.
AOAC Official Methods of Analysis (1990) Bias and Precision 681
6.3.4 If no outliers are removed for a given cycle (Cochran, single Grubbs, pair Grubbs), outlier removal is complete.
Also, stop outlier removal whenever more than 2/9 of the laboratories are flagged for removal. With a higher
removal rate, either the precision parameters must be taken without removal of all outliers or the method must be
considered as suspect.
Note: The decision as to whether a value(s) should be removed as an outlier ultimately is not statistical in nature. The
decision must be made by the Associate Referee on the basis of the indicated probability given by the outlier test and any
other information that is pertinent. (However, for consistency with other organizations adhering to the harmonized outlier
removal procedure, the estimate resulting from rigid adherence to the prescribed procedure should be reported.)
6.4 Bias (Systematic Deviation) of Individual Results
6.4.1 (Estimated) Bias = mean amount found — amount added (or known or assigned value)
Note: Error of a single value = the single value - amount added (true value)
% Recovery = (measured concentration in fortified material - measured concentration in unfortified material) X
100/(known increment in concentration)
The amount added should be a substantial fraction of, or more than, the amount present in the unfortified material.
6.4.2 A true or assigned value is known only in cases of spiked or fortified materials, certified reference materials, or by
analysis by another (presumably unbiased) method. Concentration in the unfortified material is obtained by direct
analysis or by the method of additions. In other cases, there is no direct measure of bias, and consensus values
derived from the collaborative study itself often must be used for the reference point.
6.4.3 Notes:
(a) [Note for Youden Manual] Youden equates "true" or "pure" between-laboratory variability (not including the within-
laboratory variability) to the variability in bias (or variability in systematic error) of the individual laboratories.
Technically, this definition refers to the average squared difference between individual laboratory biases and the
mean bias of the assay.
(b) The presence of random error limits the ability to estimate the systematic error. To detect the systematic error of a
single laboratory when the magnitude of such error is comparable to that laboratory's random error, at least 15
values are needed, under reasonable confidence limit assumptions.
6.5 Precision (Random Error)
The relative basis (i.e., relative standard deviation, RSD) is the most useful measure of precision in chemical analytical
work because the RSDs are usually independent of concentration or amount of analyte over a reasonable range of con-
centrations. Therefore, the use of RSD facilitates comparision of variabilities at different concentrations. When the RSD
increases rapidly with a decrease in concentration or amount, the rise delineates the limit of usefulness of the method (limit
of reliable measurement). The most important types of precision are:
6.5.1 Reproducibility — among-laboratories (including within-laboratories) precision, designated as s R .
Note: This component is not obtained merely by calculating the standard deviation of all the data (except when there
are no replicates) since this term must be corrected by a replication term (Youden, 8.1, p. 19). The correction term
must be extracted by an analysis of variance technique (Steiner, 8.1, p. 78-81). However, this crude, overall cal-
culation of the standard deviation of all the data can serve as a check on the arithmetic, since the 2 values are
usually fairly close.
6.5.2 Repeatability — within-laboratory precision, designated as s r .
6.5.3 Among-laboratories (not including within-laboratory, variability). Designated as s L and used only for calculating
Sr-
6.5.4 Relationship among precision components.
(a) The relationship among the 3 precision parameters is:
S R = S L + S r
The parameters, s R 2 , s L 2 , and s r 2 , must be nonnegative, by definition. The estimate of s L 2 , however, can be negative.
This frequently occurs in practice when s r 2 is so large (poor repeatability) that it swamps out s L 2 . A negative estimate
of the s L 2 term arises from the fact that s L 2 is calculated from the difference of 2 terms, each of which is calculated
independently. If the second term is larger than the first, the difference is negative. When this occurs, s L 2 is set
equal to zero, which does result in a biased estimate of s R 2 . If this occurs with a number of the materials in the
collaborative study, the method is probably unsatisfactory due to poor replication. Otherwise, such an occasional
aberration can be tolerated.
(b) When only single determinations are performed on each material (except in the case of the split level design), there
is no rigorous basis for calculating s r 2 , and within-laboratory variability cannot be estimated directly.
(c) The ISO definitions for repeatability value (r) and reproducibility value (R) (see 8.5) are simple multiples of the
above measures of precision expressed as standard deviations. They are shown to be expressible in terms of the
corresponding standard deviations below. The ISO definitions use a prediction interval statement: the value below
682 Guidelines for Collaborative Study Procedure AOAC Official Methods of Analysis (1990)
which the absolute difference between 2, and only 2, single test results of identical test material may be expected
to lie with a specified probability (usually 95%); in other words, assuming normal distribution, when duplicate
measurements are performed, the absolute difference between the results of each of these duplicate measurements
is expected to be below r or R in 95% of the cases. The relationship between the 2 definitions is:
Repeatability value (r) = 2 ■ 2 1/2 • s r = 2.8 ■ X ■ RSD r /100
Reproducibility value (R) = 2-2 1/2 *s R = 2.8 -X -RSDr/100
The coefficient 2.8 is derived from assumptions about the distribution of the sample populations, and s r and s R are
repeatability and reproducibility standard deviations, respectively; RSD r and RSD R are the corresponding relative
standard deviations, and X is the mean of the laboratory means.
6.5.5 Confidence limits for precision terms. Standard deviations and relative standard deviations from actual collaborative
studies are merely estimates of "true values." The "confidence interval" (bounded by the confidence limits) is the
range within which the true value is expected to lie with a stated degree of confidence (customarily 95%). The
confidence intervals of precision terms are rarely given because about 200 values are required to estimate the standard
deviation to within approximately 10%; for a small study of about 30 values, the standard deviation can be estimated
to only about 25%.
6,6 Incorrect, Improper ; or Illusory Values (False Positive and False Negative Values)
These results are not necessarily outliers (no a priori basis for a decision), since there is a basis for determining their
incorrectness (a positive value on a blank material, or a zero (not found) or negative value on a spiked material). There
is a statistical basis for the presence of false negative values; In a series of materials with decreasing analyte concentration,
as the RSD increases, the percent false negatives increases from an expected 2% at an RSD = 50% to 17% at a RSD =
100%, merely from normal distribution statistics alone.
When false positives and /or false negatives exceed about 10% of all values, analyses become uninterpretable from lack
of confidence in the presence or absence of the analyte, unless all positive laboratory samples are reanalyzed by a more
reliable (confirmatory) method with a lower limit of determination than the method under study. When the proportion of
zeros (not necessarily false negatives) becomes greater than approximately 30%, the distribution can become bimodal and
even more uninterpretable (is the analyte present or absent?).
7. FinaJ Report
7.1 The final report should contain a description of the materials used, their preparation, any unusual features in their distri-
bution, and a table of all valid data, including outliers. When replication is performed, the individual values, not just
averages, must be given, unless the method requires averages (e.g., microbiological methods). Values not used for specified
reasons, such as decomposition, failure to follow method, or contamination, should not be included in the table since they
may be included erroneously in subsequent recalculations. The report should include the statistical parameters calculated
with and without specified outliers. Report the standard deviations, means, and the corresponding RSDs. Proofread tables
very carefully since errors are of typographical origin. Give the names of the participants and their organizations, if agree-
ment has been obtained for their acknowledgment.
7.2 The final report should be published in a generally accessible publication, or availability of the report from the organization
sponsoring the method should be indicated in the published method. Without public documentation, the significance of
the study is very limited.
7.3 The report should be sent to all participants, preferably at the mauscript stage, so that clerical and typographical errors
may be corrected before publication. If changes in values from the original submission are offered, they must be accom-
panied by an explanation.
7.4 Example of Table of Statistical Parameters: See Table 1.
8. References
8.1 W.J. Youden & E.H. Steiner (1975) Statistical Manual 8.3 T. Dols & B. Armbrecht (1976) J. Assoc. Off. Anal,
of the AOAC. Association of Official Analytical Chem- Chem. 59, 1204-1207.
ists, 2200 Wilson Blvd., Arlington, VA 22201 USA. The 8.4 International Organization for Standardization Guide 18,
fifth printing (1987) contains several explanatory foot- Geneva Switzerland. Available from American National
notes. Standards Institute, 1430 Broadway, New York, NY
8.2 Handbook for AOAC Members (1989). Availability as in 10018 USA and other national standards organizations.
8.1. 8.5 Ibid, ISO 5725-1986.
AOAC Official Methods of Analysis (1990)
Outlier Tests
683
Table 1 [x] collaborative tests carried out at the international
level in [year(s)] by [organ ization(s)] in which [y and
z] laboratories participated, each performing [k] rep-
licates, gave the following statistical results:
Results expressed in [units]
Material [Description and listed across the top in increasing order of mag-
nitude of means]
Number of laboratories retained after eliminating outliers
Number of outlying laboratories removed
Mean
True or accepted value, if known
Repeatability standard deviation (s r )
Repeatability relative standard deviation (RSD r )
Repeatability value, r(2.8 x s r )
Reproducibility standard deviation (s R )
Reproducibility relative standard deviation (RSD R )
Reproducibility value, R(2.8 x s B )
The repeatability and reproducibility values may also be expressed as a rel-
ative value (as a percentage of the determined mean value), when the
results so suggest.
If the recovery and precision values are more or less constant for all ma-
terials or for groups of materials, an overall average value may be pre-
sented. Although such averaging may not have statistical validity, it does
have practical value.
Appendix A-1 Critical Values for the Cochran Test (Abbrevi-
ated from ISO 5725-1986, P = 0.01)
L = number of laboratories at a given level (concentration)
r = number of replicates per laboratory
L
r - 2
r = 3
r = 4
r = 5
r = 6
3
0.993
0.842
0.883
0.834
0.793
4
0.968
0.864
0.781
0.721
0.676
5
0.928
0.788
0.696
0.633
0.588
6
0.883
0.722
0.626
0.564
0.520
7
0.838
0.664
0.568
0.508
0.466
8
0.794
0.615
0.521
0.463
0.423
9
0.754
0.573
0.481
0.425
0.387
10
0.718
0.536
0.447
0.393
0.357
11
0.684
0.504
0.418
0.366
0.332
12
0.653
0.475
0.392
0.343
0.310
13
0.624
0.450
0.369
0.322
0.291
14
0.599
0.427
0.349
0.304
0.274
15
0.575
0.407
0.332
0.288
0.259
16
0.553
0.388
0.316
0.274
0.246
17
0.532
0.372
0.301
0.261
0.234
18
0.514
0.356
0.288
0.249
0.223
19
0.496
0.343
0.276
0.238
0.214
20
0.480
0.330
0.265
0.229
0.205
21
0.465
0.318
0.255
0.220
0.197
22
0.450
0.307
0.246
0.212
0.189
23
0.437
0.297
0.238
0.204
0.182
24
0.425
0.287
0.230
0.197
0.176
25
0.413
0.278
0.222
0.190
0.170
26
0.402
0.270
0.215
0.184
0.164
27
0.391
0.262
0.209
0.179
0.159
28
0.382
0.255
0.202
0.173
0.154
29
0.372
0.248
0.196
0.168
0.150
30
0.363
0.241
0.191
0.164
0.145
35
0.325
0.213
0.168
0.144
0.127
40
0.294
0.192
0.151
0.128
0.114
Appendix A-2 Critical Values for the Grubbs Single Value and
Pair Value Tests Expressed as the Percent Re-
duction in the Standard Deviation Caused by
Removal of the Suspect Value(s) (See 6.3.2 for
calculating the Grubbs statistics.)
L = number of laboratories at a given level (concentration)
Single value test
Pair value test
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
35
40
91.3
80.7
71.3
63.6
57.4
52.3
48.1
44.5
41.5
38.9
36.6
34.6
32.8
31.2
29.8
28.5
27.3
26.2
25.2
24.3
23.4
22.7
21.9
21.2
20.6
20.0
19.5
17.1
15.3
99.7
95.4
88.3
81.4
75.0
69.4
64.6
60.5
56.8
53.6
50.8
48.3
46.0
44.0
42.1
40.4
38.9
37.4
36.1
34.9
33.7
32.7
31.7
30.8
29.9
29.1
28.3
25.0
22.5
Source: Patrick Kelly, Canada Packers, Toronto, Ontario, Canada. Single
critical values calculated from available formulas; pair critical val-
ues from simulation and fitting and should be accurate to 0.1%
absolute. (Submitted for publication to Technometrics.)
Cochran statistic = (largest individual within-laboratory variance) /(sum of all
the within-laboratory variances).
684 Guidelines for Collaborative Study Procedure
AOAC Official Methods of Analysis (1990)
Appendix A-3 Flowchart Complies with IUPAC-1987 Recommendations
IUPAC — 1987
HARMONIZED STATISTICAL PROCEDURE
SCREEN OUT
NO N- VALID DATA J
START OF LOOP
CALCULATE
PRECISION MEASURES
DROP LAB UNLESS
OVERALL FRACTION OF
LABS DROPPED WOULD
EXCEED 2/9
DROP LAB UNLESS
OVERALL FRACTION OF
LABS DROPPED WOULD
EXCEED 2/9
DROP LABS UNLESS
OVERALL FRACTION OF
LABS DROPPED WOULD
EXCEED 2/9
£
Subject Index
AACC-AOAC methods
niacin and niacinamide in foods,
drugs, and feeds, 1056-1057
riboflavin in foods and vitamin
preparations, 1053-1054
Absorbance
nomograph relating concentration,
absorptivity, and, 672
Absorption indicator method
quaternary ammonium compounds in
formulations, 228
Absorptivity
nomograph relating concentration,
absorbance, and, 672
Acenocoumarol, 565-566, 577
Acephate
in lettuce, strawberries, and
tomatoes, 282-283
Acetaminophen, 553-555, 577
Acetanilid, 538, 545, 555, 577, 606
Acetic acid
in bread, 791-793
in cake, 795
safe handling of, 650, 1224
in seafood, 874
Acetic anhydride
safe handling of, 650, 1224
Acetic serum method
water (added) in milk, 818
Acetone
in distilled liquors, 701-702
in drugs, 498-499
extract in fish meal, 80
safe handling of, 652, 1226
Acetone extraction method
fat in fish meal, 871
Acetone peroxides
in baking premixes, 1165-1166
Acetonitrile
safe handling of, 652, 1226
Acetyl value
of oils and fats, 955
2-AcetyIaniino-5-nitrothiazole
in feeds, 91
Acetylation methods
hair preparations, 366
hydroxyl value of oils and fats, 955
phenylephrine HCL in drugs, 516
Acetylcarbromal, 558, 577
Acid fuchsin D, 1121
Acid hydrolysis methods
copper- reducing substances in spices,
1000
fat in bread, 793
fat in eggs, 854
fat in flour, 780
fat in macaroni products, 796
fat in seafood, 871
filth in baked goods, 386
filth in corn flour, 385
phenacetin and phenyl salicylate in
drugs, 556
starch in baking powders, 687
Acid value
of butterfat, 837
Acid-detergent fiber
in feeds, 82-83
in feeds and forages, 74-76
Acid-insoluble residue method
soil in frozen spinach, 396
Acidic drugs, 548-549
Acidity
in nonalcoholic beverages, 751
of beer, 711-712
of brewing sugars and sirups, 734
of cheese, 842-843
of corn sirups and sugars, 1040-
1041
of eggs, 858
of feeds, 84
of food dressings, 1005
of fruit products, 918
of honey, 1033
of milk, 805
of prepared mustard, 1003
of roasted coffee, 758
of tragacanth drugs, 604
of water, 313
of wines, 744-745
see also Fat acidity
Acids
in beer, 712
in bread, 792-793
in butter, 840
in canned vegetables, 993
in color additives, 1136
in cordials and liqueurs, 706
in cream, 831
in distilled liquors, 697
in hops, 732-733
in nonalcoholic beverages, 751
in oils and fats, 958
polyunsaturated, in oils and fats,
960-963
safe handling of, 650-651, 1224^
1225
solution strengths, 658
in vinegars, 1008
see also Fatty acids
Aconitine, 534, 545, 598, 606
Acrylamide disc electrophoresis
method
identification of fish species, 883-
884
Acrylonitrile
in food, 1181-1182
Adamkiewicz test
protein in feeds, 70
Adipose tissue
hexachlorobenzene and mirex
residues in, 302-303
Adrenocortico steroids, 613-618
Adulterants
meat and poultry adulteration of
meat products, 948
in processed Florida orange juice,
929-930
in spices, 1002
Aerobic plate counts
bacteria in chilled, frozen,
precooked, or prepared foods,
429-430
microorganisms in dairy products,
435-436
microorganisms in foods, 431-434
Aerosol products
antiperspirants, 362
Affinity quantitative determination
p-lactam antibiotics in milk, 825
Aflatoxin B„ B 2
in cottonseed products, 1192-1195
in foods and feeds, 1187-1188
in mixed feed, 1193-1195
in peanuts and peanut products,
1188-1190
Aflatoxin G l9 G 2
in cottonseed products, 1192-1193
in foods and feeds, 1187-1188
in peanuts and peanut products,
1188-1190
Aflatoxin Mj
in cheese, 843
in dairy products, 1199-1201
in fluid milk, 1203-1205
in liver, 1201-1203
in milk and cheese, 1200-1201
in milk and powdered milk, 821
Anatoxins
in cocoa beans, 1190-1191
in coconut, copra, and copra meal,
1191
in corn, 1191
in corn and peanuts, 1188
in cottonseed products, 1192-1193
in cottonseed products and mixed
feed, 1193-1195
in foods and feeds, 1187-1188
in green coffee, 1195
identification of , 1187, 1197-1199
in peanuts and peanut products,
1188-1190
in pistachio nuts, 1195
in soybeans, 1195
sampling of, 1185
standards for, 1185-1187
see also specific aflatoxins
Agar
in meat, 945
Air oven method
moisture in cereal adjuncts, 730
1-1
1-2
Subject Index
AOAC Official Methods of Analysis (1990)
solids and moisture in flour, 777
solids and moisture in macaroni
products, 796
solids in yeast, 736
Akloinide
in feeds, 91-92
Alachlor
in formulations, 174-175
Alaska King Crab Marketing and
Control Board-AOAC method
drained weight of frozen crabmeat,
867
Alba red, 1122
Albumin
in evaporated milk, 833
in milk, 809
Alcohol
in almond extract, 903-904
in anise and nutmeg extracts, 905
in beer, 710-711
in cassia, cinnamon, and clove
extracts, 905
in cordials and liqueurs, 705
in distilled liquors, 691-694
in drugs, 498-499
extract of spices, 1000
in flavor extracts and toilet
preparations, 905
in flavors, 890
in fruit products, 912
in ginger extract, 905
handling of, 1184
in lemon, orange, and lime extracts,
898
in nonalcoholic beverages, 751
in peppermint, spearmint, and
wintergreen extracts, 905
percentages by volume in mixtures
of ethyl alcohol and water,
1250-1255
percentages by weight in mixtures of
ethyl alcohol and water, 1256
precipitate in fruit products, 917
in sirups used in confectionery, 1025
solution strengths, 658
in vanilla extract, 890
in vinegars, 1008
in wines, 739-741
see also Ethyl alcohol
Alcohol method
solids in yeast, 736
Alcoholic beverages
certified reference materials for, 646,
1220
see also Beer; Cordials and liqueurs;
Distilled liquors; Malt beverages
and brewing materials; Wines
Alcoholic products
denaturants in, 232
Alcohols (higher)
and ethyl acetate in distilled liquors,
700-701
Aldehydes
as acetaldehyde in frozen vegetables,
996-997
in cordials and liqueurs, 705
in distilled liquors, 697, 698-699
in lemon and orange oils, 902-903
in lemon, orange, and lime extracts,
900-901
in wines, 747
Aldicarb
in formulations, 212
in grapes and potatoes, 292-294
Aldrin
in formulations, 175-176
multiresidue methods, 279
Alfalfa leaves
filth in, 398-399, 400
Alginates
in chocolate beverages, 756
in chocolate frozen desserts, 852
in chocolate products, 775-776
in food dressings, 1007
Alimentary pastes
filth in, 387
Alizarin cyanine green F, 1120
Alizurol purple, 1122
Alizurol purple SS, 1122
Alkalies
safe handling of, 651-652, 1225-
1226
solution strengths, 658
Alkalimeter method
carbon dioxide in liming materials, 3
Alkalimetric ammonium
molybdophosphate method
phosphorus in feeds, 87
Alkalimetric methods
phosphorus in fertilizers, 13-14, 15,
16-17
Alkaline hydrolysis method
phenacetin and phenyl salicylate in
drugs, 556
Alkaline phosphatase detection
method
filth in corn meal, 382-383
Alkaline phosphatase test
mammalian feces, 412-413
Alkalinity
of ash of cacao products, 763-764
of ash of dry skim milk, 835
of ash of vinegars, 1007
of ash of wines, 742
of water, 313-314
Alkaloid drugs
chichona, 592-593
ephedra, 586-588
ergot, 588-590
ipecac, 584-586
microchemical tests, 533-536
opiates, 579-583
optical crystallographic properties,
659-660
physostigmine, 590-592
rauwolfia, 593-598
refractive indices, 668
titration factors, 580
tropane, 583-584
xanthine, 541, 584
see also specific drugs
Alkaloids
in ephedra, 586
sabadilla, in pesticide formulations,
173
in tobacco, 65-66
Alkanolamines
norepinephrine in epinephrine
preparations, 515-516
phenylalkanolamine salts in elixirs
and sirups, 519-520
phenylephrine HCl in drugs, 516-
518
phenylpropanolamine HCl in drugs,
519
Alkoxyl groups
microchemical determination, 347-
348
Allergenic extracts
in drugs, 604-605
Allethrin
technical and formulations, 164-166
Allobarbital, 537, 538
Allopurinol, 575-576, 577
Allspice
extract, 905-906
filth in, 398-399, 400-401
foreign matter in, 398
tannin in, 1000
5-A!ly!-5-(2-cyclopenten-l-yl)
barbituric acid, 537
Almond extract
alcohol in, 903-904
benzaldehyde in, 904
benzoic acid in, 904
hydrocyanic acid, 904
nitrobenzene in, 905
Aloin, 602, 606
a-Amylase
in flour, meal, and malted cereals,
786
in malt, 729
Alphazurine FG, 1119
Alternating current spark excitation
method
metals in plants, 41
Aluminum
in baking powders, 687-689
in deodorants, 361, 362
in face powders, 365
in liming materials, 4, 6
in plants, 40, 43
in soil acidifiers, 36
in water, 322, 324
Aluminum oxide
in face powders, 365
in liming materials, 4
Aluminum sulfate
aluminum in, 36
Amaranth, 1115
American Association of Cereal
Chemists, see AACC
American Conference of
Governmental Industrial
Hygienists-AOAC method
antimony in food, 242-243
American Oil Chemists' Society, see
AOCS
AOAC Official Methods of Analysis (1990)
Subject Index
American Society of Enologists-
AOAC method
acidity of wines, 744
American Spice Trade Association-
AOAC method
piperine in pepper preparations,
1002-1003
Amines
in color additives, 1126
Amino acids
in vitamin preparations, 1087-1089
Aminobarbital, 538
Aminobenzoates
antipyrine with benzocaine in drugs,
521-522
benzocaine in drugs, 521-522
butacaine sulfate in drugs, 523-524
procainamide HC1 in drugs, 524-525
procaine in drugs, 524
p-Aminobenzoic acid
in feeds, 92
Aminocarb
technical and formulations, 212-213
2-Amino-5-nitrothiazole
in feeds, 92
Aminophylline, 560, 577
Aminopyrine, 538, 545, 556, 557-
558, 577
p- Aminosalicylic acid, 549, 577
Amitriptyline, 525-526, 545
Amitrole
in formulations, 223-224
Ammonia
anhydrous, sampling of, 9-10
in baking powders, 688-689
in crab meat, 869
safe handling of, 651, 1225
solution strengths, 658
solutions, sampling of, 9
in water, 319-320
Ammoniacal silver nitrate
safe handling of, 652, 1226
Ammonium, see Quaternary
ammonium compounds
Ammonium bromide
in elixir, 504-505
Ammonium glycyrrhizinate
in flavor extracts, 907-908
Ammonium hydroxide
safe handling of, 651, 1225
Ammonium thiocyanates, 640, 1214
Amobarbital, 537, 545, 577
Amobarbital sodium, 559
Amphetamines
chemical name, 545, 624
enantiomers in bulk drugs, syrups,
and capsules, 622-623
in drugs, 520-521
rnicrochemical tests, 540
Amprolium
in feeds, 92-93
Amyl alcohol
in distilled liquors, 700-701
Amyl jp-dimethylaminobenzoate
in suntan preparations, 367-368
Amylocaine, 534, 545
Analgesics/antipyretics
acetaminophen in drug preparations,
553-555
acetanilid in drugs, 555
aminopyrine in drugs, 556, 557-558
aspirin in drugs, 555-557
with caffeine in drugs, 555-556
with codeine in drugs, 555
with morphine in drugs, 555
phenacetin in drugs, 555-556
with phenobarbital in drugs, 556,
557
with phenolphthalein in drugs, 557
phenyl salicylate in drugs, 556
quinine in drugs, 555
salicylamide in drugs, 554-555
sodium salicylate in drugs, 555
Anilazine
in formulations, 213-214
Aniline
safe handling of, 652, 1226
Animal fats
organochlorine pesticide residues in,
284
in vegetable fats and oils, 976-977
Animal feeds, see Feeds
Animal tissues
certified reference materials for, 646,
1220
copper in liver, 356-357
in feeds, identification of, 89-90
see also Drugs and feed additives in
animal tissues
Anion-exchange method
phytate in foods, 800-801
Anise
extract, 905
filth in, 398-399
foreign matter in, 398
Annatto
filth in, 398-399, 401
in macaroni products, 798-799
Anodic stripping voltammetric
method
cadmium and lead in food, 238,
239-241
lead in evaporated milk, 256-257
ANOT
in animal tissues
Anthocyanins
in fruit juices, 923
Antibiotics
P-lactam in milk, 825-829
optical crystal lographic properties,
660-661
refractive indices, 668-669
see also specific antibiotics
Antibiotics in feeds
bacitracin, 118, 129-130
chemical methods for, 129-131
chlortetracycline, 130
chlortetracycline HCI, 119-120
common and chemical names of,
132
erythromycin, 120-121
griseofulvin, 130-131
hygromycin, 121
lasalocid, 121-122, 131
lincomycin, 122-123
monensin, 123-124
novobiocin, 125
nystatin, 125-126
oleandomycin, 126
oxy tetracycline, 126-127
procaine penicillin, 127
spectinomycin, 127-128
streptomycin, 128
tylosin, 90, 128-129
Anticoagulants, 565-567
Antiemetics, 542-543
Antifungals, 543-544
Antihistamines
with aspirin, phenacetin, and
caffeine in drugs, 515
chlorpheniramine maleate in drug
tablets, 513-514
codeine in presence of, 579-580
in dosage forms, 512-520
drug combinations, 512
hydrocodone in presence of, 581
meperidine in drugs, 512
mephentermine sulfate in drugs,
512-513
methapyrilene in expectorants, 513
optical crystallographic properties,
662
pheniramine maleate in dosage
forms, 515
pyrilamine in cough sirup, 513
refractive indices, 669
thonzylamine HCI, 515
Antihypertensives, 545
Antimicrobial drugs
in milk, 829-830
Antimony
in food, 242-243
Antioxidants
butylated hydroxyanisole and
butylated hydroxytoluene in
cereals, 1139-1140
in corn and rice breakfast cereals,
791
in food, 1137-1141
in oils and fats, 1138-1139
propyl gallate in food, 1140-1141
Antiparkinsonian drugs, 544-545
Antiperspirants
boric acid in, 362-363
zirconium in, 362
Antipyretics, see Analgesics/
antipyretics
Antipyrine, 521-522, 545
Antiseptics
thymol in, 553
AOAC-AOCS method
fatty acids (crude) in oils and fats,
957
see also AOCS-AOAC methods
AOAC-ASBC method
aphids in hops, 374-375
see also ASBC-AOAC methods
AOAC-ASTM methods
1-4
Subject Index
AOAC Official Methods of Analysis (1990)
cadmium and lead in earthenware,
241-242
cation exchange capacity for peat, 39
see also ASTM-AOAC methods
AOAC-CIPAC methods
alachlor in formulations, 174-175
anilazine in formulations, 213-214
azinphos-methyl in formulations,
198-199
butachlor in formulations, 181
captan in formulations, 181-182
chJoramben in formulations, 182
chlordane, technical and
formulations, 182-183
chlordimeform in formulations, 184
copper in fungicides, 156
dicamba-MCPA and dicamba-2,4-D
in formulations, 187-188
diquat in formulations, 225
fentin in fentin-maneb formulations,
157
folpet in formulations, 192-193
isofenphos, technical and
formulations, 205-206
mercury in organic mercurial seed
disinfectants, 163
metolachlor in formulations, 195
organochlorine pesticide
contamination of pesticide
formulations, 152-153
oxythioquinox in formulations, 226-
227
propachlor in formulations, 196
triazines in formulations, 222-223
see also CIPAC-AOAC methods
AOAC-Food Chemicals Codex
Method
neutralizing value of sodium
aluminum phosphate, 686
AOAC-IUPAC methods
aflatoxin B t in cottonseed products
and mixed feed, 1193-1195
fatty acids in oils and fats, 963-964
methyl esters of fatty acids in oils
and fats, 964-965
AOAC -Office International du
Cacao et du Chocolat method
alkalinity of ash of cacao products,
763
ash of cacao products, 763
AOCS-AOAC methods
crude fiber in feeds, 80-81
hydrocarbons (saturated) in
glycerides, 980-981
hydroxyl value of oils and fats, 955
1 -monoglycerides and monoglyceride
concentrates, 983-984
peroxide value of oils and fats, 956
polyunsaturated acids in oils and
fats, 960-963
trans isomers in margarines and
shortenings, 969-970
see also AOAC-AOCS method
APC drugs, 555, 581
Aphids
in hops, 374-375
Apomorphine, 534, 545, 606
Apomorphine HCI, 580
Apple butter
filth in, 391
mold in, 418
Apple chops
filth in, 391
Apple juice
corn syrup in, 926
patulinin, 1209-1210
quinic, malic, and citric acids in,
920-921
Apples
azinphos-methyl pesticide residues
on, 294
captan pesticide residues in, 296
carbaryl pesticide residues in, 297-
298
dodine pesticide residues in, 299
fluorine on, 248-249
glyodin pesticide residues in, 302
lead on, 254-255
iV-methylcarbamate residues on,
291-292
naphthyleneacetic acid pesticide
residues in, 306-307
nicotine residues in, 307-308
organochlorine pesticide residues in,
284-285
organophosphorous pesticide residues
on, 286-289
thiram pesticide residues in, 309-
311
Applesauce
ethylenethiourea pesticide residues
in, 300-302
Apricots
azinphos-methyl pesticide residues
on, 294
Aprobarbital, 537, 545
Arabinose
in sugars and sirups, 1019
Arachidonic acid
in oils and fats, 960-963
Arecoline, 534, 545, 580, 606
Arprinocid
in feeds, 93
in premixes, 94
Arsanilic acid
in feeds, 92, 94
Arsenic
in animal tissues, 626
in baking powders, 688-689
in Bordeaux mixtures, 149, 158, 159
in cacodylate injections, 501
in calcium arsenate formulations,
149, 155
in feeds, 94-95, 356
in food, 237-239, 243-245
in iron methylarsenate, 501
in iron-arsenic tablets, 501
in lead arsenate formulations, 149,
154
in magnesium arsenate, 149
in meat, 935
in meat and poultry, 245-246
in pesticide formulations, 147-149,
154
in plants, 50
in sodium cacodylate, 501
in water, 331, 332
in zinc arsenite, 149
Arsenic oxide
in lead aresenate formulations, 154
Arsenious oxide
in Bordeaux mixtures, 158
in calcium arsenate formulations,
155
in lead aresenate formulations, 154
in pesticide formulations, 153-154
standard solutions, 640, 1214
in zinc arsenite formulations, 155
Arsenic trioxide
safe handling of, 654, 1228
Arsenious oxide titration method
chlorine in calcium hypochlorite and
bleaching powder, 161
sodium hypochlorite in pesticide
formulations , 1 60- 1 6 1
(S-Asarone
in wines, 750
ASBC-AOAC methods
calcium in beer, 715, 716
ethanol in beer, 711
extract of barley for malting, 723
glycerol in beer, 711
moisture in malting barley, 723
A L nitrosodimethylamine in beer,
719-722
specific gravity of beer and wort,
709-710
see also AOAC-ASBC methods
Asbestos
safe handling of, 654, 1228
Ascorbic acid, see Vitamin C
Ash
of baked products, 795
in baking powders, 688-689
of beer, 713
of bread, 793
of brewing sugars and sirups, 734
of butter, 837
of cacao products, 763-764, 770
of canned vegetables, 991
of cereal adjuncts, 731
of cheese, 842
of confectionery, 1024
of cordials and liqueurs, 706
of corn sirups and sugars, 1040
of cream, 832
of distilled liquors, 694-695
of dried milk, 835
of evaporated milk, 833
of feeds, 70
of flour, 777-778
of fruits and fruit products, 915
of grains, 788
of honey, 1026
of lemon, orange, and lime extracts,
901
of macaroni products, 796
of maple products, 1034
of meat extracts and similar
products, 947
of meat, 932
AOAC Official Methods of Analysis (1990)
Subject Index
I-5
of milk, 807
in mineral oil-soap emulsions, 162
of molasses, 1022
of nonalcoholic beverages, 751
of nuts and nut products, 949
in peat, 37
of plants, 40
of roasted coffee, 758
of seafood, 868
of soybean flour, 791
of spices, 1000
of sugars and sirups, 1012
of sweetened condensed milk, 834
of tea, 761
of vanilla extract, 894
of vinegars, 1007
of wines, 742
Aspirin
antihistamines in drugs with, 515,
545
chemical name, 557, 577
microchemical tests, 538
and phenacetin and caffeine in
drugs, 555-556
and phenacetin and phenyl salicylate
in drugs, 556
and phenobarbital in drugs, 557
and phenolphthalein in tablets, 557
ASTM-AOAC methods
extractives from flexible barrier
materials, 1178-1180
see also AOAC-ASTM methods
ASTM-Intersociety Committee-
AOAC methods
fluoride in plants, 52-56
Atomic absorption
spectrophotometric methods
aluminum in baking powders, 687-
688
aluminum in soil acidifiers, 36
arsenic, selenium, and zinc in food,
238-239
biuret in fertilizers, 23
boric acid in food, 1147
cadmium and lead in earthenware,
241-242
cadmium in food, 247-248
cadmium in water, 324-325
calcium in fertilizers, 27-28
chromium in water, 324-325
copper and nickel in tea, 242
copper in distilled liquors, 695
copper in fertilizers, 27-28, 31
copper in fungicides, 156
copper in liver, 356-357
copper in serum, 357
copper in water, 324-325
dibutyltin dilaurate in feeds, 97
iron in distilled liquors, 696
iron in fertilizers, 27-28, 31
iron in iron chelate concentrates,
31-32
iron in water, 324-325
lead in evaporated milk, 255-256
lead in fish, 257
lead in food, 258
lead in paint, 233
lead in water, 324-325
magnesium in fertilizers, 27-28, 32
magnesium in water, 324-325
manganese in fertilizers, 27-28, 33
mercury in mercury -containing
drugs, 508-509
mercury in water, 326-327
metals in plants, 42
minerals in ready-to-feed milk-based
formulas, 1110
nickel in tea, 269
nutrients in fertilizers, 27-28, 31,
32-33
potassium in beer, 722-723
potassium in water, 327-328
roxarsone in feeds and premixes,
109-110
silver in water, 324-325
sodium in beer, 723
sodium in fertilizers, 34
tin in food, 270-271
zinc in fertilizers, 35
zinc in food, 272-273
zinc in water, 324-325
Atomic absorption
spectrophotometric-flame
photometric method
calcium, potassium, and sodium in
electrolyte replenishers,
503-504
Atomic weights, 657
Atropine sulfate, 580
Atropine, 534, 545, 580, 583, 606
Automated methods
acenocoumarol, dicumarol,
phenprocoumon, warfarin
potassium, and warfarin sodium
in drugs, 565-566
carbon, hydrogen, and nitrogen
determination, 341
2,4-D in formulations, 184-185
digoxin in drugs, 600-601
fat in milk, 814-816
fluoride in plants, 52-56
lysine in nutritional supplements,
1100-1101
methenamine and methenamine
mandelate in drugs, 531-532
niacin and niacinamide in cereal
products, 1055-1056
niacin and niacinamide in foods,
drugs, and feeds, 1056-1057
nitrogen in meat, 935-937
nitrogen in plants, 59-60
phenylephrine HCL in drugs,
517-518
phosphorus in fertilizers, 14-15
phosphorus in meat, 933-935
phosphorus in water, 329-330
potassium in fertilizers, 25-26
protein in feeds, 72-74
protein in grains, 788
riboflavin in foods and vitamin
preparations, 1053-1054
Azide method
oxygen (dissolved) in water,
315-316
Azinphos-methyl
in formulations, 197-199
residues on cole-type crops and
fruits, 294
Babcock method
essential oil in flavor extracts and
toilet preparations, 905
fat in cream, 832
fat in raw milk, 812-814
fat in seafood, 871
Bacillus anthracis
differentiation from B. cereus,
466-467
Bacillus cereus
differentiation from other strains,
466-467
enumeration and confirmation in
foods, 464-466
Bacillus stearothermophillus
in sugars, 458
Bacillus stearothermophilus disc
methods
p- lactam antibiotics in milk,
825-827
Bacillus subtilis qualitative field disk
assay
(3 -lactam antibiotics in milk, 829
Bacillus thuringiensis
differentiation from B. cereus,
466-467
Bacitracin
in feeds, 118-119, 129-130
Bacon
Af-nitrosamines (volatile) in,
938-940
iV-nitrosopyrrolidine in, 940-941
Bacteria
in foods and cosmetics, 431
population of maple sap, 1038-1039
Bacterial colony counts
dairy products, 435-436
milk, 435
Bacteriostatic activity
of disinfectants, 133-137, 143-145
of laundry additives, 143-145
Bailey-Andrew method
caffeine in roasted coffee, 758
caffeine in tea, 761
Baked products
acetic and propionic acids in cake,
795
ash of, 795
extraneous materials in, 385-387
fat in, 795
fat in fig bars and raisin filled
crackers, 795-796
fiber (crude) in, 795
filth in, 386
mineral oil in, 794-795
moisture in fig bars and raisin filled
crackers, 795
pH of, 795
protein in, 795
solids in, 795
sterols (as cholesterol) in, 795
sugars in, 795
1-6
Subject Index
AOAC Official Methods of Analysis (1990)
Baking chemicals
neutralizing values, 685-686
preparation of sample, 685
Baking powders
aluminum in, 687-688
carbon dioxide in, 685
metaJs and other constituents in,
688-689
phosphorus in, 689
preparation of sample, 685
starch in, 687
tartaric acid in, 687
Baking premixes
acetone peroxides in, 1165-1166
Barbital, 537, 538
Barbiturates
in drugs, 535, 537, 559
optical crystallographic properties,
663
refractive indices, 669-670
Barbituric acid condensation method
citral in flavor extracts and toilet
preparations, 906
Barium
in plants, 40
in water, 327
Barium-140
in milk, 353-355
Barium hydroxide treatment
acidity of wines exclusive of S0 2 ,
744-745
Barium sulfate
in face powders, 365
Barley
filth in cereals, 388
for malting, 723
ochratoxins in, 1207-1208
piperonyl butoxide pesticide residues
in, 309
sterigmatocystin in, 1210-1211
Bathocuproine method
copper in fungicides, 156
Bay leaves
filth in, 398-399
Beans
hydrocyanic acid in, 1.213
weevils in, 394
see also Green beans
Beef
adulteration of meat products, 948
calcium in mechanically separated
meat, 941
filth in ground beef or hamburger,
390-391
preservatives in ground meat, 1 144
virus in, 494-495
see also Meat and meat products
Beer
acidity of, 711-712
acids (volatile), 712
alcohol in, 710-711
ash of, 713
bitterness of, 718-719
calcium in, 715-716
caloric content, 71.1
caramel in, 718
carbohydrate content, 712
carbon dioxide in, 713-714
chlorides in, 718
color of, 708
copper in, 716-717
dextrin, 712
diacetyl in, 712-713
ethanol in, 711
extract, 710
fermentation of, 7 1 1
foam collapse rate, 714-715
glycerol in, 711
haze after chilling, 708-709
iron in, 717
nitrogen (free amino) in, 713
A^-nitrosodimethylamine in, 719-722
pH of, 712
phosphorus in, 713
potassium in, 722-723
protein in, 713
proteolvtic chillproofing enzymes in,
719
sample preparation, 708
sodium in, 723
specific gravity, 709
starch (unconverted) in, 713
sugars (reducing), 712
sulfur dioxide in, 718
viscosity, 710
Belladonna, 583
Bellier test
peanut oil in olive, cottonseed, corn,
and soybean oils, 978-979
Bemegride, 537, 545
Bendiocarb, 214
Bendrofiumethiazide, 570, 577
Benedict solution method
lactose in meat, 945
Benfluralin, 179-180
Benomyl, 214-215
Benzaldehyde
in almond extract, 904
in cordials and liqueurs, 707
in nonalcoholic beverages, 751-752
Benzene
safe handling of, 652, 1226
Benzene hexachloride
in formulations, 179
residues, 294
Benzocaine, 521-522, 538, 545
Benzoic acid
in almond extract, 904
chemical name, 538, 548
in flour, 784
in food, 1141-1144
in nonsolid food and beverages,
1141-1142
Benzoquinone method
hair preparations, 366
Benzoyl peroxide bleach
in flour, 784
Benzo[a]pyrene
in food, 1176-1178
Benzthiazide, 572-573, 577
Benztropine mesylate, 583-584, 606
Benzy [morphine, 534
Berberine, 534, 545
Berlin Institute method
invert sugar in sugars and sirups,
1017
Berry fruits
seeds in, 914
Beta particle counting method
strontium-90 in water, 349-351
Betaine
in orange juice, 917-918
Beverages
benzoic acid in, 1142
quaternary ammonium compounds
in, 1153-1154
salicyclic acid in, 1155-1156
see also Alcoholic beverages;
Distilled liquors; Malt beverages
and brewing materials;
Nonalcoholic beverages and
concentrates; and specific
beverages
BF method
aflatoxins in peanuts and peanut
products, 1190
a-BHC
in lettuce, strawberries, and
tomatoes, 282-283
7-BHC
in formulations, 176-179
Bicarbonate
in water, 322
Bifuran
in feeds, 100
Bioassay methods
chick edema factor in oils and fats,
981
eye irritants in cosmetics, 360-361
paralytic shellfish poison, 881-882
vitamins and other nutrients, 1091 —
1099
Biochemical identification kit method
E. coli in foods, 474-475
Enter obacteriaceae in foods, 474-
475
Salmonella sp. in foods, 473-475
Biochemical oxygen demand
of water, 314-315
Biological samples
copper in serum, 357
Biphenyl
in citrus fruits, 295-296
Bismuth
in drugs, 502
Bismuthate method
manganese in fertilizers, 33
manganese in water, 331
Bitartrate method
tartaric acid in fruit and fruit
products, 918
Bithionol
in feeds, 95
Bitterness
of beer, 718-719
Bitterness units method
bitterness of beer, 718-719
Biuret
in fertilizers, 22-23
Biuret test
protein in feeds, 70
AOAC Official Methods of Analysis (1990)
Subject Index
I-7
Blackberries
insects in frozen products, 391-392
mold in, 418
Bleaching powder
chlorine in, 161
Block digestion method
protein (crude) in meat, 937
Blueberries
maggots in, 392
Bone fertilizers, 11
Borates
in food, 1144-1145
Bordeaux mixtures
arsenic in, 149
arsenious oxide in, 158
with calcium arsenate, 149-150, 159
carbon dioxide in, 158, 159
copper in, 149-150, 158, 159
with lead arsenate, 149-150, 158-
159
lead in, 149, 155
moisture in, 158, 159
with Paris green, 149-150, 158
with zinc arsenate, 149-150
Boric acid
in antiperspirants and deodorants,
362-363
in face powders, 365
in food, 1144-1147
in meat, 1145-1146
in water, 331, 332
Boron
in caviar, 1145
in fertilizers, 28-30
in plants, 40, 42, 50
Boron triffluoride method
fatty acids in oils and fats, 963-964
Breaded fish products
fish content, 864-865
Breading
filth in, 387
Breads
acetic and propionic acids in, 791-
792
acids (volatile) in, 792-793
ash of, 793
calcium in, 793
chloride in ash of, 793
citric acid in, 793
fat and fat number, 791
fat in, 793
fiber (crude) in, 793
fiber (total dietary), 791
filth in, 385-387
iron in, 793
lactose in, 794
pH of, 794
protein in, 793
sample preparation, 790-791
solids (total) in, 791
sterols (as cholesterol) in, 793
sugars in, 794
thiamine in, 1051-1052
vitamins in enriched bread, 793
Breakfast cereals
extraneous materials in, 387-388
see also Cereal foods
Brewer's grits, 381
Brewers' grains, 737-738
Brewing materials, see Malt
beverages and brewing materials
Brillant lake red R, 1121
Brilliant blue FCF, 1115, 1118
Brine flotation method
filth in corn meal, 382
Brine saturation method
filth in sage, 403
Broccoli
filth in canned products, 395
Brodifacoum, 224
Bromate titration
resorcinol in hair lotions, 366
salicylic acid in hair lotions, 366
Bromates
in white and whole wheat flour,
784-785
Bromides
in dosage forms, 504-505
in water, 331-332
Brominated vegetable oils
in nonalcoholic beverages, 1166-
1167
Bromination methods
benzocaine in drugs, 522
dinitrophenol in drugs, 550
phenolsulfonates in deodorants, 364
phenolsulfonates in drugs, 552
sterols in macaroni products, 797
thiouracil in drugs, 574
Bromine
microchemical methods, 337-339
safe handling of, 652, 1226
in water, 331
Bromisovalum, 558, 577
Bromophenol blue methods
quaternary ammonium compounds in
food and beverages, 1153-1154
quaternary ammonium compounds in
milk, 1151
Bromoxynil octanoate
in formulations, 180-181
Brucine, 534, 545
Brucine colorimetric method
nitrogen (nitrate) in water, 320
Bufencarb
in grapes and potatoes, 292-294
Buffer solutions
for calibration of pH equipment,
640-641, 1214-1215
for colorimetric pH comparisons,
641-642, 1215-1216
Buquinolate
in feeds, 95
Bushel weight
malt, 724
Butabarbital sodium, 537, 545, 561-
562, 577
Butacaine sulfate, 523-524, 545
Butachlor, 181
Butalbital, 537, 545
Butethal, 537, 545
Butter
acids (volatile) in, 840
butyric acid in, 839-840
casein, ash and salt in, 837
critical temperature of dissolution of
oil from, 838
examination of fat, 837
fat in, 837
fatty acids in, 838-840
filth in, 378-379
moisture in, 837
mold in, 420, 840
phosphatase in, 840
preservatives in, 840
salt in, 837
sample preparation, 836-837
sampling, 803-804, 836
Butter oil
p-sitosterol in, 975-976
Butterfat
acid value of, 837
color additives in, 838
lactic acid in, 838
refrective index of, 837
vegetable fats in, 973-975
w-Butyl alcohol
in distilled liquors, 700
Butylate
in herbicide formulations, 221
Butylated hydroxyanisole
in cereals, 1139-1140
in foods, 1137-1138
in oils and fats, 1138-1139
Butylated hydroxytoluene
in cereals, 1139-1140
in foods, 1137-1138
in oils and fats, 1138-1139
2-(p-ter*-Butylphenoxy)-l-methyl
2-chloroethyl sulfite (aramite)
residues, 294
Butyric acid
in bread, 792-793
in butter, 839-840
in fat, 958-960
in seafood, 874
Cabbage
N- methyl carbamate residues on,
291-292
nicotine residues in, 307-308
Cacao bean and its products
aflatoxins in cocoa beans, 1190-
1191
alginates in chocolate beverages, 756
alginates in, 775-776
alkalinity of insoluble and soluble
ash of, 763-764
ash of, 763-764, 770
caffeine in, 776
chocolate liquor, cacao mass, 770
crude fiber in, 764
fat in, 770-772
filth in, 373-374
fructose, glucose, lactose, maltose,
and sucrose in milk chocolate,
773
glucose in, 773-775
lactose in milk chocolate, 772
milk fat in milk chocolate, 772
moisture in, 763
Subject Index
AOAC Official Methods of Analysis (1 990)
nitrogen in, 764
pectic acid in, 765-766
pH of, 764
protein in milk chocolate, 772
Salmonella in chocolate, 470-471,
478-480
sample preparation, 763
shell in, 764
spiral vessel count of, 766-767
starch in, 775
stone cell and group count of, 767-
770
sucrose in, 773-774
threobromine in, 776
Cacao fat
coconut and palm kernel oils in
cocoa butter and fat from milk
chocolate, 772
critical temperature for dissolution in
acetic acid, 772
index of refraction, 771
iodine absorption number, 771
lecithin in, 772
melting point, 771
milk fat in milk chocolate, 771
Reichert-Meissl and Polenske values,
771
saponification number, 772
separation of, 771
silver number for coconut and palm
kernel oils, 772
unsaponifiable matter in cocoa
butter, 771
Cacodylate injections
arsenic in, 501
Cadmium
in earthenware, 232
in feeds, 95
in food, 237-241, 246-248
in food ware, 241-242
in water, 324^325
Caffeine
acetanilid in drugs with, 555-556
aminopyrine in drugs with, 556
antihistamines in drugs with, 515
aspirin in drugs with, 555
in cacao products, 776
codeine in drugs with, 555
in green coffee, 757
microchemical tests, 541
morphine in drugs with, 555
in nonalcoholic beverages, 752-754
phenacetin in drugs with, 555-556
phenobarbital in drugs with, 556
with potassium bromide in drugs,
504
quinine in drugs with, 555
in roasted coffee, 758
in tea, 761-762
Caffeine, 577, 606
Calcium
in baking powders, 688-689
in beer, 715-716
in bread, 793
in calcium arsenate formulations, 155
in canned vegetables, 991-992
in face powders, 365
in feeds, 84-85
in fertilizers, 27-28, 30
in flour, 779
in fruits and fruit products, 916
in infant formulas, 1106-1107, 1110
in lime sulfur formulations, 160
in liming materials, 4-5
in magnesic limestone, 1-2
with magnesium in drugs, 502-503
in mechanically separated poultry
and beef, 941
with phosphorus and iron in vitamin
preparations, 504
with potassium and sodium in
electrolyte replenishers, 503-
504
in plants, 40, 42, 43
safe handling of, 651, 1225
in salt, 333-334
in water, 324
Calcium arsenate
Bordeaux mixtures with, 149-150,
159
formulations, 149, 154-155
Calcium bromide, 504-505
Calcium carbonate
in calcium silicate slags, 3
Calcium cyanide formulations, 159
Calcium cyclamate
in canned fruit, 1168-1169
in nonalcoholic beverages, 1 167—
1168
Calcium gluconate, 503
Calcium hypochlorite
chlorine in, 161
Calcium oxalate precipitate method
oxalic acid in canned vegetables,
993-994
Calcium oxide
in liming materials, 2, 4-5
safe handling of, 651, 1225
Calcium pantothenate
in vitamin preparations, 1079
Calcium salycilate drugs, 584
Calcium silicate slags, 3-4
Calculation method
protein efficiency ratio, 1096-1098
Calomel
mercury in, 509-510
in ointments, 511
Caloric content
of beer, 711
of wines, 742
Cambridge filter pads
nicotine on, 66-67
Camphor, 602, 606
Canadian lead number
of maple products, 1035
Candling procedures
parasites in fish muscle, 882-883
Candy
filth in, 393-394
Salmonella in, 476-478
Cannabinoi, 621, 624
Canned food containers
microleak detection, 456-458
Canned foods
low-acid, commercial sterility, 455-
459
tin in, 270-271
see also specific foods and food
groups
Capillary tube method
melting point of fats and fatty acids,
954
Capsicums
filth in, 398-399, 401
in ginger extract, 905
Captan
in formulations, 181-182
residues in firm fruits, 296
Caramel
in beer, 718
in cordials and liqueurs, 706
Caraway extract, 905-906
Caraway seed
filth in, 398-400
foreign matter in, 398
Carbadox
in feeds, 95-96
Carbamate pesticides
aldicarb, 212, 292-292
arninocarb, 212-213
bendiocarb, 214
benomyl, 214-215
bufencarb, 292-292
carbanolate, 291-292
carbaryl, 291-294
carbofuran, 215-216, 291-294
dithiocarbamates, 217-218
methiocarb, 218-219, 292-294
methomyl, 292-294
N-methylcarbamate , 29 1 -294
oxamyl, 292-294
pirimicarb, 219-220
propoxyl, 291-292
thiocarbamates in herbicide
formulations, 221
thiram, 222
Carbanolate
on apples, cabbage, collards, corn
kernels, green beans, kale, and
turnip tops, 291-292
Carbanthrene btue, 1120
Carbaryl
on apples, cabbage, collards, corn
kernels, green beans, kale, and
turnip tops, 291-292
in apples and spinach, 296-298
in grapes and potatoes, 292-294
Carbidopa, 544-545, 545
Carbofuran
on apples, cabbage, collards, corn
kernels, green beans, kale, and
turnip tops, 291-292
in formulations, 215-216
in grapes and potatoes, 292-294
Carbohydrate content
of beer, 712
of fruit juices, 922
of wines, 742
Carbon
in fertilizers, 30
and hydrogen and nitrogen,
AOAC Official Methods of Analysis (1990)
Subject Index
I-9
microchemical determination,
341
and hydrogen, microchemical
determination, 339-341
Carbon column cleanup method
organophosphorus pesticide residues,
286-288
Carbon dioxide
in baking powders, 685
in beer, 713-714
in Bordeaux mixtures, 158, 159
correction factors for gasometric
determination of, 1258-1259
density, 1257
in liming materials, 3
in self-rising flour, 779-780
in wines, 747-749
Carbon disulfide
in fumigant mixtures, 164
safe handling of, 652, 1226
Carbon disulfide evolution method
dithiocarbamates in formulations,
217-218
Carbon ratio mass spectrometric
methods
corn sirups and sugars in honey,
1032-1033
corn syrup and cane sugar in maple
syrup, 1035
corn syrup in apple juice, 926
corn syrup in orange juice, 926-927
Carbon tetrachloride
in drugs, 499
in fumigant mixtures, 164
safe handling of, 653, 1227
in wheat and com grain, 290-291
Carbonates
in fertilizers, 30
in water, 322
Carbophenothion
on apples and green beans, 286-287
on apples, carrots, endive, kale,
lettuce, potatoes, and
strawberries, 287-289
Carbromal, 558-559, 577
Carcinogens
safe handling of, 654, 1228
Cardamon
filth in, 398-399, 401
foreign matter in, 398
Carius combustion method
bromine, chlorine, or iodine
determination, 337-338
sulfur determination, 345-346
Carmel
in wines, 747
Carotenes
in dried plant materials and mixed
feeds, 1048-1049
in fresh plant materials and silages,
1048
in macaroni products, 798-799
in plants, 63
Carotenoids
in eggs, 853
in macaroni products, 798-800
Carrots
organophosphorus pesticide residues
on, 287-289
Casein
in butter, 837
in evaporated milk, 833
in malted milk and chocolate malted
milk, 835
in milk, 809
phosphatase in, 824-825
Salmonella in, 470-471
Casein coagulation method
proteolytic chillproofing enzymes,
719
Cassia extract
alcohol in, 905
oil in, 905
Cat-eye bioassay method, 584
Catalase
in frozen vegetables, 997-998
Cataylic combustion method
sulfur determination, 347
Cation exchange capacity
for peat, 39
Catsup
benzoic acid in, 1142
Caustic value
liming materials, 2
Caviar
boron in, 1145
Cayenne pepper
filth in, 401
CB method
aflatoxins in cocoa beans, 1190-1191
aflatoxins in peanuts and peanut
products, 1188-1190
Celery
thiram pesticide residues in, 309-311
Celery leaves
filth in, 398-399
Celery seed
filth in, 398-399, 401
foreign matter in, 398
Cellulose acetate strip method
identification of fish species, 883-
888
Cephaeline, 585-586, 606
Ceramic fiber filter method
crude fiber in feeds, 80-81
Cereal adjuncts, brewing materials,
730-731
Cereal foods
antioxidants in corn and rice
breakfast cereals, 791
baked products, 794-796
bread, 790-794
butylated hydroxy anisole and
butylated hydroxytoluene in,
1139-1140
glucose, fructose, sucrose, and
maltose in presweetened cereals,
789-790
macaroni, egg noodles, and similar
prodicts, 796-801
niacin and niacinamide in, 1055-
1056
starch in, 789
wheat nour, 777-778
wheat, rye, corn, buckwheat, rice,
barley, and soybeans and
products, 788-790
Certified reference materials, 645-
648, 1219-1222
Cesium- 137
in milk and other foods, 353-355
Charcoal column chromatographic
method
separation of sugars in honey, 1028-
1030
Cheese
acidity of, 842-843
anatoxin Mj in, 843, 1200-1201
ash of, 842
chloride in, 842
citric acid in, 845-846
color additives in, 843
dehydroacetic acid in, 1147-1148
fat in, 844
filth in, 378-379
gelatin in cottage cheese, 847
gums in soft curd cheese, 846-847
lactose in process cheese, 846
moisture in, 841-842
nitrate and nitrite in, 843-844
nitrogen in, 842
phosphatase in, 847-850
Salmonella in powders, 478-480
salt in, 842, 933
sample collection, 840
sample preparation, 840-841
sampling, 804
sorbic acid in, 1156-1157
sorbic acid in cottage cheese, 847
tartaric acid in, 844-845
titanium in, 271, 842
Chelated iron
in iron chelate concentrates, 31-32
Chelometric methods
liming materials, 5
Chemical hazards
safe handling of, 652-654, 1226-
1228
Chemical oxygen demand
of water, 316-317
Chemical preservatives
in foods, 1141-1163
Chenopodium oil, 602
Cherries
azinphos-methyl pesticide residues
on, 294
maggots in, 392
Chervil
filth in, 398-399
Chewing gum
filth in, 394
Chick bioassay
vitamin D 3 in poultry feed
supplements, 1094-1095
Chick edema factor
in oils and fats, 981-982
Chick embryo bioassay
toxicity of aflatoxin B l5 1199
Chicory
filth in, 374
in roasted coffee, 759
1-10
Subject Index
AOAC Official Methods of Analysis (1990)
Chili powder
filth in, 401
Chinchona alkaloids, 592-593
Chives
filth in, 398-399
Chloral hydrate, 562, 577
Chloramben
in formulations, 182
Chloramine T pesticide formulations,
161
Chloramines
in milk, 820-821
Chloramphenicol
in milk, 829-831
Chlordane, AG
alpha and gamma isomers in, 183
granular formulations, 184
heptachlor in, 184
technical , hexachlorocyclopentadiene
in, 183
technical, in formulations, 182-184
Chlordimeform
in formulations, 184
Chloride
in ash of bread, 793
in beer, 718
in cheese, 842
in calcium cyanide formulations, 159
in deodorants, 363
in distilled liquors, 696-697
in eggs, 856
in meat extracts and similar
products, 947
in mi Ik- based infant formula, 1113
in plants, 50-51
in prepared mustard, 1003
in roasted coffee, 759
in tobacco, 64
in water, 320-321
in water-soluble color additives,
1135-1136
in wines, 743
see also Salt; Sodium chloride
Chlorinated hydrocarbons
in drugs, 499
Chlorine
as sodium chloride in seafood, 870
in bleaching powder, 161
in calcium hypochlorite, 161
in chloramine T, 161
in disinfectants, 137-138
in fat of flour, 783
in feeds, 85
in fertilizers, 30
in fruit products, 917
in meat and meat products, 933
microchemical methods, 337-339
in organohalogen pesticides, 175
safe handling of, 652, 1226
in sodium hypochlorite, 160-161
Chlorine methods
chlordane, technical and
formulations, 182-183
DDT in formulations, 186
heptachlor in formulations, 193
Chlorobutanol
in drugs, 499-500
chemical name, 524, 545
Chloroform
in drugs, 499
safe handling of, 653, 1227
in wheat and corn grain, 290-291
Chloroform extraction
2-isopropyl-4-pentenoyl urea, 563
Chloroform-methanol extraction
method
fat in foods, 1100
Chlorogenic acid
in green coffee, 757
in roasted coffee, 759
p-Chlorophenyl phenyl sulfone
residues, 298
Chlorophyll
in plants, 62-63
Chloroplatinate methods
potassium in fruits and fruit
products, 915
Chlorothiazide, 570-573, 577
Chlorotoluron, 216-217
Chloroxuron, 216-217
Chlorpheniramine maleate, 513-515,
545, 587-588, 606
Chlorpropamide, 567, 577
Chlorpyrifos
in formulations, 199
in lettuce, strawberries, and
tomatoes, 282-283
Chlortetracycline
in feeds, 130
Chlortetracycline HC1
in feeds, 119-120
Chocolate and chocolate products
alginates in frozen desserts, 852
cacao mass of chocolate liqueur, 770
casein in chocolate malted milk, 835
see also Cacao bean and its
products; Milk chocolate
Cholecalciferol, see Vitamin D
Cholesterol
animal fats in vegetable oils and
fats, 976-977
in baked products, 795
in bread, 793
in eggs, 855-856
in macaroni products, 797-798
in multicomponent foods, 1103-
1105
Choline, 534, 545
Cholinesterase inhibition method
organophosphorus pesticide residues,
289-290
Chromatographic methods
acetic and protionic acids in bread,
791-792
aspirin, phenacetin, and caffeine in
drugs, 555-556
benzoic and salicylic acids in drugs,
548
codeine in APC drug tablets, 581
codeine with antihistamines in drugs,
579-580
ergotamine in drugs, 589
fatty acids (volatile) in seafood,
872-875
fatty acids and butyric acid in butter,
839-840
glucose in honey, 1031
glycerides in shortening, 985-986
ipecac alkaloids in drugs, 585-586
isocitric acid in fruits and fruit
products, 919
morphine in opium and paregoric,
581-582
neostigmine in drugs, 591
physostigmine salicylate and
physostigmine sulfate in drug
dosage forms, 590-591
polar components in frying fats,
968-969
vanillin, ethyl vanillin, and coumarin
in vanilla extract, 892-893
see also Column chromatographic
methods; Gas chromatographic
methods; Liquid
chromatographic methods
Chromatographic-spectrophotometric
method, 758-759
Chromic acid
safe handling of, 650, 1224
Chromium
in water, 324-325
Chromotropic acid colorimetric
method
methanol in distilled liquors, 702
Cigarette filler
glycerol in, 65
menthol in, 67-68
Cinchonidine, 534, 545
Cinchonine, 534, 545
Cinchophen, 538, 545, 599, 606
Cinnamon
alcohol in extracts, 905
filth in, 398-399, 401-402
oil in extracts, 905
CIPAC-AOAC methods
aminocarb technical and
formulations, 212-213
chlorotoluron, chloroxuron, or
metoxuron in formulations,
216-217
cyhexatin technical and formulations,
225
cypermethrin in pesticide
formulations, 166-167
dichlobenil in formulations, 188-189
diflubenzuron in formulations, 190—
191
endosulfan in formulations, 191-192
formothion in fonnulations, 204-205
7-BHC in technical BHC,
formulations, and lindane
shampoos and lotions, 178-179
methiocarb technical and
formulations, 218-219
permethrin in pesticide formulations,
167-168
propoxur technical and formulations,
220
temefos in formulations, 211
terbuthylazine in formulations, 220-
221
AOAC Official Methods of Analysis (1990)
Subject Index
1-11
tetradifon (technical) in formulations,
197
thiram in formulations, 222
triadimefon technical and
formulations, 228-229
see also AOAC-CIPAC methods
Citral
in flavor extracts and toilet
preparations, 906
in lemon and orange extracts, 901
Citric acid
in bread, 793
in cheese, 845-846
in cordials and liqueurs, 706
in dried milk, 835
in fruits and fruit products, 918-921
in milk, 805-806
in nonalcoholic beverages, 751
in wines, 746
Citrus fruit
biphenyl pesticide residues in, 296
fith in canned juices, 392-393
mold in juices, 418, 420-421
see also specific fruits
Citrus Red No! 2, 1119
CBams
shell in canned products, 390
shucked, volume of, 867
Clopidol, 626-628, 636
Clostridium botulinum
and toxins in foods, 459-461
Clostridium nigrificans
in sugars, 458
Clostridium perfringens
in foods, 462-464
Clostridium thermosaccharolyticum
in sugars, 458
Cloves
alcohol in extracts, 905
filth in, 398-399
foreign matter in, 398
oil in extracts, 905
tannin in, 1000
Coating and glazing substances
in roasted coffee, 759
CobaBamin
assays, 1080-1082
in milk-based formulas, 1110-1112
in vitamin preparations, 1082-1083
Cobalt
in feeds, 85-86
in fertilizers, 30
in plants, 43-45
Cocaine, 606
HC1, 580, 606, 620, 624
microchemical tests, 534
with procaine in drugs, 524
titration factors, 580
Cocoa butter
coconut and palm kernel oils in,
772
unsaponifiable matter in, 771
Cocoa, see Cacao bean and its
products
Coconut
aflatoxins in, 1191
filth in, 379-380
Salmonella in, 476-478
shredded, glycerol in, 950
Coconut oil
in cocoa butter and in fat extracted
from milk chocolate, 772
silver number for detection of, 772
Cod fish
minced flesh in mixed fillet-minced
cod blocks, 868
Cod liver oil
in emulsions, 602
Codeine
with acetanilid in drugs, 555
with antihistamines, 579-580
in APC drug tablets, 581
with caffeine in drugs, 555
chemical name, 545, 577
microchemical tests, 534
phosphate, 580
with procaine in drugs, 524
sulfate, 580
and terpin hydrate in elixirs, 580-
581
titration factors, 580
Codex Alimentarius Draft European
Regional Standard for Vinegar-
AOAC method
solids in vinegar, 1008-1009
Coffee
filth in, 374
Coffee (green)
aflatoxins in, 1195
caffeine in, 757
chlorogenic acid in, 757
coloring matter in, 757
macroscopic examination, 757
ochratoxin A in, 1208-1209
Coffee (roasted)
acidity of, 758
ash of, 758
caffeine in, 758
chicory in, 759
chlorides in, 759
chlorogenic acid in, 759
coating and glazing substances in,
759
dextrin in, 759
fiber (crude) in, 760
gelatin in, 759
glucose (commercial) in, 759
macroscopic examination, 757-758
moisture in, 760
petroleum ether extract of, 760
sample preparation, 758
solids (soluble) in, 760
starch in, 760
sugars in, 760
Colchicine, 588
Coie-type crops
azinphos-methyl pesticide residues
on, 294
Coliform counts
milk, 435
Coliform organisms
in chilled, frozen, precooked, or
prepared foods, 429-430
in dairy products, 434, 435-436
in eggs in egg products, 428
in foods, 437-438
in shellfish growing waters, 436-437
in tree nut meats, 430-431
see also Fecal coliforms
Collaborative study guidelines, 673-
684
Collards
filth in, 395-396
N-methylcarbamate residues on,
291-292
Color
of beer, 708
of black malt, 727
of brewing sugars and sirups, 733
of caramel malt, 727
of distilled liquors, 690, 691
of egg yolks, 853
in oils and fats, 982
of raw cane sugars, 1010
in spices, 999
in vanilla extract, 894-895
in vinegars, 1008
of white wines, 739
Color additives
in butterfat, 838
in cheese, 843
in cream, 833
dye (pure) in, 1123-1125
in evaporated milk, 833
extracts of, 1125
FD&C, in foods, 1117-1119
free acid in, 1136
halogens in, 1135-1136
in ice cream and frozen desserts,
852
inorganic salts in, 1136
insoluble matter in, 1125
intermediates in , 11 26- 1132
in macaroni products, 798-799
melting point of, 1136
metals and other elements in, 1 132—
1135
in milk, 821
natural coloring matters, 1118
oil-soluble, in foods, 1116-1117
pigments and lakes, 1115
separation and identification, 1115-
1126
soluble matter in, 1136
soluble, and their lakes, 1115
specifications and methods for colors
appearing in previous editions,
1123
subsidiary and lower sulfonated
dyes, 1132
synthetic organic, analysis, 1118-
1123
synthetic organic, in foods, 1115-
1116, 1123
volatile matter in, 1125
yellow AB in macaroni products,
798-799
see also specific colors
Color classification
of honey, 1026
of maple products, 1034
-12
Subject Index
AOAC Official Methods of Analysis (1990)
Colorimetric methods
aldehydes in lemon, orange, and
lime extracts, 900-901
ammonia in crabmeat, 869
arsenic in feeds, 94-95
benzene hexachloride pesticide
residues, 294
benzocaine in drugs, 521-522
bifuran in feeds, 100
bromide in water, 331-332
2-(p-fm-butylphenoxy)- 1 -methyl 2-
chloroethyl sulfite (aramite)
pesticide residues, 296
calcium gluconate in drugs, 503
carbaryl pesticide residues, 296-298
carotenoids in macaroni products,
799-800
chlordane, technical and
formulations, 183
chlorophyll in plants, 62
citral in lemon and orange extracts,
901
citric acid in cheese, 845-846
cobalt in feeds, 85-86
cobalt in fertilizers, 30
color in vanilla extract, 894-895
copper in distilled liquors, 695
copper in feeds, 86
copper in food, 248
copper in milk and milk products,
821
copper in plants, 45-46
creatinine in meat, 941
DDT pesticide residues, 298
diacetyl in beer, 712-713
disulfram in drug tablets, 574
P-estradiol in drugs, 607
ethopabate in feeds, 98-99
fluoride in water, 321-322
fluorine in feeds, 86
fluorine on apples and pears, 248-
249
furazolidone in feeds, 100-101
glycarbyl amide in feeds, 100
hypochlorites and chloramines in
milk, 820-821
indole in crabmeat, oysters, and
shrimp, 877-878
iodide in water, 331-332
iron in beer, 717
iron in distilled liquors, 696
iron in plants, 46
iron in water, 323
ketosteroids in drugs, 607
lactic acid in eggs, 859-860
lead on apples and pears, 254-255
liming materials, 6-8
malathion pesticide residues, 303
manganese in feeds, 87
manganese in fertilizers, 33
manganese in plants, 46-47
menthol in cigarette filler, 67
mercury in foods, 264-266
methanol in distilled liquors, 702
methoxychlor pesticide residues, 304
methyl anthranilate in nonalcoholic
beverages, 755
molybdenum in plants, 47
niacin and niacinamide in drugs,
foods, and feeds, 1054-1055
nihydrazone in feeds, 105
nitrate and nitrite nitrogen in feeds,
77-78
nitrites in cured meat, 938
nitrofurazone in feeds, 100
nitrogen (ammonia) in water, 319—
320
nitrogen (nitrate) in water, 320
nitrogen (total) in water, 319
nitrogen in beer, 713
parathion in formulations, 208
parathion pesticide residues, 308
phenol in hazardous substances, 236
phenylephrine HCL in drugs, 516-
517
phosphorus in fruits and fruit
products, 916
phosphorus in wines, 743-744
piperonyl butoxide in formulations,
170
propyl gallate in food, 1140-1141
protein in feeds, 73-74
pyrogallol in hair dyes, 366
salicyclic acid in food and
beverages, 1155-1156
Salmonella in foods, 480-486
sodium cyclamate and calcium
cy clam ate in canned fruit,
1168-1169
sodium lauryl sulfate in egg white,
1163-1164
sorbic acid in wines, 949
sulfamethoxine in feeds, 110-111
sulfur dioxide in beer, 718
sulfurous acid in dried fruit, 1158-
1159
thiourea in orange peel, 1 162-1163
a-tocopherol and a-tocopheryl
acetate in foods and feeds,
1071-1074
a-tocopheryl acetate (supplemental)
in foods and feeds, 1075-1076
trimethylamine nitrogen in seafood,
869-870
urea in feeds, 76-77
vitamin A in mixed feeds, premixes,
and foods, 1045-1047
vitamin D in vitamin preparations,
1061-1064
zinc in fertilizers, 34-35
zinc in food, 272
zirconium in antiperspirants, 362
Coloring matter
in distilled liquors, 690
in green coffee, 757
in macaroni products, 798-799
see also Color additives
Column chromatographic methods
butyric acid in fat, 958-960
color additives in foods, 1115-1116
fatty acids in eggs, 860
glycerides in monoglyceride
concentrates, 982-983
hydrocarbons (saturated) in
glycerides, 980-981
see also Chromatographic methods;
Gas chromatographic methods;
Liquid chromatographic methods
Combustion methods
bromine, chlorine, or iodine
determination, 337-339
carbon and hydrogen determination,
339-341
sulfur determination, 345-347
Concentration
nomograph relating absorptivity,
absorbance, and, 672
Condensed milk
filth in, 378-379
sampling, 803
Condimental seeds
filth in, 398-399
Conductivity value
of maple sirup, 1036
Confectionery
alcohol in sirups used in, 1025
ether extract of, 1024-1025
paraffin in, 1025
shellac in, 1025
starch in, 1024
Convection oven method
moisture in frozen french-fried
potatoes, 998
Cooking
seafood products, 865
Copper
in beer, 716-717
in Bordeaux mixtures, 149-150,
158, 159
in copper carbonate formulations,
155
in copper naphthenate formulations,
155
in distilled liquors, 695
in feeds, 84, 86
in fertilizers, 27-28, 31
in food, 248
fungicides, 156
in infant formulas, 1106-1107, 1110
in liver, 356-357
in milk, 821
in pesticide formulations, 149-150,
154
in plants, 40, 42, 45-46
in serum, 357
in tea, 242, 762
in water, 324-325
in wines, 743
Copper carbonate formulations, 155
Copper catalyst method
protein in feeds, 74
Copper naphthenate formulations,
155
Copper salts
in feeds, 84
Copper serum method
water (added) in milk, 818-819
Copper-reducing substances
in spices, 1000
Coprostanol
in heat-processed materials, 413-414
AOAC Official Methods of Analysis (1 990)
Subject Index
1-13
Cordials and liqueurs
acids in, 706
alcohol by volume, 705
alcohol in, 705
aldehydes in, 705
ash in, 706
benzaldehyde in, 707
caramel in, 706
fusel oil in, 705
glycerol in, 706
hydrazino-7-undecalactone properties
in, 707
methanol in, 705
phosphorus in, 706
physical examination, 704
solids (total) in, 705-706
specific gravity, 705
sucrose in, 706
thujone in, 707
7-undecalactone in, 707
volatile esters, 706-707
Coriander
filth in, 398-399, 401
foreign matter in, 398
Corn
anatoxins in, 1188, 1191
breakfast cereals, antioxidants in,
791
fat acidity, 788-789
field corn in mixtures of field and
sweet corn, 994
filth in corn cereals and corn chip
products, 387-388
filth in flour, 385
foreign matter in canned products,
395
fumigant residues in grain, 290-291
iron in degerminated, bolted whole
meal, 788
mold in cream-style canned
products, 422
W-methylcarbamate residues on
kernels, 291-292
thiram pesticide residues in, 309-
311
zearalenone in, 1211-1213
see also Cereal foods; Grains
Corn grits
extract of, 731
sorting, 730
Corn Industries Research
Foundation-AOAC methods
moisture in corn sirups and sugars,
1039-1040
saccharides (major) in corn sirups
and sugars, 1042-1043
saccharides (minor) in corn sirups
and sugars, 1044
Corn meal
filth in, 382-383
Corn oil
peanut oil in, 978-979
Corn sirups and sugars
acidity of, 1040-1041
in apple juice, 926
ash of, 1040
dry substance in, 1040-1041
glucose in, 1042
in honey, 1032-1033
in maple syrup, 1035
moisture in, 1039-1040
nitrogen in, 1042
in orange juice, 926-927
saccharides (major) in, 1042-1043
saccharides (minor) in, 1044
sample preparation, 1039
sugars (reducing) in, 1042
Cortisone acetate, 608-609, 619
Cosmetics
bacteria in, 431
deodorants and antiperspirants, 361-
365
depilatories, 365
eye irritants in, 360-361
face powders, 365
hair preparations, 366-367
propylene glycol in, 360
suntan preparations, 367-368
vanishing cream, 368
water and ethyl alcohol in, 359
Cotarnine, 534, 545
Cottage cheese
gelatin in, 847
sorbic acid in, 847
Cottonseed oil
in oils and fats, 977-978
peanut oil in, 978-979
Cottonseed products
aflatoxins in, 1192-1193
Coumarin
in vanilla extract, 892-893
in wines, 750
Crabmeat
ammonia in, 869
filth in canned products, 389-390
frozen, drained weight of, 867
generic identification of, 886, 889
indole in, 877-878
shell in canned products, 389
Cracking flotation method
insects in grains and seeds, 381
Cranberry juice cocktail
quinic, malic, and citric acids in,
920-921
Cranberry sauce
mold in, 418
Cream
acids (volatile) in, 831
ash of, 832
color additives in, 833
fat in, 832
fatty acids in, 831
filth in, 378-379
gelatin in, 832
lactic acid in, 831
lactose in, 832
nitrogen (total) in, 832
phosphatase in, 833
preservatives in, 833
pressurized, 831
sample collection, 831
sample preparation, 831
solids (total) in, 831
water (added) in, 832
Cream of tartar
and free tartaric acid in tartrate
powders, 686-687
Creatine
in meat extracts and similar
products, 947
Creatinine
in meat extracts and similar
products, 947
in meat, 941
Cresidine sulfonic acid
in FD&C Red No. 40, 1127-1128
Cryoscopic method
water (added) in milk, 819
Crystallographic methods
p-ionone in flavor extracts and toilet
preparations, 906-907
rotenone in derris and cube powder,
168-169
Cucumbers
organochlorine pesticide residues in,
284-285
Cube powder, 168-169
Culture media
eggs and egg products, 427-428
Salmonella, 467-470
Cumin
filth in, 398-399
foreign matter in, 398
Cundiff-Markunas method
alkaloids in tobacco, 66
Cuprethol method
copper in beer, 717
Curculio larvae
in pecan pieces, 379
Cured meats
nitrites in, 938
Curry powder
filth in, 398-399
foreign matter in, 398
CuS0 4 /Ti0 2 mixed catalyst method
protein in feeds, 70
Cyanamide
nitrogen in, 20
Cyanide
in calcium cyanide formulations, 159
distilled liquors, 703-704
safe handling of, 653, 1227
in wines, 749
Cyanide method
formaldehyde in formulations, 226
Cyanogenetic glucosides
in feeds, 90, 1213
Cyclamates
cyclohexylamine in, 1169-1171
Cycloate
in herbicide formulations, 221
Cycf ©barbital, 537, 546
Cyclohexane
safe handling of, 653, 1227
Cyclohexanol test
colors in distilled liquors, 691
Cyclohexylamine
in cyclamates and artificially
sweetened products , 1 1 69- 1171
Cyclohexylsulfamate salts
in nonalcoholic beverages, 1168
-14
Subject Index
AOAC Official Methods of Analysis (1990)
Cyclopropene
in oils, 978
Cyhexatin
technical and formulations, 225
Cylinder plate method
bacitracin in premix feeds, 118
Cyperniethrin
in pesticide formulations, 166-167
2,4-D
in formulations, 184-185, 187-188,
196
Dairy products
aflatoxin M, in, 1199-1201
alcohol by volume in alcoholic
products, 705
barium- 140 in milk, 353-355
butter, 836-840
cesium- 137 in milk, 353-355
cheese, 840-850
coliforms in, 434, 435-436
cream, 831-833
dried milk, nonfat dry milk, and
malted milk, 834-836
ethylenethiourea pesticide residues in
milk, 300-302
evaporated and condensed milk,
833-834
filth in, 378-379
ice cream and frozen desserts, 850-
852
iodine-131 in milk, 353-355
lead in evaporated milk, 255-257
milk, 804-831
sampling, 802-804
sediment in, 375-378
somatic cells in milk, 496-497
sorbic acid in, 1156
strontium-89 and -90 in milk, 351-
353
titanium in cheese, 271
see also specific dairy products
Dalapon
magnesium salt in formulations,
185-186
sodium salt in formulations, 185-
186
D&C colors
Blue No. 3, 3 122
Blue No. 4, 1119
Blue No. 6, 1119
Blue No. 9, 1120
Brown No. 1, 1120
Green No. 5, 1120
Green No. 8, 1120
Orange No. 4, 1120
Orange No. 5, 1120
Orange No. 11, 1120
Orange No. 17, 1121
Red No. 6, 1120, 1132
Red No. 7, 1121, 1132
Red No. 8, 1121
Red No. 9, 1121
Red No. 17, 1121
Red No. 19, 1121
Red No. 21, 1121
Red No. 22, 1121
Red No. 27, 1121
Red No. 28, 1121
Red No. 30, 1121
Red No. 31, 1121
Red No. 33, 1121
Red No. 34, 1121
Red No. 36, 1121
Red No. 39, 1122
Violet No. 2, 1122
Yellow No. 7, 1122
Yellow No. 8, 1122
Yellow No. 10, 1122
Yellow No. 11, 1122
DCPA
in formulations, 186
DDE
multiresidue methods, 279
DDT
in formulations, 186-187
in lettuce, strawberries, and
tomatoes, 282-283
multiresidue methods, 279
residues, 298
DDVP
in formulations, 199-200
Decoquinate, 96-97, 628, 636
Deep maroon, 1121
Dehydroacetic acid
in cheese, 1147-1148
Denaturants
in alcoholic products, 232
Deniges-Tourrou test
dulcin in food, 1171
Densitometric method
alcohol by volume in distilled
liquors, 692-693
alcohol in alcoholic dairv products,
705
Deodorants
aluminum in, 361, 362
boric acid in, 362-363
chlorides in, 363
hexachlorophene in, 363-364
methenamine in, 364
phenolsulfonates in, 364-365
sulfides in, 363
urea in, 365
zinc in, 361-362
Deoxynivalenol
in wheat, 1205-1207
Depilatories
sulfides in powders, 365
Derris and cube powder
ether extract of, 1.69
rotenone in, 168-169
Desoxyephedrine, 540
Dessert powders
gelatin, 929
starch, 930
Dessert preparations, see Gelatin
Detergent method
fat in raw milk, 814
Devarda method
nitrogen in fertilizers, 19
Dexamethasone, 614-616
Dexamethasone acetate, 616, 619
Dexamethasone phosphate, 614, 619
Dextran
in raw cane sugar, 1020-1021
Dextrin
in beer, 712
in honey, 1029
in roasted coffee, 759
in vinegars, 1008
Dextroamphetamine, 540, 546
Dextrose
in corn sirups and sugars, 1042-
1043
Diacetyl
in beer, 712-713
Diacetylmorphine, 534, 620
2,5-Diaminotoluene
in hair dyes and rinses, 366
Diastase method
starch in feeds, 83
Diastatic activity
of flour, 785-786
of honey, 1033
Diastatic power
of malt sirups, 733-734
of malt, 727-729
Diazepam, 620-621
Diazinon
confirmatory method, 289-290
Diazinon
on apples, carrots, endive, kale,
lettuce, potatoes, and
strawberries, 287-289
in formulations, 200-201
4,4'-(Diazoamino)bis(5-methoxy-2-
methyl-benzenesulfonic acid
in FD&C Red No. 40, 1127-1128
4,4'-(Diazoamino)dibenzenesulfonic
acid
in FD&C Yellow No. 6, 1129-1132
Dibromofluorescein, 1120
Dibutyltin dilaurate
in feeds, 97
Dicamba
in formulations, 187-188
Dichlobenil
in formulations, 188-189
Dichlone
in fresh fruits and vegetables, 298-
299
Dichlorimide method
hair preparations, 366
Dichloroacetic acid
safe handling of, 654, 1228
2,6-Dichloroindophenol titrimetric
method
vitamin C in ready-to-feed milk-
based formulas, 1108-1109
vitamin C in vitamin preparations
and juices, 1058-1059
Dichlorophen, 529-530, 546
Dichromate oxidation
alcohol in wines, 739-741
Dichromate oxidation method
ether in drugs, 499
glycerol in beer, 711
Dicofol
in formulations, 189-190
Dicumarol, 565-566, 577
AOAC Official Methods of Analysis (1990)
Subject Index
1-15
Dieldrin
in formulations, 175-176
in lettuce, strawberries, and
tomatoes, 282-283
multiresidue methods, 279
Dienestrol in drugs, 609-610, 619
Diethyl ether
safe handling of, 653, 1227
Diethyiamine
safe handling of, 653, 1227
Diethylcarbonate
in food, 1 147
in wines, 750
Diethylpropion HCI, 576-577, 577
Diethylstilbestrol, 97, 609, 619
Differential pulse polarographic
methods
arsenic in cacodylate injections, 501
iodine in thyroid drug tablets, 618-
619
saccharin in food, 1173-1174
sulfites in food, 1159-1160
Diflubenzuron
in formulations, 190-191
Difluoroacetic acid
safe handling of, 654, 1228
Digestion methods
fiber in plants, 59
mercury in fish, 264
metals at trace levels in food, 238,
243-244
nitrogen (nitrite) in flour, 783-784
phosphorus determination, 344-345
protein in flour, 781-782
proteolytic activity of flour and
malted wheat flour, 786-787
shell in canned clams and oysters,
390
shell in canned crabmeat, 389
see also Kjeldahl methods; and other
specific methods
Digital density meter method
specific gravity of beer and wort,
709-710
Digitalis, 599-601
Digitonin method
sterols in macaroni products, 797-
798
Digitoxin, 599-600, 606
Digitoxosides, 601
Digoxin, 600-601, 606
Dihydroanethole, 754-755
Dihydrosafrole, 754-755
Dill
filth in, 398-399
Dill seed
foreign matter in, 398
Dilution method
sucrose in sugars and sirups, 1016
Dimethisterone, 612, 619
Dimethylformamide
safe handling of, 653, 1227
Dimetridazole, 97-98
Dinitrophenol, 538, 550
Dinitrophenylhydrazine method
santonin in drug mixtures, 603
santonin in santonica, 604
Dioxins
in oils and fats, 981-982
Diphenhydramine HCI, 538, 546
Diquat
in formulations, 225
Direct acid hydrolysis
starch in feeds, 83
Direct current arc excitation method
metals in plants, 41
Direct reading spectrographic
method
metals in plants, 41
Direct sieving method
filth in tea, 375
Disc electrophoresis method
oyster detection, 865-866
Disinfectants
bacteriostatic activity, 133-137,
143-145
chlorine in, 137-138
fungicidal activity, 138
germicidal activity, 137-141
laundry additive, 143-145
phenol coefficient methods, 133-135
sanitizing activity of, 138-140
for seeds, 162-163
sporicidal activity, 141-142
spray products, 140-141
for swimming pools, 145-146
tuberculocidal activity, 142-143
use-dilution methods, 135-137
see also Seed disinfectants
Dispersion microscopy
glass fragment characterization and
characterization, 637-638
Distillation methods
alkaloids in tobacco, 65-66
arsenic in dosage forms, 501
arsenic in pesticide formulations,
147-148
fluoride in plants, 52-56
fluorine in food, 250-253
fluorine in pesticide formulations,
152
meperidine in drugs, 512
moisture in cheese, 841-842
moisture in feeds, 69
moisture in hops, 732
moisture in spices, 999
neostigmine in drugs, 591
thiram in formulations, 222
Distilled liquors
acetone, other ketones, isopropanol,
and tertiary butyl alcohol in,
701-702
acids in, 697
alcohol by volume, 691-694
alcohol by weight, 694
alcohols (higher) and ethyl acetate
in, 700-701
aldehydes in, 697, 698-699
ash of, 694-695
chloride in, 696-697
color of, 690
coloring matter (natural and artificial
organic and water-soluble) in,
690
colors (artificial) in, 691
copper in, 695
cyanide in, 703-704
esters in, 697-698
ethyl acetate and alcohols (higher)
in, 700-701
extract of, 694
furfural in, 701
fusel oil in, 699-700
iron in, 696
isopropanol, acetone and other
ketones, and tertiary butyl
alcohol in, 701-702
methanol in, 702-703
phosphorus in, 695
physical examination, 690
potassium in, 695
sodium in, 695
specific gravity, 691
spirits, 690-704
tannin in, 703
tertiary butyl alcohol, acetone and
other ketones, and isopropanol
in, 701-702
see also Cordials and liqueurs
Disulfoton
in formulations, 201
Disulfram, 574, 577
Dithiocarbamates
in formulations, 217-218
Dithiodiglycolic acid
in cold permament waves,
366-367
Dithizone method
cadmium in food, 246-247
lead in food, 258-262
mercury in foods, 264-266
DNA colony hybridization methods
detection of E. coli heat- labile
enterotoxins, 441-444
enterotoxigenic E. coli, AAA-AA^>
Salmonella in foods, 488-490
2,4-DNT
in wastewater and groundwater,
334-336
Docosenoic acid
in oils and fats, 967
Dodine
in apples, peaches, pears, pecans,
and strawberries, 299
in formulations, 225-226
Dressings for food, see Food dressings
Dried fruits
moisture in, 912
sulfurous acid in, 1158-1159
Dried milk and its products
ash of, 835
citric acid in, 835
crude fat and ether extract in, 80
fat in, 835
filth in, 378-379
lactic acid in, 835
moisture in, 834
protein in, 834
sample preparation, 834
sampling, 803, 834
starch in, 84
1-16
Subject Index
AOAC Official Methods of Analysis (1990)
Drugs
acids, 548-549
adrenocortico steroids, 613-618
alkaloids and related amines, 533-
536, 541
alkanolamines, 515-520
aminobenzoates, 521-525
analgesics and antipyretics, 553-558
anticoagulants, 565-567
antiemetics, 542-543
antifungals, 543-544
antihistamines, 512-520
antihypertensives, 545
antiparkinsonian, 544-545
antiseptics, 553
barbiturate, 559
certified reference materials for, 647,
1221
chemical and common names, 545-
546, 577-578, 606, 619, 624
chinchona alkaloids, 592-593
digitalis, 599-601
ephedra alkaloids, 586-588
ergot alkaloids, 588-590
estrogens, natural, 607-609
estrogens, synthetic, 609-612
fluoride in tablets and solutions, 505
foreign matter in leafy crude
materials, 406
general directions, 498
halogenated, 499-501
hypnotics, 558-565
illicit, 620-624
inorganic, 501-512
ipecac alkaloids, 584-586
microchemical tests, 533-541
microscopy, 541-542
natural products, 602-605
niacin and niacinamide in, 1054-
1055, 1056-1057
nitrites in tablets, 512
opium alkaloids, 579-583
optical crystallographic properties,
659-667
phenolic, 549-553
phenyJethylamines, 520-521
physostigmine alkaloids, 590-592
progestational steroids, 612-613
rauwolfia alkaloids, 593-598
refractive indices, 668-671
RRR- or a//-raoa-tocopherol in,
1074-1075
sampling, 498
sedatives, 558-565
silver protein in, 512
solvents in, 498-499
sulfonamides, 567-570
sulfur-containing, 573-577
sympathomimetic, 536, 540
synthetics, 525-533, 536, 538-541
thiazides, 570-573
thyroid, 618-619
tropane alkaloids, 583-584
vasoconstrictors, 524
vitamin E in, 1078-1079
xanthine alkaloids, 541, 584
see also specific drugs and drug
groups
Drugs and feed additives in animal
tissues
ANOT, 625
arsenic, 626
chemical and common names, 636
clopidol, 626-628
decoquinate, 628
ethoxyquin, 628-629
melengestrol acetate, 629-631
nalidixic, 631
sulfamethazine in swine, 631-634
sulfonamide, 634-635
zoalene, 635
Drugs in feeds
2-acetylamino-5-nitrothiazole, 91
alkomide, 91-92
p-aminobenzoic acid, 92
2-amino-5-nitrothiazole, 92
amprolium, 92-93
antibiotics , 115-131
arprinocid, 92-93
arsanilic acid, 94
arsenic, 94-95
bifuran, 100
bithionol, 95
buquinolate, 95
cadmium, 95
carbadox, 95-96
common and chemical names of,
132
decoquinate, 96-97
dibutyltin dilaurate, 97
diethylstilbestrol, 97
dimetridazole, 97-98
ethopabate, 98-99
furazolidone, 99-101
glycarbylamide, 100
ipronidazole, 100
melengestrol acetate in feed
supplements, 101-103
nequinate, 103
nicarbazin, 103
nicotine, 103-104
nifursol, 104
nihydrazone, 105
nitarsone, 105
nithiazide, 105
nitrodan, 106
nitrofurazone, 100
nitromide, 106
nitrophenide, 106
phenothiazine, 106
piperazine, 106-107
pyrantel tartrate, 107
racephenicol, 108
reserpine, 108
ronnel, 108
roxarsone, 108-110
sulfaguanidine, 1 1 1
sulfamethazine, 111
sulf amethoxine , 110-111
sulfanitran, 111-112
sulfaquinoxaline, 112
sulfonamides, 112-115
zoalene, 101, 115
see also Antibiotics in feeds
Drugs in premixes
furazolidone, 99-101
roxarsone, 108-110
Drupelet berries
mold in, 418
Dry ash methods
calcium in feeds, 84
Dry column- thermal energy
analyzer method
iV-nitrosopyrrolidine in fried bacon,
940-941
Dry rehydratable film method
bacteria and coliform organisms in
milk, 435
Drying methods
moisture in feeds, 69
moisture in hops, 732
solids in yeast, 736
Dulcin
in food, 1171-1172
in nonalcoholic beverages, 1167-
1168
Dumas method
protein in feeds, 71-72
Duquenois-Levine qualitative test
cannabinol in drug powders, 621
Dye binding method
protein in dried milk, 834
protein in milk, 808-809
Dyes
in color additives, 1123-1125
oil-soluble, 1115
subsidiary and lower sulfonated,
1132
water-soluble, 1115
Dyphyline, 541, 546
EDTA titrimetric method
calcium and magnesium in liming
materials, 5
hardness of water, 323
magnesium in fertilizers, 32
Egg noodles, see Macaroni products
Eggs and egg products
acidity of ether extract of, 858
aflatoxin Bi in, 1195-1197
carotenoids in, 853
chloride in, 856
cholesterol in, 855-856
color of yolks, 853
ethoxyquin residues in, 628-629
fat in, 854
fatty acids (volatile) in, 860-861
fatty acids (water-soluble) in, 862
filth in, 388-389
glucose and sucrose in, 856-857
glycerol in, 857
p-hydroxy butyric, lactic, and
succinic acid in, 858-859
lactic acid in, 858-860
lipids and lipid phosphorus in, 854-
855
microbiological methods, 427-428
nitrogen in, 854
phosphorus in, 856
pyoverdine in, 862-863
quaternary ammonium compounds
in, 863
Salmonella in, 470-471, 476-480
AOAC Official Methods of Analysis (1990)
Subject Index
1-17
sampling of, 853
sodium laury] sulfate in egg white,
1163-1164
solids (total) in, 853
succinic acid in, 861-862
Electrolyte replenishes
calcium, potassium, and sodium in,
503-504
Electrolytic methods
copper in Bordeaux mixtures, 158,
159
copper in copper carbonate
formulations, 155
copper in copper naphthenate
formulations, 155
copper in Paris green pesticide
formulations, 154
Electrometric titration method
acidity in corn syrup, 1041
Elemental analyses
liming materials, 4-8
Elevated temperature enrichment
method
Vibrio cholerae in oysters, 492-494
Elmslie-Caldwell method
iodine in mineral mixed feeds, 87
Emetine, 585-586, 606
Emetine HC1, 580
Emission spectrographic methods
boric acid in food, 1147
metals in plants, 40
Emulsifying agents
in foods, 1163-1164
Endive
organophosphorus pesticide residues
on, 287-289
Endosulfan
in apples and cucumbers, 284-285
in formulations, 191-192
Endosulfan sulfate
in apples and cucumbers, 284-285
Endrin
in formulations, 175-176
multiresidue methods, 279
Enterobacteriaceae
identification in foods, 474-475
Enterotoxins
E. coli, detection, 441-444, 448-
449
staphylococcal, in foods, 451-455
Enzymatic methods
carbon dioxide in wines, 748
citric acid in wine, 746
extract of corn grits, 731
glucose and fructose in wine, 741-
742
lactose in milk, 810-811
Enzymatic-gravimetric method
fiber (dietary) in foods, 1105-1106
Enzyme digestion method
filth in pork sausage, ground beef,
or hamburger, 390-391
Enzyme immunoassay
Salmonella in foods, 480-488
Enzyme-linked immunosorbent
assays
aflatoxin Bi in cottonseed products
and mixed feed, 1193-1195
soy protein in raw and heat-
processed meat products, 942-
945
Enzymes
in foods, 1164
Eosin yellowish method
quaternary ammonium compounds in
aqueous solutions and milk,
1154-1155
Eosin YS, 1121
Ephedra alkaloids, 586-588
Ephedrine
chemical name, 546
in drug inhalants, 586
in drug tablets and capsules, 586
HC1, 580, 606
microchemical tests, 534, 540
in solid dosage drugs, 586-587
sulfate, 519-520, 546, 580, 586-
587, 606
titration factors, 580
in water-soluble jellies, sirups, and
solutions of ephedrine salts, 586
^/-Ephedrine, 535
Epinephrine, 515-516
Epinephrine bitartrate, 515-516, 546
Epinephrine HC1, 515-516, 540
EPN
on apples and green beans, 286-287
EPTC
in herbicide formulations, 221
Equipment hazards, 649-650, 1223-
1224
Ergocalciferol, see Vitamin D
Ergot alkaloids, 588-590
Ergotamine, 589-590, 606
Erucic acid
in oils and fats, 965-967
Erythrityl tetranitrate, 527, 546
Erythromycin
in feeds, 120-121
in milk, 829-831
Erythrosine, 1115, 1119
Erythrosine yellowish NA, 1120
Escherichia coli
in chilled or frozen foods, 438-439
in chilled, frozen, precooked, or
prepared foods, 429-430
detection of heat-labile enterotoxins,
441-444
enterotoxigenic, identification, 444-
448
enterotoxin detection, 448-449
in foods, 437-438
identification in foods, 474-475
invasiveness of mammalian cells,
439-441
in shellfish growing waters, 436-437
in tree nut meats, 430-431
Essential oils
in emulsion, 906
in flavor extracts and toilet
preparations, 905
in fruits and fruit products, 923
in nonalcoholic beverages, 752
Esters
in distilled liquors, 697-698
in lemon oil, 903
in nonalcoholic beverages, 751
p-Estradiol, 607, 619
Estradiol valerate, 607, 619
Estrogens
conjugated, 607
cortisone acetate, 608-609
dienestrol, 609
P-estradiol, 607
estradiol valerate, 607
ethinyl estradiol, 607-608
with ethynodiol, 611-612
hexestrol, 610
ketosteroids, 607
mestranol, 611-612
Ethanol
in beer, 711
in canned salmon, 880-881
safe handling of, 653, 1227
Ethchlorvynol, 563, 577
Ether
in drugs, 499
Ether extract
cereal adjuncts, 731
of confectionery, 1024-1025
of derris and cube powder, 169
in dried milk products, 80
in feeds, 79
of plants, 59
of prepared mustard, 1003
Ether extraction methods
fat in fig bars and raisin filled
crackers, 795-796
phenolphthalein in tablets, 552
succinic acid in eggs, 861-862
sulfonmethane or sulfonethylmethane
in drugs, 563
unsaponifiable matter in cocoa
butter, 771
unsaponifiable residue of oils and
fats, 971-972
Ethinyl estradiol, 607-608, 619
Ethion
on apples, carrots, endive, kale,
lettuce, potatoes, and
strawberries, 287-289
in formulations, 201-202
Ethisterone, 613, 619
Ethopabate, 98-99
Ethoxyquin, 90, 628-629, 636
Ethyl acetate
and alcohols (higher) in distilled
liquors, 700-701
in distilled liquors, 700-701
safe handling of, 653, 1227
Ethyl alcohol
apparent specific gravity, 1231-1249
in distilled liquors, 700
and water in cosmetics, 359
see also Alcohol; Alcoholic
beverages
Ethyl parathion, 209-210
Ethyl vanillin, 891-893
Ethylan (perthane) residues, 298
Ethylene dibromide
in fumigant mixtures, 164
in grains and grain products, 299-
300
in wheat and corn grain, 290-291
1-18
Subject Index
AOAC Official Methods of Analysis (1990)
Ethylene dichioride
in fumigant mixtures, 164
in spice oleoresins, 1175
Ethylenethiourea
in applesauce, milk, potatoes, and
spinach, 300-302
Ethylhydrocupreine, 534, 546
Ethylmorphine, 534, 546, 580, 606
Ethynodiol diacetate, 611-612
European DecaiTeination
Association-AOAC method
solids (soluble) in roasted coffee,
760
Evaporated milk
albumin in, 833
ash of, 833
casein in, 833
color additives in, 833
fat in, 833
filth in, 378-379
gelatin in, 833
lactic acid in, 833
lactose in, 833
lead in, 255-257, 833
nitrogen (total) in, 833
preservatives in, 833
sample preparation, 833
sampling, 803, 833
solids (total) in, 833
Excreta
in condiment seeds, 402
in flour, 384
in flour, 415
on foods and containers, 414-415
see also Mammmalian feces
Ext. D&C Violet No. 2, 1122
Ext. D&C Yellow No. 7, 1122
Extract, see Ether extract
Extraction methods
extractives from flexible barrier
materials, 1178-1180
lighter-than-water solvents for drugs,
498
meperidine in drugs, 512
phenylpropanolamine HC1 in drugs,
519
Staphylococcal enterotoxin in foods,
454-455
starch in feeds, 83
water in fertilizers, 12
Extraneous materials
animal excretions, 407-415
aphids in hops, 374-375
in baked goods, 385-387
in breakfast cereals, 387-388
in dairy products, 375-379
in eggs and egg products, 388-389
in fruits and fruit products, 391-393
in grains and their products, 380-
385
isolation techniques, 369-372
mold and rot fragments, 416-423
in nuts and nut products, 379-380
in peanut butter, 380
plant material in vanilla extract,
895-896
in poultry, meat, fish, and other
marine products, 389-391
shell in cacao products, 764
in snack products, 393
in spices and other condiments, 397-
407
in sugars and sugar products, 393-
394
in vegetables and vegetable products,
394-397
see also Filth
Eye irritants
and water in cosmetics, 360-361
Eylenol method
nitrates and nitrites in meat, 937-
938
Face powders, 365
Falling number determination
method
a-amylase in flour, meal, and malted
cereals, 786
Fanchon maroon, 1121
FAO/WHO-AOAC method
thawing frozen vegetables, 995
Fast green FCF, 1115, 1119
Fat
in baked products, 795
in bread, 793
in butter, 837
in cacao products, 770-772
in cereal adjuncts, 731
in cheese, 844
in cream, 832
critical temperature for dissolution in
acetic acid, 772
in dried milk, 835
in eggs, 854
or ether extract in flour, 780
or ether extract in grains, 788
in evaporated milk, 833
in fig bars and raisin filled crackers,
795-796
in fish meal, 871-872
in flour, 780
in food dressings, 1005
in foods, 1100
(foreign) containing tristearin in lard,
979-980
in ice cream and frozen desserts,
851
in macaroni products, 796
in malted milk, 835
in meat and meat products, 931-932
in milk, 811-818
from milk chocolate, 772
in nuts and nut products, 949
in seafood, 871
separation from ice cream, 851
in sweetened condensed milk, 834
see also Animal fats; Oils and fats
Fat (crude)
in dried milk products, 80
in feeds, 79
in fish meal, 80
in pet foods, 79-80
Fat acidity
corn, 788-789
flour, 780
grains, 788
Fat number, 791
Fatty acids
in butter, 838-840
in cream, 831
cyclopropene in oils, 978
melting point of, 953-954
methyl estersin oils and fats, 964-
965
cis, cw-methylene interrupted
polyunsaturated, in oils, 969
in oils and fats, 963-964
saturated and unsaturated in oils and
fats, 960
trans isomers in margarines, 970-
971
Fatty acids (crude)
in crude and refined oils, 957
in oils and fats, 957
Fatty acids (volatile)
in eggs, 860-861
in seafood, 872-875
Fatty acids (water-soluble)
in eggs, 862
FD&C colors
Blue No. 1, 1117-1118, 1126
Blue No. 2, 1117-1118
in foods, 1117-1119
Green No. 3, 1117-1119
Red No. 2 (former), 1117-1118
Red No. 3, 1117-1119
Red No. 4, 1119
Red No. 40, 1117-1119, 1127-1128
Yellow No. 5, 1117-1119, 1128-
1129, 1132
Yellow No. 6, 1117-1119, 1129-
1132
Fecal coliforms
in foods, 437-438
in shellfish growing waters, 436-437
Feed additives in animal tissues, see
Drugs and feed additives in animal
tissues
Feeds
2-acetyIamino-5-nitrothiazo1e in, 91
acid-detergent fiber in, 74-76, 82-
83
acidity of, 84
aflatoxin B, in, 1193-1195
aflatoxins in, 1187-1188
albuminoid nitrogen in, 77
alkomide in, 91-92
p-aminobenzoic acid, 92
2-amino-5-nitrothiazole in, 92
ammoniacal nitrogen in, 76
amprolium in, 92-93
animal tissues in, 89
antibiotics in, 115-131
arprinocid in, 93
arsanilic acid in, 94
arsenic in, 94-95, 356
ash of, 70
bifuran in, 100
bithionol in, 95
buquinolate in, 95
AOAC Official Methods of Analysis (1990)
Subject Index
1-19
cadmium in, 95
calcium in, 84-85
carbadox in, 95-96
carotenes and xanthophylls in dried
plant materials and mixed feeds,
1048-1049
carotenes in fresh plant materials and
silages, 1048
certified reference materials for, 646,
1220
chlorine in, 85
cobalt in, 85-86
copper in, 86
cyanogenetic glucosides in, 90, 1213
decoquinate in, 96-97
dibutyltin dilaurate in, 97
diethyl stilbestrol in, 97
dimetridazole in, 97-98
ether extract in, 79
ethopabate in, 98-99
ethoxyquin in, 90
fat (crude) in, 79
fiber (crude) in, 80-82
fluorine in, 86
furazolidone in, 90, 99-101
galactan in, 84
glycarbylamide in, 100
hydrocyanic acid in, 90
hygromycin, 121
in mixed feeds, premixes, and pet
foods, 1069-1070
ipronidazole in, 100
iron in, 84
lignin in, 82-83
manganese in, 84, 87
melengestrol acetate in feed
supplements, 101-103
menadione sodium bisulfite in
premixes, 1079-1080
microscopic examination of, 88-90
mineral salts in, 84
minerals in, 84-88, 89-90
mixed mineral, iodine in, 87
moisture in, 69-70
nequinate in, 103
niacin and niacinamide in, 1054-
1055, 1056-1057
nicarbazin in, 103
nicotine in, 103-104
nifursol in, 104
nihydrazone in, 105
nitarsone in, 105
nithiazide in, 105
nitrate and nitrite nitrogen in, 77-78
nitrodan in, 106
nitrofurazone in, 100
nitromide in, 106
nitrophenide in, 106
pentosans in, 84
phenothiazine in, 106
phosphorus in, 87-88
piperazine in, 106-107
protein in, 70-76
protein, pepsin digestibility of, 78-
79
pyrantel tartrate in, 107
racephenicol in, 108
reserpine in, 108
ronnel in, 108
roxarsone in, 108-110
sample preparation, 69, 91
sampling of, 69, 91
sucrose in, 83
sugars in, 83
sulfaguanidine in, 111
sulfamethazine in, 111
sulfamethoxine in, 110-111
sulf anitran in , 111-112
sulfaquinoxaline in, 112
sulfonamides in, 112-115
a-tocopherol and a-tocopherol
acetate in, 1071-1074
RRR- or a//-raoa-tocopherol in feed
supplements, 1074-1075
a-tocopheryl acetate (supplemental)
in, 1075-1076
urea in, 76-77
vegetable tissues in, 89
vitamin A in mixed feeds and
premixes, 1045-1047
vitamin D in concentrates, 1091-
1094
vitamin D 3 in poultry feed
supplements, 1094-1095
zinc in, 84
zoalene in, 90, 101, 115
see also Drugs and feed additives in
animal tissues
Fehling solution method (modified)
sugars in feeds, 83
Fenitrothion, 202-203
Fennel
filth in, 398-399
Fennel seed
foreign matter in, 398
Fensulfothion, 203-204
Fentin, 156-157
Fenugreek
filth in, 398-399
foreign matter in, 398
Fermentation
of beer, 711
Ferric ammonium citrate
in drugs, 506-507
Ferric glycerophosphate
in drugs, 506-507
Ferric oxide
in liming materials, 4
Ferric pyrophosphate
in drugs, 506-507
Ferricyanide method
quaternary ammonium compounds in
commercial preservatives , 1151-
1152
Ferrous ammonium sulfate
in drugs, 506-507
Ferrous fumarate
in drugs, 506-507
Ferrous gluconate
in drugs, 506-507
Ferrous salts
in feeds, 84
Ferrous sulfate
in drugs, 506-508
Ferrous sulfate-zinc-soda method
nitrogen in fertilizers, 19
Fertilizers
acid-forming or nonacid-forming
quality of, 36
aluminum in, 36
ash (acid-insoluble), 11
biuret in, 22-23
bone, 11
boron in, 28-30
calcium in, 27-28, 30
carbon (carbonate) in, 30
certified reference materials for, 647,
1221
chlorine in, 30
cobalt in, 30
copper in, 27-28, 31
iron in, 27-28, 31
lanthanum in, 27-28
liquid, 9
magnesium in, 27-28, 32-33
manganese in, 27-28, 33
mechanical analysis of, 11
methyleneureas in, 21-22
nitrates in, 17, 20
nitrogen activity of, 20-21
nitrogen in, 9-10, 17-23
nutrients in, 27-28
in pesticide formulations, 147
phosphate rock, 1 1
phosphorus in, 12-17
platinum recovery procedure for, 23
potassium in, 23-27
sample preparation, 10-1 1
sampling of, 9
slag, 11
sodium in, 33
solid, 9
sulfur in, 34
tankage, 11
triamino-s-triazine in, 22
urea in, 21-22
urea-formaldehyde , 2 1
water in, 11-12
zinc in, 27-28, 34-35
Fiber (crude)
in baked products, 795
in bread, 793
in cacao products, 764
in cereal adjuncts, 731
in feeds, 80-82
in flour, 780
in grains, 788
in macaroni products, 796
in nuts and nut products, 949
in plants, 59
in prepared mustard, 1004
in roasted coffee, 760
in soybean flour, 791
in spices, 1000
in tea, 762
see also Acid-detergent fiber
Fiber (dietary)
in flour, 780
in foods, 1105-1106
Fig paste
filth in, 392
-20
Subject Index
AOAC Official Methods of Analysis (1990)
Fifth
in alimentary pastes, 387
in apple butter, 391
in apple chops, 391
in baked goods with fruit and nut
tissue, 386
in barley, oatmeal, and mixed dry
infant cereal, 388
in beverages and beverage materials,
373-375
in breading of frozen foods, 387
in broccoli (canned), 395
in candy, 393-394
in canned crabmeat, 389-390
in canned fish and fish products, 390
in canned shrimp, 390
in chewing gum, 394
in chicken giblet paste, 389
in citrus and pineapple juices, 392-
393
in coconut, 379-380
in coffee and coffee substitutes, 374
in corn and rice cereals, 387-388
in corn flour, 385
in corn meal, 382-383
in cracked wheat and flours, 381-
382
in dairy products, 378-379
in eggs and egg products, 388-389
in fig and fruit paste, 392
in flour, 384
in grain products, 383
in grains and seeds, 380
in green leafy vegetables, 395-396
in high bran content breads, 385-
386
in high-fat products, 386-387
in jam and jelly, 392
light and heavy, 372-373
in mushrooms, 396-397
in peanut butter, 380
in pecans, 379
in popcorn, 393
in pork sausage, ground beef, or
hamburger, 390-391
in potato chips, 393
in potato products, 397
in pureed infant food, 396
in raisins, 393
in rice flours, products, and paper,
384-385
in sauerkraut, 397
in shelled nuts, 379
in sirups, molasses, and honey, 394
in soy flour, 385
in spices and other condiments, 397-
406
in starch, 385
in sugars, 394
in tea, 375
in tomato products, 397
in wheat germ, 383-384
in wheat gluten, 385
in white breads, 386-387
in whole wheat cereals, 388
Filtration methods
filth in food dressings
filth in sirups, molasses, and honey,
394
filth in sugars, 394
pepsin digestibility of protein feeds,
78-79
Fingerprint detection, 637
Fish and other marine products
acetic acid in seafood, 874
ammonia in crabmeat, 869
ash of seafood , 868
boron in caviar, 1 145
butyric acid in seafood, 874
cooking seafood products, 865
crude fat and acetone extract in fish
meal, 80
detection of frozen and thawed
shucked oysters, 865-866
drained liquid from shucked oysters,
867
drained weight of crabmeat, 867
drained weight of frozen shrimp and
crabmeat, 867
ethanol in canned salmon, 880-881
fat in fish meal, 871-872
fat in seafood, 871
fatty acids (volatile) in seafood,
872-875
filth in canned crabmeat, 389-390
filth in canned fish and fish
products, 390
fish content of frozen breaded fish
products, 864-865
fish oil and marine animal oil
detection, 980
formic acid in seafood, 872-873
histamine in seafood, 875-877
identification of canned Pacific
salmon, 889
identification of cooked and frozen
crabmeat, 886, 889
identification of fish species, 883-
888
indole in crabmeat, oysters, and
shrimp, 877-878
indole in shrimp, 878-879
lead in fish, 257-258
mercury in fish, 263-264
methyl mercury in fish and shellfish,
266-269
minced fish flesh in mixed fillet-
minced cod blocks, 868
net contents of frozen seafood, 864
nitrogen (total) in seafood, 868
organochlorine pesticide residues in,
283-284
paralytic shellfish poison, 881-882
parasites in fish muscle, 882-883
PCBs in, 283-284
poliovirus 1 in oysters, 495-496
propionic acid in seafood, 874
salt in seafood, 870, 933
sample treatment and preparation,
864
seafood in seafood cocktail, 868
shell in canned clams and oysters,
390
shell in canned crabmeat, 389
shrimp in shrimp cocktail, 867-868
sodium and potassium in seafood,
870-871
solids (total) in seafood, 868
trimethylamine nitrogen in seafood,
869-870
valeric acid in seafood, 874
Vibrio cholerae in oysters, 492-494
volume of shicked oysters, clams, or
scallops, 867
Flame photometric methods
potassium and sodium in wines, 744
potassium and/or sodium in plants,
47-48
potassium in distilled liquors, 695
potassium in fertilizers, 23-26
potassium in fruits and fruit
products, 915
potassium in tobacco, 64™65
sodium and potassium in seafood,
870-871
sodium in distilled liquors, 695
sodium in fertilizers, 33-34
sodium in fruits and fruit products,
915-916
Flameless atomic absorption
spectrophotometry; methods
mercury in fish, 263-264
mercury in food, 262-263
mercury in water, 326-327
Flaming red, 1121
Flammable solvents
safe handling of, 652, 1226
Flavor extracts and toilet
preparations
(3-ionone, 906
alcohol in, 905
ammonium glycyrrhizinate in, 907-
908
citral, 906
essential oil in, 905-906
see also Flavors
Flavoring additives
in vanilla extract, 896
Flavors
alcohol in, 890
almond extract, 903-905
(3-asarone in wines, 750
cassia, cinnamon, and clove extracts,
905
coumarin in wines, 750
extracts and toilet preparations, 905-
909
lemon, orange, and lime extract,
flavors, and oils, 898-903
vanilla extract and its substitutes,
890-898
in wines, 750
Flaxseed oil
identification, 986
Flexible barrier materials
extractives from, 1178-1180
Flotation methods
filth in alimentary pastes, 387
filth in apple butter, 391
filth in breading of frozen foods, 387
filth in breads (high-bran), 385-386
AOAC Official Methods of Analysis (1990)
Subject Index
1-21
filth in candy, 393-394
filth in canned broccoli, 395
filth in canned crabmeat, 389-390
filth in canned fish and fish
products, 390
filth in canned shrimp, 390
filth in cocoa, chocolate, and press
cake, 373-374
filth in cracked wheat and flours,
381-382
filth in dehydrated potato products,
397
filth in flour, 384
filth in grains and seeds, 380
filth in gums, 406
filth in nuts, 379
filth in papain, 406
filth in potato chips, 393
filth in rice flours, products, and
paper, 384-385
filth in soy flour, 385
filth in spices and condiments, 397-
404
filth in tea, 375
filth in wheat germ, 383-384
filth in wheat gluten, 385
filth in white breads and high-fat
products, 386-387
filth in whole wheat cereals, 388
foreign matter in canned corn, 395
insects in frozen blackberries and
raspberries, 391-392
weevils in beans and peas, 394
see also Extraneous materials
Flour
excreta in, 415
filth in, 381-382, 384
filth in corn flour, 385
filth in rice flour, 384-385
insect eggs in, 384
insect excreta in, 384
starchy, in meat, 941
Fiuazifop-butyl, 192
Flucytosine, 543-544, 546
Fluometuron, 218
Fluorescein, 1122
Fluorescein sodium, 532-533, 546
Fluorescence quenching method
fluorides in food, 1148
Fluorescent antibody screening test
Salmonella in foods, 476-478
Fluoride
in drug tablets and solutions, 505
in food, 1148-1149
in hazardous substances, 232-233
in plants, 51-56
in water, 321—322
Fluoride-selective electrode method
fluoride in drug tablets and
solutions, 505
Fluorine
on apples and pears, 248-249
in feeds, 86
in food, 250-253
in hazardous substances, 232-233
microchemical determination, 341-
342
in pesticide formulations, 150-152
Fluor ogenic assay
for Escherichia coli in chilled or
frozen foods, 438-439
Fluorogenic monoclonal enzyme
immunoassay screening method
Salmonella in foods, 486-488
Fluorometric methods
amprolium in feeds, 93
buquinolate in feeds, 95
decoquinate in animal tissues, 628
decoquinate in feeds, 96-97
ethoxyquin in feeds, 90
fluroescein sodium in drugs, 532-
533
histamine in seafoods, 876-877
pyoverdine in eggs, 862-863
quinacrine HC1 in drugs, 593
reserpine in drugs, 595
riboflavin in foods and vitamin
preparations , 1 052- 1 053
riboflavin in ready-to-feed milk-
based formulas, 1 107
selenium in food, 269-270
selenium in plants, 57
sterols in macaroni products, 798
thiamine in bread, 1051-1052
thiamine in foods, 1049-1051
thiamine in grain products, 1051
thiamine in milk-based formula,
1113-1114
Foam collapse rate, 714-715
Foam flashing method
foam collapse rate of beer, 715
Folic acid
assays, 1080-1082
in vitamin preparations, 1083-1084
Foipet
in formulations, 192-193
Food additives (direct)
acetone peroxides in baking
premixes, 1165-1166
antioxidants, 1137-1141
brominated vegetable oils in
nonalcoholic beverages, 1 166—
1167
chemical preservatives , 1141 — 11 63
cyclohexylamine in cyclamates and
artificially sweetened products.
1169-1171
cyclohexylsulfamate salts in
nonalcoholic beverages, 1168
dulcin in food, 1171-1172
emulsifying agents, 1163-1164
enzymes, 1164
ethylene dichloride and
trichloroethylene in spice
oleoresins, 1175
fumaric acid in food, 1137
monosodium glutamate in food,
1174-1175
5-nitro-2-propoxyaniline in food,
1172
nonnutritive sweeteners in
nonalcoholic beverages, 1 167—
1168
saccharin in food, 1172-1174
saccharin in nonalcoholic beverages,
1174
sodium cyclamate and calcium
cyclamate in canned fruit,
1168-1169
sorbitol in food, 1167
see also specific analyte
Food additives (indirect)
acrylonitrile in food , 1181-1182
benzo[a]pyrene in food, 1176-1178
extractives from flexible barrier
materials, 1178-1180
extractives from rubber articles,
1180
iV-nitrosamines in baby bottle rubber
nipples, 1182-1183
polycyclic aromatic hydrocarbons,
1176-1178
see also Metals and other elements;
specific analyte
Food and Agriculture Organization/
World Health Organization, see
FAO/WHO
Food Chemicals Codex-U.S.
Pharmacopeia-AOAC method
tocopherol isomers in mixed
tocopherol concentrate, 1076
see also AOAC-Food Chemicals
Codex Method
Food dressings
acidity of, 1005
alginates in, 1007
filth in, 405
nitrogen (total) in, 1005
polysorbate 60 in, 1 163
sample preparation, 1004
solids (total) in, 1004
sucrose in, 1005
sugars (reducing) in, 1005
Food extracts
vitamin B 6 in, 1089-1091
Food packaging
bird excrement, 414-415
urine on, 407-409, 411-412
Foodware
cadmium and lead in, 241-242
Forages
acid -detergent fiber in, 74-76
nitrate in, 357-358
Foreign matter
in brewer's grits, 381
in corn (canned), 395
in leafy, crude drugs, 406
in spices and condiments, 398
Forensic sciences
fingerprint detection, 637
glass fragment characterization and
matching, 637-638
mineral wool insulation properties,
638-639
voice print identification, 639
Formaldehyde
in food, 1149
in formulations, 226
in maple sirup, 1037-1038
safe handling of, 653, 1227
in seed disinfectants, 226
1-22
Subject Index
AOAC Official Methods of Analysis (1990)
Formaldehyde titration method
nitrogen in fertilizers, 19
Formic acid
in food, 1149
safe handling of, 650, 1224
in seafood, 872-873
Formothion
in formulations, 204-205
technical and formulations, 205-206
Fowler modification
Canadian lead number of maple
products, 1035
French dressing
filth in, 405
gums in, 1005-1006
Fritted glass crucible method
crude fiber in feeds, 81—82
Frog legs
Salmonella in, 476-478
Frozen desserts, see Ice cream and
frozen desserts
Frozen foods
Escherichia coli in, 438-439
filth in breading of, 387
fish content of breaded fish products,
864-865
insects in blackberries and
raspberries, 391-392
microbiological methods, 429-430
net contents of seafood, 864
soil in fruits and vegetables, 393
Frozen fruits
drained weight of, 911
fill of container of, 910-911
net contents of, 910
sugar mixtures, fruit content of, 911
thawing of, 911
Fructose
in corn sirups and sugars, 1042-
1043
densities of solutions, 1272
Hammond table for calculating,
1286-1294
in honey, 1028, 1030
in milk chocolate, 773
in plants, 59
in presweetened cereals, 789-790
refractive indices of solutions, 1281
in sugars and sirups, 1018-1019
in wines, 741-742
Fruits and fruit products
acidity (titratable) of fruit products,
918
acidity (volatile) of fruit products,
918
adulteration of processed Florida
orange juice, 929-930
alcohol in fruit products, 912
alcohol precipitate in fruit products,
917
anthocyanins in fruit juices, 923
ash of, 915
betaine in orange juice, 917-918
calcium in, 916
carbohydrates in fruit juices, 922
chlorine (total) in fruit products, 917
citric acid in, 918-921
corn syrup in apple juice, 926
corn syrup in orange juice, 926-927
drained weight of frozen fruits, 911
extraneous materials in, 391-393
fill of container of frozen fruits,
910-911
filth in citrus and pineapple juices,
392-393
filth in fruit paste, 392
fluorine on apples and pears, 248-
249
fruit content of frozen fruit-sugar
mixtures, 911
glucose (commercial) in, 922
isocitric acid in, 919
lactic acid in, 921
laevo-malic acid in, 919
lead on apples and pears, 254-255
lead in juices, 256-257
lemon juice in, 925-926
magnesium in, 916
malic acid in, 918, 919, 920-921
malvidin glucosides in grape juice,
923-924
manganese in, 916
moisture in dried fruits, 912
moisture in prunes and raisins, 912-
914
mold in processed products, 418-
419
net contents of frozen fruits, 910
oil (essential) in, 923
oil (recoverable) in, 923
organic acids (foreign) in fruit
juices, 921-922
pectic acid in fruit products, 917
phosphorus in, 916-917
potassium in, 915
protein in fruit products, 917
quinic acid in cranberry juice
cocktail and apple juice, 920-
921
sample preparation, 911
sampling of, 910
seeds in berry fruits, 914
sodium in, 915-916
sodium cyclamate and calcium
cyclamate in canned fruit,
1168-1169
soil in frozen products, 393
solids (soluble) in, 915
solids (total) in, 914
solids (water-insoluble) in, 914
starch in, 922-923
sucrose in, 922
sugars (reducing) in, 922
sulfur in ash of, 917
sulfur (total) in fruit products, 917
sulfurous acid in dried fruit, 1 158-
1159
tartaric acid in, 918
thawing of frozen fruits, 911
thiourea in, 1160-1163
viscosity of fruit products, 912
see also specific fruits
Fumaric acid
in food, 1137
Fumigants
mixed formulations, 164
residues in grain, 290-291
Fuming acids
safe handling of, 651, 1225
Fungi
in eggs and egg products, 428
Fungicidal activity
of disinfectants, 138
Fungicides
copper in, 156
Furazolidone
in feeds, 90
in feeds and premixes, 99-101
Furfural
in distilled liquors, 701
Fusel oil
in cordials and liqueurs, 705
in distilled liquors, 699-700
Fusion method
dye in color additives, 1125
Galactan
in feeds, 84
Galactose
in sugars and sirups, 1019
Gamma-ray spectroscopic method
iodine-131, barium- 140, and cesium-
137 in milk and other foods,
353-355
Garlic
filth in, 399, 402
Garlic powder
Salmonella in, 470-471
Gas chromatographic methods
acetone and alcohols in drugs, 498-
499
acrylonitrile in food, 1181-1182
alachlor in formulations, 174-175
alcohol in flavors, 890
alcohol in lemon, orange, and lime
extracts, 898
alcohol in wines, 741
alcohols (higher) and ethyl acetate in
distilled liquors, 700-701
d-trans -attsthrin in pesticide
formulations, 165-166
ammonium glycyrrhizinate in flavor
extracts, 907-908
amphetamine in drugs, 520-521
animal fats in vegetable oils and
fats, 976-977
(3-asarone in wines, 750
benfluralin in formulations, 180
benzoic acid and sorbic acid in food,
1143-1144
7-BHC in technical BHC,
formulations, and lindane
shampoos and lotions, 178-179
brominated vegetable oils in
nonalcoholic beverages, 1166—
1167
bromoxynil octanoate in
formulations, 180-181
butachlor in formulations, 181
butylated hydroxyanisole and
AOAC Official Methods of Analysis (1990)
Subject Index
-23
butylated hydroxytoluene in
cereals, 1139-1140
caffeine in instant tea, 761-762
camphor in drugs, 602
captan in formulations, 181
carbohydrates in fruit juices, 922
chick edema factor in oils and fats,
981-982
chlordimeforrn in formulations, 184
chlorobutanol in drugs, 499-500
cholesterol in multicomponent foods,
1103-1105
clopidol in animal tissues, 626-628
cocaine HC1 in drug powders, 620
coumarin in wines, 750
cypermethrin in pesticide
formulations, 166-167
DCPA in formulations, 186
denaturants (volatile) in alcoholic
products, 232
deoxynivalenol in wheat, 1205-1207
dexamethasone in drug substance
and elixirs, 614, 615-616
diazinon in formulations, 200-201
dichlobenil in formulations, 188-189
diethylcarbonate in wines, 750
dihy droanethole , dihydrosafrole ,
isosafrole, methyl salicylate,
and safrole in nonalcoholic
beverages, 754-755
disulfoton in formulations, 201
docosenoic acid in oils and fats, 967
endosulfan in formulations, 191-192
endosulfan, endosulfan sulfate,
tetradifon, and tetrasul
pesticidue residues in apples and
cucumbers, 284-285
erucic acid in oils and fats, 965-967
ethanol in beer, 711
ethchlorvynol in drugs, 563
ethyl parathion in formulations, 209-
210
ethylan (perthane) pesticide residues,
298
ethylene dibromide pesticide
residues, 299-300
ethylene dichloride and
trichloroethylene in spice
oleoresins, 1 175
ethylenethiourea pesticide residues,
300-302
fatty acids (volatile) in seafood, 875
fatty acids in eggs, 860-861
trans fatty acid isomers in
margarines, 970-971
fenitrothion, technical and
formulations, 202-203
fensulfothion in formulations, 203-
204
fentin in fentin-maneb formulations,
157
fluazif op-butyl in formulations, 192
fluometuron in formulations, 218
for polymers and oxidation products
of heated vegetable oils, 967-
968
formothion in formulations, 204-205
fumigant mixtures, 164
fumigant residues in grain, 290-291
glycerol, propylene glycol, and
triethylene glycol in cigarette
filler and tobacco, 65
heptachlor in AG chlordane, 184
heptachlor in formulations, 193-194
hexachlorobenzene pesticide residues
in fatty products, 302-303
|3-hydroxybutyric, lactic, and
succinic acids in eggs, 858-859
indole in shrimp, 878-879
ipronidazole in feeds, 100
isofenphos, technical and
formulations, 205-206
malathion in formulations, 206-207
melengestrol acetate in animal
tissues, 629-631
melengestrol acetate in feed
supplements, 101-103
menadione sodium bisulfite in feed
premixes, 1079-1080
menthol in cigarette filler, 67-68
methanol in distilled liquors, 702-
703
methanol in hazardous substances,
233-234
methaqualone in drug powders, 621-
622
JV-methyl carbamate residues on
fruits and vegetables, 291-292
methyl esters of fatty acids in oils
and fats, 964-965
methyl mercury in fish and shellfish,
266-269
methyl parathion in formulations,
208-210
metolachlor in formulations, 195
metribuzin in formulations, 219
mirex pesticide residues in fatty
products, 302-303
nicotine on Cambridge filter pads,
66-67
nikethamide in drugs, 599
N-nitrosamines in baby bottle rubber
nipples, 1182-1183
A^nitrosodibutylamine in latex infant
pacifiers, 234-236
N-nitrosodimethylamine in beer,
719-722
A^nitrosodimethylamine in nonfat
dry milk, 835-836
nonvanillin vanilla volatiles in
vanilla extract, 898
JV-octyl bicycloheptene
dicarboximide, 172-173
organic acids in vanilla extract, 897-
898
organochlorine and
organophosphorus pesticide
residues, 275-277, 280, 282-
283
organochlorine pesticide residues in
fish, 283-284
paraldehyde in drugs, 563-564
parathion in formulations, 207
PCBs in fish, 283-284
PCBs in foods, 280
PCBs in paper and paperboard, 285-
286
PCNB in formulations, 195
pentachlorophenol pesticide residues,
308-309
perrnethrin in pesticide formulations,
167-168
phencyclidene in drug powders, 623
phenothiazine in drugs, 574-575
pirimicarb in formulations, 219-220
propachlor in formulations, 196
propoxur technical and formulations,
220
propylene glycol in cosmetics, 360
pyrethrin and piperonyl butoxide in
formulations, 170-172
ronnel in feeds, 108
P-sitosterol in butter oil, 975-976
sorbitol in food, 1167
sporeformers in low-acid canned
foods, 458-459
sulfamethazine residues in swine
tissues, 633-634
sulprofos in formulations, 210-211
terbuthylazine in formulations, 220-
221
tetradifon (technical) in formulations,
197
thiocarbamates in herbicide
formulations, 221
tocopherol isomers in mixed
tocopherol concentrate, 1076
tocopheryl acetate in supplemental
vitamin E concentrates, 1077-
1078
triazines in formulations, 222-223
trifluralin in formulations, 180
triglycerides in oils and fats, 972-
973
vegetable fats in butterfat, 974-975
vitamin E in drugs, 1078-1079
water and ethyl alcohol in cosmetics,
359
Gas chromatographic-mass
spectrometric method
sulfamethazine residues in swine
tissues, 631-633
Gases
Certified Reference Materials, 648,
1222
Gasometric determination
carbon dioxide in baking powders,
685
Gel permeation chromatographic
method
organochlorine pesticide residues in
animal fats, 284
Gelatin
in cottage cheese, 847
in cream, 832
dessert powders, 929
in evaporated milk, 833
in ice cream and frozen desserts,
852
jelly strength of, 929
in milk, 820
1-24
Subject Index
AOAC Official Methods of Analysis (1990)
pentachlorophenol pesticide residues
in, 308-309
phosphorus in, 929
in roasted coffee, 759
sucrose in, 930
Geotrichum mold counting, 420-422
Germicidal activity
of disinfectants, 137-141
Ginger
extract, 905
filth in, 398-399, 401
foreign matter in, 398
volatile oil and resin in, 1001
Glass
fragment characterization and
matching, 637-638
in meat scraps, 389
Glucoamylase method
starch in cereals, 789
Glucose
in brewing sugars and sirups, 734
in cacao products, 774-775
in corn sirups and sugars, 1042-
1043
in eggs, 856-857
Hammond table for calculating,
1286-1294
in honey, 1029-1030
in milk chocolate, 773
in plants, 59
in presweetened cereals, 789-790
refractive indices of solutions, 1281
in sugars and sirups, 1017-1018
Glucose (commercial)
in confectionery, 1024
in fruits and fruit products, 922
in honey, 1031
in maple products, 1035
in nonalcoholic beverages, 756
in roasted coffee, 759
in sugars and sirups, 1016
in wines, 741
Glucose oxidase methods
glucose in corn sirups and sugars,
1042
Glucuronidase
in chilled or frozen foods, 438-439
Glycarbylamide
in feeds, 101
Glycerides
in monoglyceride concentrates, 982-
983
saturated hydrocarbons in, 980-981
in shortening, 985-986
Glycerol
in beer, 711
in cigarette filler and ground
tobacco, 65
in cordials and liqueurs, 706
in eggs, 857
in lemon, lime, and orange extracts,
899
in shredded coconut, 950
in vanilla extract, 890
in vanishing cream, 368
in vinegars, 1008
in wines, 741
Glycyrrhizic acid or acid salts
in licorice products, 908-909
Glyodin
in apples and pears, 302
Glyoxyiic acid test
protein in feeds, 70
Giyphosate, 205
Grains
ash of, 788
brewers', 737
crude fat or ether extract, 788
extraneous materials in, 380-385
fat acidity, 788
fiber (crude) in, 788
filth in grain products, 383
fumigant residues in grain, 290-291
light filth in, 380
moisture in, 788
protein in, 788
sample preparation, 788
thiamine in grain products, 1051
urine on, 410-411
see also specific grains
Grapes
azinphos-methyl pesticide residues
on, 294
malvidin glucosides in juice, 923-
924
Af-methylcarbamate residues in, 292-
294
Gravimetric cobaltinitrite method
potassium in fruits and fruit
products, 915
Gravimetric methods
alcohol extract of spices, 1000
aloin in drugs, 602
aluminum in deodorants, 361, 362
aluminum in water, 322, 324
amphetamine drugs, 520
ash of cheese, 842
ash of milk, 807
ash of spices, 1000
aspirin and phenolphthalein in
tablets, 557
barbiturates in drugs, 559
barium in water, 327
benzaldehyde in almond extract, 904
bismuth compounds in drugs, 502
calcium in water, 324
camphor in spirits, 602
carbromal and pentobarbital in
drugs, 559
chloride in plants, 50
chlorides in deodorants, 363
chlorobutanol in drugs, 499
citric acid in cheese, 845
citric acid in milk, 805-806
dyes in color additives, 1124
ether extract of prepared mustard,
1003
ether extract of, 59
extractives from rubber articles,
1180
fat (crude) in nuts and nut products,
949
fat (crude) in pet food, 79-80
fatty acids (water-insoluble) in
butter, 838-839
iodoform in drugs, 500-501
iron in water, 322
jalap in drugs, 604
lactose in cream, 832
lactose in milk, 810
lead in pesticide formulations, 149
liming materials, 4-5
magnesium in fertilizers, 32
magnesium in plants, 46
magnesium in water, 324
mercury in drug dosage forms, 509-
510
mercury in organic mercurial seed
disinfectants, 163
methyl anthranilate in nonalcoholic
beverages, 755
methylene chloride extract of spices,
1000
moisture in cacao products, 763
moisture in fig bars and raisin filled
crackers, 795
moisture in malt, 725
moisture in tobacco, 64
oxygen determination, 343-344
phenolphthalein in drug preparations,
552
phosphorus in baking powders, 689
phosphorus in fertilizers, 13, 15, 16,
17
phosphorus in plants, 56
piperazine in drugs, 533
podophyllum in drugs, 602
polysorbate 60 in shortening, oils,
and dressings, 1163
potassium and sodium in plants, 47
sabadilla alkaloids in formulations,
173
saccharin in food, 1173
sand and silica in plants, 40
selenium in plants, 57
silica in water, 322
soil in frozen fruits and vegetables,
393
solids (alcohol-insoluble) in canned
peas, 990
solids (alcohol-insoluble) in frozen
peas, 996
solids in prepared mustard, 1003
solids in vinegar, 1008-1009
solids (insoluble) in canned
vegetables, 990
solids (total) in canned vegetables,
989
solids (total) in frozen spinach, 995
solids (total) in ice cream and frozen
desserts, 851
solids (total) in seafood, 868
sulfate in water, 331
sulfides in deodorants, 363
sulfur determination, 345-346, 347
sulfur in fertilizers, 34
sulfur in fruit products, 917
sulfur in lime sulfur, 159-160
AOAC Official Methods of Analysis (1990)
Subject Index
I-25
theophylline in drugs, 584
tin in food, 270
volatile matter in color additives,
1125
zinc in deodorants, 361-362
zinc in fertilizers, 34
zinc in pesticide formulations, 150
Green beans
fibrous material in frozen products,
996
7V-methylcarbamate residues on,
291-292
organophosphorous pesticide residues
on, 286-287
Green vegetables
captan pesticide residues in, 296
Griseofulvin
in feeds, 130-131
Ground beef
preservatives in, 1144
Groundwater
TNT, RDX, HMX, and 2,4-DNT in,
334-336
Growth hioassay
thiamine HC1 in vitamin
preparations, 1091
Guaiacol, 550, 577
Guaifenesin, 550, 577
Guinea green B, 1115
Gums
in drugs, 604
filth in, 406
in ice cream and frozen desserts,
851-852
in mayonnaise and French dressing,
1005-1006
in salad dressing, 1006-1007
in soft curd cheese, 846-847
Gustafson method
ash of flour for phosphated and self-
rising flour, 778
Gutzeit method
arsenic in food, 244
Hair preparations, 366-367
Hallucinogens
optical crystallographic properties,
663
refractive indices, 670
Halogens
in pure colors, 1135-1136
Halphen test
cottonseed oil in oils and fats, 977-
978
cyclopropene in oils, 978
Hanus method
iodine absorption number of oils and
fats, 955-956
Hardness
of water, 323
Hazardous substances
cadmium and lead in earthenware,
232
carbonate and hydroxide in soda lye,
232
denaturants (volatile) in alcoholic
products, 232
fluorides in, 232-233
lead in paint, 233
methanol in, 233-234
N-nitrosodibutylamine in latex infant
pacifiers, 234-236
phenol in, 236
see also specific analyte or matrix
Headspace gas chromatographic
method
ethanol in canned salmon, 880-881
Hehner number
in oils and fats, 957
HeLa cell culture
E. coli invasiveness of mammalian
cells, 439-441
Helindone pink CN, 1121
Helium leak test
low-acid canned food containers,
456-458
Heptabarbital, 537, 546
Heptachlor
in AG chlordane, 184
in formulations, 193-194
multiresidue methods, 279
Heptachlor epoxide
multiresidue methods, 279
Herbicides
volatility, 153
Heroin, 524, 534, 546, 620
Hexachlorobenzene
in fatty products, 302-303
Hexachlorocyclopentadiene
in technical chlordane, 183
Hexachlorophene
in deodorants, 363-364
Hexane
safe handling of, 653, 1227
Hexane distillation
fatty acids (crude) in oils and fats,
957
Hexestrol, 610, 619
Hexofoarbital sodium, 537, 546
Hexylresorcinol, 551, 577
High fructose corn sirup
in honey, 1031-1032
Hiltner method
aldehydes in lemon and orange oils,
902
Histamine
in seafood, 875-877
HMX
in wastewater and groundwater,
334-336
Holaday-Velasco minicolumn method
aflatoxins in corn and peanuts, 1188
Homatropine, 534, 546
Homatropine HC1, 580, 606
Homatropine hydrobromide^ 580, 606
Honey
acidity (free, lactone, and total) of,
1033
ash of, 1026
color classification of, 1026
corn sirup products in, 1032-1033
dextrin in, 1029
diastatic activity of, 1033
filth in, 394
fructose in, 1028, 1030
glucose in, 1029-1031
high fructose corn sirup in, 1031 —
1032
hydroxy methylfurfural in, 1031
moisture in, 1026
nitrogen in, 1026
polarization of, 1027
proline in, 1026-1027
reducing disaccharides as maltose,
1029
refractive index and water content,
1027
sample preparation, 1025-1026
separation of sugars in, 1028-1030
sucrose in, 1028-1030
sugars (reducing) in, 1027-1028
Hops
acids in, 732-733
aphids in, 374-375
moisture in, 732
preparation of sample for chemical
analysis, 732
sampling of, 731-732
Horseradish
filth in, 405
Hortvet method
water (added) in cream, 832
water (added) in milk, 819
Hot leach atomic absorption method
cadmium and lead in cookware, 241
Howard moid counting, 416-419
Hydralazine HC1, 545, 546
Hydrastine, 534, 546
Hydrastinine, 534, 546
Hydrazine method
nitrites in tablets, 512
Hydrazine sulfate distillation method
arsenic in pesticide formulations,
147-148
Hydrazino-7-undecalactone
properties
in cordials and liqueurs, 707
Hydrocarbons (saturated)
in glycerides, 980-981
Hydrochloric acid
solution strengths, 658
standard solution, 642-643, 1216-
1217
Hydrochloric acid inversion
sucrose in plants, 59
Hydrochlorothiazide, 572-573
chemical name, 546, 577
in drug tablets, 570-571
microchemical tests, 538
Hydrocodone bitartrate, 580, 581,
606
Hydrocodone HC1, 580, 606
Hydrocortisone, 613-614, 619
Hydrocyanic acid
in almond extract, 904
in beans, 1213
in feeds, 90
1-26
Subject Index
AOAC Official Methods of Analysis (1990)
Hydroflumethazide, 572-573, 577
Hydrofluoric acid
safe handling of, 651, 1225
Hydrofluoric acid test
fluorides in food, 1148
Hydrogen
and carbon, microchemical
determination, 339-341
carbon, and nitrogen, microchemical
determination, 341
Hydrogen peroxide
in milk, 1149
Hydrogen peroxide method
formaldehyde in formulations, 226
Hydrogen sulfide
safe handling of, 653, 1227
in water, 322
Hydrolysis method
sulfanilamide in drugs, 569
Hydrometer method
alcohol by weight in distilled
liquors, 692, 694
Hydromorphone, 534, 546
Hydrophobic grid membrane filter
method
coliforms and Escherichia coli in
foods, 437-438
microorganisms in foods, 431-433
Salmonella in foods, 478-480
p -Hydroxy butyric acid
in eggs, 858-859
Hydroxyl value
of oils and fats, 955
Hydroxylamine method
aldehydes in lemon oil, 902-903
Hydroxymethylfurfural
in honey, 1031
8-Hydroxyquinoline sulfate, 538, 546
Hygromycin
in feeds, 121
Hyoscine, 535
Hyoscyamine, 534, 546
Hypnotics and sedatives
acetylcarbromal in drugs, 558
aminophylline in drugs, 560
amobarbital sodium in drugs, 559
barbiturate drugs, 559
barbiturates in drugs, 559
bromisovalum in drugs, 558
butabarbital sodium in drugs, 561-
562
carbromal in drugs, 558-559
chloral hydrate in drugs, 562
ethchlorvynol in drugs, 563
(2-isopropyl-4-pentenoyl) urea in
drugs, 563
meprobamate in drugs, 564-565
paraldehyde in drugs, 563-564
pentobarbital in drugs, 559
phenaglycodol in drugs, 564
phenobarbital in drugs, 558, 560-561
phenytoin in drugs, 560-561
phenytoin sodium in drugs, 562
secobarbital sodium in drugs, 559
suJfonmethane or sulfonethylmethane
in drugs, 563
theobromine in drugs, 561
Hypochlorites
in milk, 820-821
Hypophosphites
in sirups, 505-506
Hypophosphorus acid
safe handling of, 653, 1227
Ice cream and frozen desserts
alginates in chocolate frozen
desserts, 852
color additives in, 852
fat in, 851
gelatin in, 852
gums in, 851-852
lactic acid in, 851
phosphatase in, 852
protein in, 851
sample preparation, 851
separation of fat from ice cream,
851
solids (total) in, 851
weight per unit volume of packaged
ice cream, 850-851
IDF-ISO-AOAC methods
acid value of butterfat, 837
aflatoxin Mj in cheese, 843
aflatoxin M : in milk, 821
chloride in cheese, 842
citric acid in cheese, 845-846
copper in milk and milk products,
821
fat in butter, 837
fat in cheese, 844
fat in cream, 832
fat in dried milk, 835
fat in evaporated milk, 833
fat in ice cream and frozen desserts,
851
fat in milk, 811
fat in sweetened condensed milk,
834
fat in whey cheese, 844
moisture in butter, 837
mold in butter, 840
nitrogen (total) in milk, 807-808
organochlorine and
organophosphorus pesticide
residues, 821
protein in milk, 808-809
refractive index of butterfat, 837
salt in butter, 837
solids in cream, 831
solids in evaporated milk, 833
solids (total) in milk, 807
sucrose in sweetened condensed
milk, 834
vegetable fats in butterfat, 973-975
water (added) in milk, 819-820
Immersion refractometer method
methanol in distilled liquors, 702
Immiscible solvent method
color additives in foods, 1115
Immunodiffusion screening method
motile Salmonella in foods, 490-492
Incubation methods
biochemical oxygen demand of
water, 314-315
Index of refraction
of cacao fat, 771
of oils and fats, 952-953
Indicating strip method
salt (chlorine as sodium chloride) in
meat, fish, and cheese, 933
salt (chlorine as sodium chloride) in
seafood, 870
Indicator titrimetric methods
acidity of beer, 711-712
liming materials, 1-2
Indicators
for colorimetric pH comparisons,
641-642, 1215-1216
Indigo, 1119
Indigotine, 1115, 1118
Indole
in crabmeat, oysters, and shrimp,
877-878
in shrimp, 878-879
Inductively coupled plasma
spectroscopic methods
calcium, copper, iron, magnesium,
manganese, phosphorus,
potassium, sodium, and zinc in
infant formulas, 1106-1107
metals and other elements in plants,
42
Industrial chemicals
PCBs in foods, 280
PCBs in paper and paperboard, 285-
286
Infant foods
filth in dry cereal, 388
filth in purees, 396
mold in purees, 419
Infant formulas
calcium in, 1106-1107, 1110
cobalamin in ready-to-feed milk-
based formulas , 1110-1112
copper in, 1106-1107, 1310
iron in, 1106-1107, 1110
magnesium in, 1106-1107, 1110
manganese in, 1106-1107, 1110
minerals in ready-to-feed milk-based
formulas, 1110
niacin and niacinamide in ready-to-
feed milk-based formulas, 1109
nutrients in ready-to-feed milk-based
formulas, 1107
phosphorus in, 1106-1107, 1112-
1113
potassium in, 1106-1107, 1110
riboflavin in ready-to-feed milk-
based formulas, 1107
sodium in, 1106-1107, 1110
vitamin B 6 in ready- to-feed milk-
based formulas, 1107-1108
vitamin C in ready-to-feed milk-
based formulas, 1108-1109
vitamins and other nutrients in,
1106-1114
zinc in, 1106-1107, 1110
AOAC Official Methods of Analysis (1990)
Subject Index
I-27
Infant products
latex pacifiers, N-nitrosodibutylamine
in, 234-236
yV-nitrosamines in baby bottle rubber
nipples, 1182-1183
Infrared analyzer method
organic carbon in water, 317-318
Infrared spectroscopic methods
acetyl carbromal and bromisovalum
in drugs, 558
AG chlordane in formulations, 184
aldicarb in formulations, 212
aldrin in formulations, 175-176
alpha and gamma isomers in AG
chlordane technical, 183
atropine in drug tablets, 583
azinphos-methyl in formulations,
197-198
benzene hexachloride in
formulations, 179
chlorinated hydrocarbons in drugs,
499
chlorobutanol in drugs, 499-500
cyclohexylamine in cyclamates and
artificially sweetened products,
1169-1171
2,4-D in formulations, 187-188
DCPA in formulations, 186
DDT in formulations, 186-187
DDVP in formulations, 199-200
dicamba in formulations, 187-188
dieldrin in formulations, 175-176
endrin in formulations, 175-176
ethisterone in drugs, 613
gums in ice cream and frozen
desserts, 851-852
hydrocortisone in drugs, 613-614
MCPA in formulations, 187-188
meprobamate in drugs, 564-565
methazole in formulations, 194-195
methimazole in drugs, 573-574
mineral oil in baked products, 794-
795
nitrate esters in drugs, 527
nitroglycerin in drugs, 527
pentaerythrityl tetranitrate and
meprobamate in drugs, 528-529
phenaglycodol in drugs, 564
rotenone in derris and cube powder,
169
santonin in drug mixtures, 604
trans isomers in margarines and
shortenings, 969-970
Infusion method
chicory in roasted coffee, 759
Inorganic material (added)
in phosphated flour, 778
Inorganic residues
in nuts and nut products, 950
Inorganic salts
in color additives, 1136
Insects
in apple chops, 391
eggs in flour, 384
excrement on foods and containers,
414-415
excreta in flour, 384
fly eggs and maggots in fruit juices.
392-393
fly eggs and maggots in pureed
infant foods, 396
fly eggs and maggots in tomato
products, 397
in frozen blackberries and
raspberries, 391-392
maggots in blueberries and cherries,
392
in mushrooms, 396-397
penetration thru packaging materials,
406-407
in vegetables and vegetable products,
394
in wheat, 380
Insoluble acids
in oils and fats, 957
Insoluble matter
in color additives, 1125
Intermediates
in color additives, 1126-1132
International Dairy Federation, see
IDF
International Organization for
Standardization, see ISO
International Union of Pure and
Applied Chemistry, see IUPAC
Intersociety Committee, see ASTM-
Intersociety Committee- AOAC
method
Inversion methods
sucrose in plants, 59
Invert sugar
Hammond table for calculating,
1286-1294
in maple products, 1035
in molasses, 1023
refractive indices of solutions, 1282
lodates
in white and whole wheat flour,
784-785
Iodine
in drugs, 506
in iodized salt, 334
microchemical methods, 337-339
in mineral mixed feeds, 87
in ointments, 506
standard solution, 643, 1217
in thyroid drug tablets, 618-619
in water, 331-332
Iodine-131
in milk and other foods, 353-355
Iodine absorption number
of cacao fat, 771
of oils and fats, 955-956
Iodine reaction method
starch in beer, 713
starch in brewing sugars and sirups,
734
Iodoform
drug substance, 500, 546
on gauze, 501
in ointments, 500-501
lodometric methods
arsenic in pesticide formulations,
148-149
hydrogen sulfide in water, 322
Ion exchange chromatographic
methods
antihistamines in drug combinations,
512
sulfur amino acids in food and feed
ingredients, 1102-1103
tryptophan in foods and food and
feed ingredients, 1101-1102
Ion exchange methods
arsenic in pesticide formulations, 149
boric acid in antiperspirants and
deodorants, 362-363
malic acid in maple sirup, 1036-
1037
methyldopa and chlorothiazide
combination in drugs, 571-572
strontium-89 and -90 in milk, 351-
353
zinc in fertilizers, 35-36
Ion selective electrode method
fluorine in feeds, 86
sodium in foods for special dietary
use, 1099
Ion-pair column chromatographic
method
phenylephrine HCL in drugs, 518-
519
trimethobenzamide HC1 in drugs,
542-543
p-Ionone
in flavor extracts and toilet
preparations, 906
in raspberry concentrates, 907
p-Ionone-m-nitrobenzhydrazide, 907
Ipecac alkaloids, 584-586
Ipomea, 604
Ipronidazoie
in feeds, 101
Iron
in baking powders, 688-689
in beer, 717
bioavailability of, 1098-1099
in bread, 793
with calcium and phosphorus in
vitamin preparations, 504
in degerminated, bolted whole corn
meal, 788
in distilled liquors, 696
in drugs, 506-508, 592
in face powders, 365
in feeds, 84
in fertilizers, 27-28, 31
in flour, 778-779
in infant formulas, 1106-1107, 1110
in liming materials, 4, 6
in macaroni products, 796
in plants, 40, 42, 43, 46
in water, 322-323, 324-325
in wines, 743
Iron-arsenic tablets
arsenic in, 501
Iron chelate concentrates
iron in, 31-32
1-28
Subject Index
AOAC Official Methods of Analysis (1990)
Iron dextran
in drugs, 506-507
Iron methylarsenate
arsenic in, 501
Iron oxides
in face powders, 365
Iron sorbitex
in drugs, 506-507
Iso-alpha acids method
bitterness of beer, 719
ISO-AOAC methods
moisture in roasted coffee, 760
weight (apparent) per unit volume
and specific gravity of fats and
oils, 951-952
see also IDF-1SO-AOAC methods
ISO/TC34/SC11/N99-AOAC method
moisture in oils and fats, 951
see also IDF-ISO-AOAC methods
Isobutyl alcohol
in distilled liquors, 700-701
Isocitric acid
in fruits and fruit products, 919
Isoniazid, 549, 577
Isooctane
safe handling of, 653, 1227
Isopilocarpine, 598
Isopropanol
in cassia, cinnamon, and clove
extracts, 905
in distilled liquors, 701-702
in lemon and orange flavors, 899
in lemon extracts, 899
in peppermint, spearmint, and
wintergreen extracts, 905
Isoproterenol, 540, 546
IsosafroBe
in nonalcoholic beverages, 754-755
IUPAC-AOAC methods
antioxidants in oils and fats, 1 138—
1139
copper in food, 248
triglycerides in oils and fats, 972-
973
see also AOAC-IUPAC methods
Jackson-Mathews modification of
Nyns selective method
fructose in sugars and sirups, 1018
Jalap, 604
Jams and jellies
benzoic acid in, 1142
filth in, 392
Jones reduction method
nitrate and nitrite in cheese, 843-
844
Kale
filth in, 395-396
A^-methylcarbamate residues on,
291-292
organophosphorous pesticide residues
on, 287-289
Karl Fischer method
moisture in cacao products, 763
moisture in oils and fats, 951
water in dried vegetables, 995
water in fertilizers, 12
Ketosteroids, 607
Kjeldahl methods
arsenic in food, 243-244
nitrogen in eggs, 854
nitrogen in fertilizers, 17-18
nitrogen in food dressings, 1005
nitrogen in laboratory wort, 727
nitrogen in meat, 935
nitrogen in milk, 807-808
nitrogen in nonvolatile ether extract
of pepper, 1000
nitrogen in plants, 59-60
nitrogen in prepared mustard, 1003
nitrogen in spices, 1000
nitrogen in sugars and sirups, 1012
nitrogen in tobacco, 64
nitrogen (total) in water, 318-319
organic thiocyanate sprays, 228
phosphorus determination, 344-345
protein in beer, 713
protein in brewing sugars and sirups,
733
protein (crude) in nuts and nut
products, 949
protein in dried milk, 834
protein in feeds, 70, 72, 74
protein in fruit products, 917
protein in grains, 788
protein in laboratory malt, 727
see also micro-Kjeldahl method
Kleher method
aldehydes in lemon oil, 902
aldehydes in orange oil, 902
Knapheide-Lamb method
iodine in mineral mixed feeds, 87
Knorr alkahmeter method
carbon dioxide in liming materials, 3
Koettstorfer number
of oils and fats, 957
Laboratory safety, 649-655, 1223-
1229
p -Lactam antibiotics
in milk, 825-829
Lactic acid
in butterfat, 838
in canned vegetables, 994
in cream, 831
in dried milk, 835
in eggs, 858-860
in evaporated milk, 833
in fruits and fruit products, 921
in ice cream and frozen desserts,
851
in milk and milk products, 806-807
in sweetened condensed milk, 834
in wines, 746
Lactose
Hammond table for calculating,
1286-1294
in bread, 794
in cream, 832
in evaporated milk, 833
in meat, 945
in milk chocolate, 772, 773
in milk, 810-811, 816-818
in process cheese, 846
in sugars and sirups, 1019
in sweetened condensed milk, 834
purity in sugars and sirups, 1019—
1020
Laevo-malic acid
in cordials and liqueurs, 706
in fruits and fruit products, 9.19
Lake red C, 1121
Lake red CBA, 1121
Lakes (colors), 1115, 1119, 1122
Lane-Eynon volumetric method
fructose in sugars and sirups, 1016-
1017
glucose in sugars and sirups, 1016-
1017
invert sugar in sugars and sirups,
1016-1017
lactose in sugars and sirups, 1016—
1017
maltose in sugars and sirups, 1016—
1017
sugars in brewing sugars and sirups,
734
total sugars in molasses as invert
sugar, 1022-1023
Langer method
santonin in drug mixtures, 603
Lanthanum
in fertilizers, 27-28
LaParola-Mariani test
dulcin in food, 1171-1172
Lard
foreign fats containing tristearin in,
979-980
Lasalocid
in feeds, 121-122, 131
Laundry additives
bacteriostatic activity, 143-145
Lead
on apples and pears, 254-255
in Bordeaux mixtures, 149, 159
in color additives, 1132-1134
in earthenware, 232
in evaporated milk, 255-257, 833
in fish, 257-258
in food, 237-241, 258-262
in foodware, 241-242
in fruit juices, 256-257
in lead arsenate formulations, 154
in paint, 233
in pesticide formulations, 149
suitability of methods and
precautions, 253-254
in water, 324-325
Lead arsenate
Bordeaux mixtures with, 149-150,
158-159
formulations, 149, 154
Lead chlorofluoride method
fluorine in pesticide formulations,
150-151
Lead number (Wichmann)
of vanilla extract, 893-894
Lead salt-ether method
saturated and unsaturated fatty acids
in oils and fats, 960
AOAC Official Methods of Analysis (1990)
Subject Index
I-29
Lecithin
in cacao fat, 772
Lemon extract
alcohol in, 898
aldehydes in, 900-901
ash of, 901
citral in, 901
essential oil in, 905-906
glycerol in, 899
isopropanol in, 899
methanol in, 898
solids (total) in, 901
specific gravity, 898
sucrose in, 901
Lemon flavors
isopropanol in, 899
Lemon juice, 925-926
Lemon oil
aldehydes in, 902-903
esters in, 903
in extracts, 899
in oil-base flavors, 900
physical constants of 10% distillate,
902
pinene in, 903
refractive index of, 901
specific gravity of, 901
spectrophotometric absorbance
characteristics, 901-902
steam distillation residue, 902
Lettuce
organochlorine and
organophosphorus pesticide
residues in, 282-283
organophosphorus pesticide residues
on, 287-289
Levant worm seed, 604
Levodopa, 544-545
Licorice
glycyrrhizic acid or acid salts in,
908-909
sugars in extracts, 909
Light green SF yellowish, 1115
Lignin
in feeds, 82-83
in plants, 60-62
Lima beans
calcium in canned products, 991-
992
Lime extract
alcohol in, 898
aldehydes in, 900-901
ash of, 901
glycerol in, 899
methanol in, 898
solids (total) in, 901
specific gravity, 898
sucrose in, 901
Lime oil
in oil-base flavors, 900
Lime sulfur formulations, 159-160
Limestone
calcium in, 1-2
magnesium in, 1-2
sampling of, 1
Liming materials
aluminum in, 4, 6
calcium in, 4-5
carbon dioxide in, 3
caustic value, 2
elemental analysis of, 4-8
iron in, 4, 6
magnesium in, 5
manganese in, 7
mechanical analysis of, 1
neutralizing value of, 1-2
phosphorus in, 4, 7
sample preparation, 1, 4, 6
sampling of, 1
silica in, 4
silicon in, 6, 7-8
titanium in, 7
titanium oxides in, 4, 7
Lincomycin
in feeds, 122-123
Lindane
multiresidue methods, 279
residues, 294
Lindo-Gladding method
potassium in fertilizers, 23
Linoieic acid
in oils and fats, 960-963
Linolenic acid
in oils and fats, 960-963
Lipid phosphorus
in eggs, 854-855
in flour, 782-783
in macaroni products, 797
Lipids
in eggs, 854-855
in flour, 782
in macaroni products, 797
Liqueurs, see Cordials and liqueurs
Liquid chromatographic methods
acetaminophen in drug tablets, 554
aflatoxins in cottonseed products,
1192-1193
aflatoxins M] and M 2 in fluid milk,
1203-1205
allopurinol in drug tablets, 575-576
aminocarb technical and
formulations, 212-213
ami tripty line in drug dosage forms,
525-526
amphetamine enantiomers in bulk
drugs and dosage forms, 622-
623
anilazine in formulations, 213-214
antioxidants in oils and fats, 1138-
1139
arprinocid in feeds, 93
azinphos-methyl in formulations,
198-199
bacitracin in premix feeds, 129-130
bendiocarb technical and
formulations, 214
benomyl in formulations, 214-215
benzoate, caffeine, and saccharin in
soda beverages, 752
brodifacoum technical and
formulations, 224
captan in formulations, 181-182
carbofuran in formulations, 215-216
chlorpropamide in drug tablets, 567
chlorpyrifos in formulations, 199
colchicine in drugs, 588
cortisone acetate in bulk and dosage
forms, 608-609
cyhexatin technical and formulations,
225
2,4-D in formulations, 184-185,
188, 196
dalapon (magnesium and/or sodium
salt) in formulations, 185-186
dexamethasone acetate in bulk drug
and suspensions, 616
dexamethasone in drug substance
and elixirs, 614-615
diazepam in drug tablets, 620-621
dicamba in formulations, 188
dicofol in formulations, 190
dicumarol, phenprocoumon, and
warfarin sodium, 566
diethylpropion HO in drug substance
and tablets, 576-577
ditlubenzuron in formulations, 190—
191
ethion in formulations, 201-202
fensulfothion in formulations, 204
flucytosine in drug capsules, 543-
544
folpet in formulations, 192-193
furazolidone in feeds and premixes,
99-100
glucose, fructose, sucrose, and
maltose in presweetened cereals,
789-790
glycyrrhizic acid or acid salts in
licorice products, 908-909
glyphosate, technical and
formulations, 205
hydrocortisone in drugs, 613-614
intermediates and reaction
byproducts in FD&C Yellow
No. 5, 1128-1129
intermediates in FD&C Red No. 40,
1127-1128
intermediates in FD&C Yellow No.
6, 1129-1132
MCPA ester and salt in
formulations, 194
MCPP in formulations, 188
methiocarb technical and
formulations, 218-219
methocarbamol in drugs, 526
methyl parathion in formulations, 209
Af-methylcarbamate residues in
grapes and potatoes, 292-294
methyldopa, methyldopa-
hydrochlorothiazide, or
methyldopa-chlorothiazide in
drug tablets, 570-571
morphine sulfate in bulk drug and
injections, 582-583
oxazepam in drug tablets, 622
oxythioquinox in formulations, 226-
227
parathion in formulations, 208
picloram in formulations, 196
pilocarpine, isopilocarpine, and
pilocarpic acid in drugs, 598
1-30
Subject Index
AOAC Official Methods of Analysis (1990)
prednisolone in bulk drugs and
tablets, 617-618
primidone in drug tablets, 576
pseudoephedrine HC1 and triprolidine
HC1 or chlorpheniramine
maleate in drug combinations,
587-588
purity of lactose, 1019-1020
quinic, malic, and citric acids in
cranberry juice cocktail and
apple juice, 920-921
rotenone in pesticide formulations,
169-170
saccharides (major) in corn sirups
and sugars, 1042-1043
saccharides (minor) in corn sirups
and sugars, 1044
separation of sugars in honey, 1030
sugars in licorice extracts, 909
sulfamethoxazole in drug tablets, 569
sulfisoxazole in drug dosage forms,
569-570
2,4,5-T in formulations, 196-197
temefos in formulations, 211
TNT, RDX, HMX, and 2,4-DNT in
wastewater and groundwater,
334-336
triadimefon technical and
formulations, 228-229
triamino-.v-triazine in fertilizer mixes,
22
urea and methyleneureas in
fertilizers, 21-22
vitamin D in fortified milk and
milkpowder, 1068-1069
vitamin D in mixed feeds, premixes,
and pet foods, 1068-1069
vitamin D in multivitamin
preparations, 1066-1067
vitamin D in vitamin AD
concentrates, 1067-1068
vitamin D in vitamin preparations,
1064-1065
zearalenone and a-zearalenol in
corn, 1212-1213
Liquid chromatographic-fluorometric
method
indole in shrimp, 879
Lithium
safe handling of, 651, 1225
Lithium bromide
in elixir, 504-505
Lithol rubin B, 1120
Lithol rubin BCA, 1121
Liver
arlatoxin Bj in, 1201-1203
aflatoxin M, in, 1201-1203
copper in, 356-357
Loganberries
mold in, 418
Lysergic acid diethylamide, 620, 621
Lysine
in nutritional supplements, 1 100—
1101
Macaroni products
ash of, 796
carotenoids in, 799-800
coloring matter in, 798-799
fat in, 796
fiber (crude) in, 796
iron in, 796
lipid and lipid phosphorus in, 797
moisture in, 796
pH of, 797
phytate in foods, 800-801
protein in, 797
sample preparation, 796
solids (total) in, 796
sterols in, 797-798
tartrazine in, 799
unsaponifiable residue of, 797
water-soluble protein-nitrogen, 797
Mace
filth in, 398-400
foreign matter in, 398
MacMichael viscosimeter method
viscosity of acidulated flour-water
suspension, 787-788
Macroscopic method
foreign matter in canned corn, 395
Maggots
in blueberries and cherries, 392
see also Insects
Magnesium
with calcium in drugs, 502-503
in face powders, 365
in fertilizers, 27-28, 32-33
in fruits and fruit products, 916
in infant formulas, 1106-1107, 1110
in liming materials, 5, 6
in magnesic limestone, 1-2
in plants, 40, 42, 46
safe handling of, 653, 1227
in salt, 334
in water, 324-325
Magnesium acetate method
ash of flour, 778
Magnesium arsenate
arsenic in, 149
Magnesium nitrate method
sulfur in plants, 58
Magnesium oxide
in liming materials, 5
Magnesium oxide method
nitrogen in fertilizers, 19
Magnesium perchlorate
safe handling of, 653, 1227
Magnesium uranyl acetate test
urine on grain, 410
MaBathion
on apples, carrots, endive, kale,
lettuce, potatoes, and
strawberries, 287-289
confirmatory method, 289-290
in formulations, 206-207
residues, 303
Maleic hydrazide
residues, 303-304
Malic acid
in cordials and liqueurs, 706
in fruits and fruit products, 918,
919, 920-921
in maple sirup, 1036-1037
in nonalcoholic beverages, 751
in wines, 746
Malt
a-amylase in, 729
black, color of, 727
bushel weight, 724
caramel, extract and color of, 727
diastatic power, 727-729
diastatic power of sirups, 733-734
extract of, 725-727
kernels, physical characteristics,
724-725
moisture in, 725
preparation of sample, 724
protein in, 727
sampling of, 723-724
Malt beverages and brewing
materials
barley, 723
beer, 708-723
brewers' grains, 737-738
cereal adjuncts, 730-731
hops, 731-733
malt, 723-729
sugars and sirups for brewing,
733-735
yeast, 735-737
Malted milk
casein in, 835
fat in, 835
Maltose
in honey, 1029
in milk chocolate, 773
Munson and Walker values,
1286-1294
in presweetened cereals, 789-790
in sugars and sirups, 1019
Maltulose
in corn sirups and sugars, 1042-1043
Malvidin glucosides
in grape juice, 923-924
Mammalian feces
alkaline phosphatase test, 412-413
in brewer's grits, 381
in corn flour, 385
in corn meal, 382-383
in grain products, 383
in heat-processed materials, 413-414
Mandelic acid, 538, 546, 548-549,
577
Manganese
in feeds, 84, 87
in fertilizers, 27-28, 33
in food, 262
in fruits and fruit products, 916
in infant formulas, 1106-1107, 1110
in liming materials, 7
in plants, 42, 46-47
in water, 331
Mannitol hexanitrate, 527-528
Manometric methods
carbon dioxide in beer, 713-714
carbon dioxide in wines, 747-748
Maple sap, sirup, and products
ash of products, 1034
bacterial population of sap,
1038-1039
AOAC Official Methods of Analysis (1990)
Subject Index
1-31
Canadian lead number of products,
1035
color classification of products, 1034
conductivity value of sirup, 1036
corn sirup and cane sugar in syrup,
1035
formaldehyde in sirup, 1037-1038
glucose in products, 1035
malic acid in sirup, 1036-1037
moisture in products, 1034
polarization of products, 1034
sample preparation, 1034
solids in products, 1034
sucrose in products, 1034
sugars (reducing) as invert sugar in
products, 1035
Winton lead number of products,
1035-1036
yeast count for sirup, 1038
Margarines
critical temperature of dissolution of
oil from, 838
trans fatty acid isomers in, 970-971
trans isomers in, 969-970
vitamin A in, 1045
Marihuana, 621
Marine toxins
paralytic shellfish poison, 881-882
Marjoram
filth in, 399, 403
foreign matter in, 398
Marls
sampling of, 1
Marsh test
colors in distilled liquors, 691
Martins yellow, 1132
Mathers test
carmel in wines, 747
colors in distilled liquors, 691
Mayonnaise
gums in. 1005-1006
MCPA
dicamba-, in formulations, 187-188
ester and salt in formulations, 194
MCPP
in formulations, 188
Meal, see specific types of meal
Meat and meat products
agar in meat, 945
arsenic in meat, 935
arsenic in meat and poultry, 245
ash of, 932
beef and poultry adulteration of meat
products, 948
boric acid in meat, 1145-1146
calcium in mechanically separated
poultry and beef, 941
creatinine in meat, 941
fat (crude) in, 931-932
filth in pork sausage, ground beef or
hamburger, 390-391
glass in meat scraps, 389
lactose in meat, 945
meat extracts and similar products,
947
milk (nonfat, dry) in meat, 945
moisture in, 931
nitrates and nitrites in meat,
937-938
nitrites in cured meat, 938
nitrogen (amino) in meat, 941
nitrogen in meat, 935-937
7V-nitrosamines (volatile) in fried
bacon, 938-940
Af-nitrosopyrrolidine in fried bacon,
940-941
phosphorus in meat, 933-935
preservatives in ground beef, 1144
protein (crude) in meat, 937
Salmonella in, 476-480
salt (chlorine as sodium chloride) in,
933
sample preparation, 931
soy protein in raw and heat-
processed meat products,
942-945
soybean flour in meat, 942
starch in meat, 946-947
starchy flour in meat, 941
sulfites in meat, 1 159
sulfurous acid in meats, 1160
virus in beef, 494-495
water (added) in sausage, 931
see also Drugs and feed additives in
animal tissues
Mechanical analysis
fertilizers, 11
liming materials, 1
of peat, 37
Medroxyprogesterone acetate, 612,
619
Meissl-Hiller gravimetric method
invert sugar in sugars and sirups,
1017
Melengestro! acetate, 101-103,
629-631, 636
Melting point
of cacao fat, 771
of color additives, 1136
of fats and fatty acids, 953-954
Melting point method
foreign fats containing tristearin in
lard, 979-980
Membrane filter-DNA method
somatic cells in milk, 497
Menadione sodium bisulfite
in feed premixes, 1079-1080
Menadione sodium bisulfite, 567, 578
Menthol
in cigarette filler, 67-68
in drugs, 602, 606
Meperidine
in drugs, 512, 546
Mephentermine sulfate
in drugs, 512-513, 546
Meprobamate, 528-529, 546,
564-565, 578
Merbromin
in drugs, 510-511, 546
Mercuric iodide
mercury in, 509-510
Mercuric nitrate
mercury in ointments, 511
Mercuric nitrate method
chloride in water, 320-321
Mercuric oxide treatment
acidity of wines exclusive of S0 2 ,
745
Mercurous iodide
in tablets, 511
Mercury
in drug dosage forms, 508-512
in fish, 263-264
in food, 262-263, 264-266
in organic mercurial seed
di s i nf ectants , 1 62- 1 63
safe handling of, 653, 1227
in water, 326-327
Mercury reduction method
pyrethrin in formulations, 170-172
Mercury salts
safe handling of, 654, 1228
Mestranol, 611-612, 619
Metals and other elements
antimony in food, 242-243
arsenic in food, 237-239, 243-246
in baking powders, 688—689
cadmium and lead in foodware,
241-242
cadmium in food, 237-241,
246-248
in color additives, 1132-1135
copper in food, 248
fluorine in food, 250-253
fluorine on apples and pears,
248-249
lead and cadmium in foodware,
241-242
lead in evaporated milk, 255-257
lead in fish, 257-258
lead in food, 237-241, 258-262
lead in juices, 256-257
lead on apples and pears, 254-255
manganese in food, 262
mercury in fish, 263-264
mercury in food, 262-263, 264-266
methyl mercury in fish and shellfish,
266-269
in plants, 40-50
selenium in food, 237-239,
269-270
standard solutions of, 41
tin in food, 270-271
titanium in cheese, 271
trace levels in foods, 237-273
zinc in food, 237-239, 272-273
see also specific element
Methamidophos
in lettuce, strawberries, and
tomatoes, 282-283
Methamphetamine, 540, 546
Methanol
in cordials and liqueurs, 705
in distilled liquors, 702-703
in hazardous substances, 233-234
in lemon, orange, and lime extracts,
898
safe handling of, 653, 1227
in vanilla extract, 894
Methapyrilene, 513, 546, 579-580
Methaqualone, 621-622, 624
1-32
Subject Index
AOAC Official Methods of Analysis (1990)
Metharbital, 537, 546
Methazoie
in formulations, 194-195
Methenamine
chemical names, 546
in deodorants, 364
in tablets, 530-532
microchemical tests, 538
Metheneamine mandelate, 531-532,
546
Methimazole, 573-574, 578
Methiocarb
in grapes and potatoes, 292-294
technical and formulations, 218-219
Methocarbamol, 526, 546
Methomyl
in grapes and potatoes, 292-294
Methoxychlor
residues, 304
Methyclothiazide, 572-573, 578
Methyl anthranilate
in nonalcoholic beverages, 755
Methyl cellosolve
safe handling of, 653, 1227
iV-Methyicarbamate
on apples, cabbage, collards, corn
kernels, green beans, kale, and
turnip tops, 291-292
in grapes and potatoes, 292-294
Methyldopa, 570-572, 578
Methylene blue, 531-532, 546
Methylene chloride extract
of spices, 1000
Methyleneureas
in fertilizers, 21-22
Methyl esters
of fatty acids in oils and fats,
964-965
Methyl mercury
in fish and shellfish, 266-269
Methyl parathion
on apples, carrots, endive, kale,
lettuce, potatoes, and
strawberries, 287-289
confirmatory method, 289-290
in formulations, 208-210
Methyl salicylate
chemical name, 578
in drugs, 551-552
essential oil in, 905-906
in nonalcoholic beverages, 754-755
Metolachlor
in formulations, 195
Metoxuron
in formulations, 216-217
Metribuzin
in formulations, 219
Micro Bailey-Andrew method
caffeine in roasted coffee, 758
Mkro-Kjeldahl method
nitrogen determination, 341-342
Microbial receptor assay
antimicrobial drugs in milk,
829-831
Microbiological methods
antibiotics in feeds, 115-129
Bacillus, 464-467
chilled, frozen, precooked, or
prepared foods, 429-435
Clostridium, 459-464
conforms in foods, 434-449
cross reference tables, 425-427
disinfectant testing, 133-146
eggs and egg products, 427-428
niacin and niacinamide in ready-to-
feed milk-based formulas, 1109
nutmeats, 429-435
poliovirus in oysters, 495-496
Salmonella, 467-492
somatic cells, 496-497
Staphylococcus, 449-455
sterility (commercial) of canned,
low-acid foods, 455-459
Vibrio, 492-494
virus in ground beef, 494-495
vitamin B 6 in ready -to-feed milk-
based formulas, 1107-1108
vitamins and other nutrients,
1080-1091
see also specific bacteria and
methods
Microchemical methods
alkoxyl groups, determination,
347-348
bromine, chlorine, or iodine
determination, 337-339
carbon and hydrogen determination,
339-341
carbon, hydrogen, and nitrogen
determination, 341
fluorine determination, 341-342
molecular weight determination, 337
nitrogen determination, 341-342
oxygen determination, 343-344
phosphorus determination, 344-345
sulfur determination, 345-347
Microchemical tests
alkaloids and related amines in
drugs, 533-536
barbiturates in drugs, 536-537
phenothiazine drug identification,
536
sympathomimetic drugs, 536, 540
synthetic drugs, 536, 538-541
uric acid on foods and containers,
414
xanthine group alkaloid drugs, 541
Microfluorometric method
vitamin C in vitamin preparations,
1058-1060
Microscopic methods
adulterants in spices, 1002
barbiturate drugs, 559
chlortetracycline in feeds, 130
crystalline substances in drugs,
541-542
direct count of bacteria in eggs and
egg products, 428
of feeds, 88-90
fdth in raisins, 393
identification of canned Pacific
salmon, 889
insect penetration through packaging
materials, 406-407
Microslide gel double diffusion test,
451-454
Microwave drying methods
moisture in cheese, 842
moisture in meat and poultry
products, 931
solids (total) in processed tomato
products, 990
Microwave-solvent extraction method
fat (crude) in meat and poultry
products, 932
Mid-infrared spectroscopic method
fat, lactose, protein, and solids in
milk, 816-818
protein in milk, 809
Milk
acidity of, 805
allatoxin Mi in, 821, 1200-1201,
1203-1205
aflatoxin M 2 in, 1203-1205
albumin in, 809
antimicrobial drugs in, 829-830
ash of, 807
bacterial and coliform counts, 435
barium-140 in, 353-355
casein in, 809
cesium- 137 in, 353-355
chloramines in, 820-821
citric acid in, 805-806
color additives in, 821
condensed/dried milk products,
starch in, 84
copper in, 821
dried milk products, crude fat and
ether extract in, 80
ethylenethiourea pesticide residues in
milk, 300-302
fat in, 811-818
filth in, 378-379
gelatin in, 820
hydrogen peroxide in, 1149
hypochlorites in, 820-821
iodine-131 in, 353-355
fj-lactam antibiotics in, 825-829
lactic acid in, 806-807
lactose in, 810-811, 816-818
nitrogen (total) in, 807-808
organochlorine and
organophosphorus pesticide
residues in, 821
phosphatase (residual) in, 821-824
preservatives in, 820
protein in, 808-809, 816-818
protein-reducing substances in,
809-810
quaternary ammonium compounds
in, 1151
raw, fat in, 814
Salmonella in powders, 478-480
Salmonella in, 470-471, 476-478
sample collection, 804-805
sample preparation, 805
sampling, 803
sediment in, 375-378, 821
solids in, 1296-1297
solids (total) in, 807, 816-818
somatic cells in, 496-497, 805
AOAC Official Methods of Analysis (1990)
Subject Index
I-33
specific gravity of, 805, 1298
strontium-89 and -90 in, 351-353
vitamin D in fortified milk and
milkpowder, 1068-1069
vitamin D in, 1091-1094
water (added) in, 818-820
see also Dried milk and its products;
Condensed milk; Evaporated
milk; Sweetened condensed milk
Milk chocolate
coconut and palm kernel oils in fat
extracted from, 772
fructose in, 773
glucose in, 773
lactose in, 772, 773
maltose in, 773
milk fat in, 771
protein in, 772
sucrose in, 773
Milk fat
in milk chocolate, 771, 772
Millon test
protein in feeds, 70
Miner method
1 -monoglycerides in fats,
shortenings, and
monoglycerides, 984-985
Mineral oil-soap emulsions, 162
Mineral oil vacuum distillation-
thermal energy analyzer method
iV-nitrosamines (volatile) in fried
bacon, 938-940
Mineral oils
in baked products, 794-795
in fats, 980
unsulfonated residue of, 161-162
Mineral salts
in feeds, 84
Mineral wool insulation properties,
638-639
Minerals
in feeds, 84-88, 89-90
in ready-to-feed milk-based
formulas, 1110
see also specific minerals
Mint
filth in, 399
Mirex
in fatty products, 302-303
Moisture
in Bordeaux mixtures, 158, 159
in butter, 837
in cacao products, 763
in cereal adjuncts, 730
in cheese, 841-842
in confectionery, 1024
in corn sirups and sugars,
1039-1040
in dried fruits, 912
in dried milk, 834
in feeds, 69-70
in fig bars and raisin filled crackers,
795
in flour, 777
in frozen french-fried potatoes, 998
in grains, 788
in honey, 1026
in hops, 732
in lead arsenate pesticide
formulations, 154
in macaroni products, 796
in malt, 725
in malting barley, 723
in maple products, 1034
in meat and meat products, 931
in meat extracts and similar
products, 947
in mineral oil-soap emulsions, 162
in molasses, 1021-1022
in nuts and nut products, 949
in oils and fats, 951
in peat, 37
in pesticide formulations, 147, 154,
158
in plants, 40
in prunes and raisins, 912-914
in roasted coffee, 760
in salt, 332
in soaps, 162
in soybean flour, 791
in spices, 999
in sugars, 1010-1011
in tea, 762
in tobacco, 64
Moisture meter method
moisture in prunes and raisins,
912-914
Mojonnier ether extraction method
fat in milk, 811-812
Molasses and molasses products
ash of, 1022
filth in, 394
invert sugar in, 1023
moisture in, 1021-1022
nitrogen in, 1022
reducing substances in, 1023-1024
sampling of, 1021
specific gravity of, 1022
sucrose in, 1022
sugars (as invert sugar) in,
1022-1023
Mold
in butter, 420, 840
in fmits and fruit products, 418-422
in ground spices, 420
Howard mold counting method,
416-420
in infant food purees, 419
in vegetables and vegetable products,
419-420, 421-422
Molecular weight
thermoelectric-vapor pressure
method, 337
Molinate
in herbicide formulations, 221
Molybdenum
in plants, 40, 47
Molybdenum blue methods
arsenic in food, 244-245
arsenic in meat, 935
arsenic in meat and poultry,
245-246
Monensin
in feeds, 123-124
Monier- Williams method
sulfurous acid in food, 1157-1158
Monocalcium phosphate, 685-686
Monochioroacetic acid
in liquids and preservatives,
1149-1150
in nonalcoholic beverages, 752
in nonalcoholic beverages and wines,
1150-1151
Monocrotophos
in lettuce, strawberries, and
tomatoes, 282-283
Monofluoroacetic acid
residues, 305-306
Monoglycerides
concentrates, 983-984
glycerides in concentrates, 982-983
1 -monoglycerides in, 984-985
1 -Monoglycerides
in fats, shortenings, and
monoglycerides, 983-985
Monosodium glutamate
in food, 1174-1175
Monostearin, 983-985
Morphine
with acetanilid, caffeine, and quinine
in drugs, 555
chemical name, 546, 578
HCl, 580, 606
microchemical tests, 534
in opium and paregoric, 581-582
procaine in presence of, 524
sulfate, 580, 582-583, 606
titration factors, 580
Most probable number method
Bacillus cereus in foods, 464-466
bacteria in chilled, frozen,
precooked, or prepared foods,
429-430
coliform group and Escherichia coli
in tree nut meats, 430-431
Staphylococcus aureus in foods,
449-450
Mouse adrenal cell and suckling
mouse assays
E. coli enterotoxins, 448-449
Multielement methods
metals at trace levels in foods,
237-242
Multiresidue methods
organochlorine pesticide residues,
274-285
organophosphorus pesticide residues,
274-283, 286-290
Multivitamin preparations
niacin and niacinamide in, 1057™
1058
vitamin D in, 1066-1067
Munitions
TNT, RDX, HMX, and 2,4-DNT in
wastewater and groundwater,
334-336
Munson- Walker method
fructose in sugars and sirups, 1017
glucose in sugars and sirups, 1017
invert sugar in sugars and sirups,
1017
1-34
Subject Index
AOAC Official Methods of Analysis (1990)
lactose in sugars and sirups, 1017
maltose in sugars and sirups, 1017
sugars in brewing sugars and sirups,
734
sugars (reducing) in nuts and nut
products, 950
sugars in plants, 59
Mushrooms
filth in, 396-397
Mustard (dry)
foreign matter in, 398
starch in, 1000
Mustard (prepared)
acidity of, 1003
chJorides in, 1003
ether extract of, 1 003
fiber (crude) in, 1004
filth in, 406
nitrogen in, 1003
sample preparation, 1003
solids in, 1003
starch in, 1004
Mustard greens
filth in, 395-396
Mustard seed
filth in, 398-399
volatile oil in, 1001-1002
Mycotoxins
deoxynivalenol in wheat, 1205-1207
handling, 1184
in nuts and nut products, 950
ochratoxin A in green coffee, 1208
ochratoxins in barley, 1207-1208
patulin in apple juice, 1209-1210
preparation of standards for,
1185-1187
sterigmatocystin in barley and wheat,
1210-1211
a-zearalenol and zearalenone in
corn, 1212-1213
zearalenone in corn, 1211-1213
see also Aflatoxins
Mydriatic drugs, 584
Myotic drugs, 584
Nalidixic acid, 631, 636
Naphthalene red B, 1121
P-Naphthol
in color additives, 1126
Naphthol yellow S, 1115, 1122
Naphthyleneacetic acid
in apples and potatoes, 306-307
ot-Naphthylthiourea
in rodenticides, 163
Narceine, 534, 546
/-Narcotine, 535
National Cottonseed Products
Association-AOAC method
fatty acids (free) in crude and
refined oils, 957
Natural coloring matters, 1118
Near-infrared methods
acid-detergent fiber and crude
protein in feeds and forages,
74-76
piperazine in drugs, 533
water in dried vegetables, 994-995
Neocinchophen, 538, 546
Neostigmine, 591, 606
Neostigmine methylsulfate, 591-592
Nequinate
in feeds, 103
Neutralizing values
of baking chemicals, 685-686
calcium silicate slags, 3
liming materials, 1-2
sodium aluminum phosphate, 686
Niacin and niacinamide
assays, 1080-1082
in cereal products, 1055-1056
in drugs, foods, and feeds,
1054-1055, 1056-1057
in multivitamin preparations,
1057-1058
in ready-to-feed milk-based
formulas, 1109
in vitamin preparations, 1084-1085
Nicarbazin
in feeds, 103
Nickel
in tea, 242, 269, 762
Nicotine
in apples, cabbage, and spinach,
307-308
on Cambridge filter pads, 66-67
chemical name, 546
in feeds, 103-104
microchemical tests, 534
in tobacco, 65-66
in tobacco products, 173-174
Nifursol
in feeds, 104
Nihydrazone
in feeds, 105
Nikethamide, 599, 606
Nitarsone
in feeds, 105
Nithiazide
in feeds, 105
Nitrates
in cheese, 843-844
esters, 527
in fertilizers, 17, 20
in forages, 357-358
in meat, 937-938
in water, 320
Nitric acid
safe handling of, 651, 1225
solution strengths, 658
Nitrites
in cheese, 843-844
in cured meat, 938
in curing preparations, 1151
in flour, 783-784
in meat, 937-938
in tablets, 512
NStroaromatics
safe handling of, 653, 1227
Nitrobenzene
in almond extract, 905
safe handling of, 653, 1227
Nitrocresol method
cobalt in plants, 43-45
Nitrodan
in feeds, 106
Nitrofurazone
in feeds, 100
Nitrogen
activity in fertilizers, 20-21
albuminoid, in feeds, 77
amido, in feeds, 77
ammoniacal, in feeds, 76
in beer, 713
in cacao products, 764
in cheese, 842
in color additives, 1125
in confectionery, 1024
in corn sirups and sugars, 1042
in cream, 832
in cyanarnide, 20
in eggs, 854
in evaporated milk, 833
in fertilizers, 9-10, 17-23
in flour, 783-784
in food dressings, 1005
in honey, 1026
and hydrogen and carbon,
microchemical determination,
341
in macaroni products, 797
in meat, 935-937, 941
in meat extracts and similar
products, 947
microchemical determination,
341-342
in milk, 807-808
in molasses, 1022
nitrate and nitrite, in feeds, 77-78
in nonvolatile ether extract of
pepper, 1000
in peat, 38
in plants, 59
in prepared mustard, 1003
in seafood, 868
in soybean flour, 791
in spices, 1000
in sugars and sirups, 1012
in tobacco, 64
in water, 318-319
in wines, 746
in wort, 735
Nitroglycerin, 526-527, 529, 546
Nitromersol
mercury in, 509-510, 546
Nitromide
in feeds, 106
Nitrophenide
in feeds, 106
5-Nitro-2-propoxyaniline
in food, 1172
in nonalcoholic beverages,
1167-1168
TV-Nitrosamines
in baby bottle rubber nipples,
1182-1183
in fried bacon, 938-940
/V-Nitrosodibutylamine
in latex infant pacifiers, 234-236
A^-Nitrosodiethylamine
in beer, 719-722
AOAC Official Methods of Analysis (1990)
Subject Index
I-35
N-Nitrosodimethyiamine
in nonfat dry milk, 835
yV-Nitrosopyrrolidine
in fried bacon, 940-941
Nitroso-R-salt method
cobalt in plants, 45
NMKL-AOAC method
benzoic acid and sorbic acid in food,
1143-1144
Nomenclature rules
vitamin E, 1070- 1071
Nonalcoholic beverages and
concentrates
acidity in, 751
acids (dibasic) in, 751
alcohol in, 751
alginates in chocolate beverages, 756
ash of, 751
benzaldehyde in, 751-752
benzoate in, 752
brominated vegetable oils in,
1166-1167
caffeine in, 752-754
citric acid in, 751
cyclohexylsulfamate salts in, 1168
dihydroanethole in, 754-755
dihydrosafrole in, 754-755
dulcin in, 1171-1172
essential oils in, 752
esters in, 751
filth isolation from, 373-374
glucose in, 756
isosafrole in, 754-755
malic acid in, 751
methyl anthranilate in, 755
methyl salicylate in, 754-755
monochloroacetic acid in, 752,
1150-1151
nonnutritive sweeteners in,
1167-1168
quaternary ammonium compounds
in, 756
saccharin in, 752, 1174
safrole in, 754-755
sample preparation, 751
solids (total) in, 755
specific gravity of, 755
sucrose in, 756
sugars (reducing) in, 756
tartaric acid in, 751
7-undecalactone in, 756
Nonfat dry milk
alkalinity of, 835
in meat, 945
A^-nitrosodimethyl amine in, 835-836
Salmonella in, 470-471, 476-478
Nonnutritive sweeteners
in nonalcoholic beverages,
1167-1168
Nordic Committee on Food Analysis,
see NMKL
Nordihydroguaiaretic acid
in foods, 1137-1138
in oils and fats, 1138-1139
Norepinephrine, 515-516, 546
Norethindrone, 612-613
Norethindrone acetate, 612, 619
Norethynodrel, 612-613, 619
Norgestrel, 612-613, 619
Noscapine, 535, 546
Novobiocin
in feeds, 125
in milk, 829-831
Nutmeg
extract, 905
filth in, 398-399, 403-404
Nutrients
in fertilizers, 27-28
see also specific nutrients
Nutritional supplements
lysine in, 1100-1101
Nuts and nut products
anatoxins in, 1187-1191, 1195
ash of, 949
coliform group and Escherichia coli
in, 430-431
examination of peanut butter, 950
extraneous materials in, 379-380
fat (crude) in, 949
fiber (crude) in, 949
filth in, 379
glycerol in shredded coconut, 950
inorganic residue in, 950
moisture in, 949
preservation of nut sample, 949
protein (crude) in, 949
sample preparation, 949
sodium chloride in, 950
starch in peanut butter, 950
sucrose in, 950
sugars (reducing) in, 950
volume of packages nuts, 949
Nystatin
in feeds, 125-126
Oatmeal
filth in cereals, 388
Oats
piperonyl butoxide pesticide residues
in, 309
Ochratoxins
in barley, 1207-1208
in green coffee, 1208-1209
/V-Octyl bicycloheptene
dicarboximide
in formulations, 172-173
Office International du Cacao et du
Chocolat-AOAC methods
fat in cacao products, 770
nitrogen in cacao products, 764
see also AOAC-Office International
du Cacao et du Chocolat
methods
Ofner volumetric method
invert sugar in sugars and sirups,
1017
Oil red XO, 1115
Oils and fats
acetyl value of, 955
acids (polyunsaturated) in, 960-963
acids (volatile) in, 958
animal fats in vegetable fats and
oils, 976-977
antioxidants in, 1138-1139
brominated vegetable oils in
nonalcoholic beverages ,
1166-1167
butyric acids (mole per cent) in fat,
958-960
chick edema factor in, 981-982
cholesterol in, 976-977
colors (synthetic) in, 982
cottonseed oil in, 977-978
cyclopropene in oils, 978
dioxins in, 981-982
docosenoic acid in, 967
erucic acid in, 965-967
fats (vegetable) in butterfat, 973-975
fatty acids in, 963-964
fatty acids (crude) in, 957
fatty acids (free) in crude and
refined oils, 957
fatty acids (saturated and
unsaturated) in, 960
fatty acids (trans isomers) in
margarines, 970-971
fish oil detection, 980
flaxseed oil identification, 986
foreign fats containing tristearin in
lard, 979-980
glycerides in monoglyceride
concentrates, 982-983
glycerides in shortening, 985-986
hexachlorobenzene and mirex
residues in fatty products,
302-303
hydrocarbons (saturated) in
glycerides, 980-981
hydro xyl value of, 955
index of refraction of, 952-953
insoluble acids in, 957
iodine absorption number of,
955-956
marine animal oil detection, 980
melting point of fats and fatty acids,
953-954
cis, cis- methylene interrupted
polyunsaturated fatty acids in
oils, 969
methyl esters of fatty acids in,
964-965
mineral oil in fats, 980
moisture in, 951
1 -monoglycerides and monoglyceride
concentrates, 983-984
1 -monoglycerides in fats,
shortenings, and
monoglycerides, 984-985
oil in anise and nutmeg extracts, 905
oil in cassia, cinnamon, and clove
extracts, 905
oil in cereal adjuncts, 730
oil in food dressings, 1005
oil in peppermint, spearmint, and
wintergreen extracts, 905
peanut oil in, 978
peanut oil in olive, cottonseed, corn,
and soybean oils, 978-979
peroxide value of, 956
polar components in frying fats,
968-969
1-36
Subject Index
AOAC Official Methods of Analysis (1990)
polymers and oxidation products of
heated vegetable oils, 967-968
polysorbate 60 in shortening, oils,
and dressings, 1163
recoverable oils in fruits and fruit
products, 923
rosin oil in, 977
salad oils, 979
sample preparation, 951
saponification number of, 957
sesame oil in, 979
(3-sitosterol in butter oil, 975-976
soluble acids in, 957
specific gravity of oils, 951-952
squalene in, 972
tea seed oil in oilve oil, 979
thiocyanogen number of, 957
titer test for, 954-955
trans isomers (isolated) in
margarines and shortenings,
969-970
triglycerides in, 972-973
unsaponifiable residue of, 971-972
volatile matter in, 951
volatile oil in mustard seed,
1001-1002
volatile oils in spices, 1001
weight (apparent) per unit volume,
952
see also Animal fats; Essential oils;
Fat
Oleandomycin
in feeds, 126
Oleic acid
in oils and fats, 960-963
Olive oil
peanut oil in, 978-979
tea seed oil in, 979
Omethoate
in lettuce, strawberries, and
tomatoes, 282-283
Onion
filth in ground preparations, 399,
402
Onion powder
Salmonella in, 470-471
Opium
alkaloids, 579-583
morphine in, 581-582
Optical somatic cell counting method
somatic cells in milk, 496-497
Optical-crystallographic properties
p-ionone-ra-nitrobenzhydrazide , 907
quaternary ammonium compounds in
milk, 1151
Orange B, 1119
Orange extract
alcohol in, 898
aldehydes in, 900-901
ash of, 901
citral in, 901
essential oil in, 905-906
glycerol in, 899
methanol in, 898
solids (total) in, 901
specific gravity, 898
sucrose in, 901
Orange flavors
isopropanol in, 899
Orange I, 1115
Orange II, 1120
Orange juice
corn syrup, 926-927
Florida, adulteration of, 929-930
thiourea in, 1161
Orange oil
aldehydes in, 902
in extracts, 899
in oil -base flavors, 900
physical constants of 10% distillate,
902
pinene in, 903
refractive index of, 901
specific gravity of, 901
spectrophotometric absorbance
characteristics, 901-902
steam distillation residue, 902
Orange peel
thiourea in, 1162-1163
Orange SS, 1115
Oregano
filth in, 399, 404
foreign matter in, 398
Organic acids
in fruit juices, 921-922
in vanilla extract, 896-898
Organic carbon
in water, 317-318
Organic matter
in peat, 38
Organic mercurial seed disinfectants,
162-163
Organochlorine pesticide residues
aldrin, 279
a-BHC, 282-283
in animal fats, 284
chlorpyrifos, 282-283
DDE, 279
DDT, 279, 282-283
dieldrin, 279, 282-283
endosulfan sulfate, 284-285
endosulfan, 284-285
endrin, 279
in fish, 283-284
heptachlor epoxide, 279
heptachlor, 279
in apples and cucumbers, 284-285
lindane, 279
multiresidue methods, 274-285
TDE, 279
tetradifon, 284-285
tetrasul, 284-285
Organochlorine and
organophosphorus pesticide
residues
in milk, 821
Organochlorine pesticides, see
Organohalogen pesticides
Organohalogen pesticides
alachlor, 174-175
aldrin, 175-176
benfluralin, 179-180
benzene hexachloride, 179
7-BHC (lindane), 176-179
bromoxynil octanoate, 180-181
butachlor, 181
captan, 181-182
chloramben, 182
chlordane, 182-184
chlordimeform, 184
chlorine in, 175
contamination of pesticide
formulations, 152-153
2,4-D, 184-185, 187-188, 196
dalapon, 185-186
DCPA, 186
DDT, 186-187
dicamba in formulations, 187-188
dichlobenil, 188-189
dicofol, 189-190
dieldrin, 175-176
diflubenzuron, 190-191
endosulfan, 191-192
endrin, 175-176
fluazifop-butyl, 192
folpet, 192-193
heptachlor, 184
heptachlor, 193-194
hexachlorocyclopentadiene in
technical chlordane, 183
MCPA, 187-188, 194
methazole, 194-195
metolachlor, 195
PCNB, 195
picloram, 196
propachlor, 196
sodium TCA. 197
2,4,5-T, 196-197
tetradifon (technical), 197
trifluralin, 179-180
Organophosphorus pesticide residues
acephate, 282-283
carbophenothion, 286-289
diazinon, 287-290
EPN, 286-287
ethion, 287-289
malathion, 287-290
methamidophos, 282-283
methyl parathion, 290
monocrotophos, 282-283
multiresidue methods, 274-283,
286-290
omethoate, 282-283
paraoxon, 286-287
parathion, 286-290
Organophosphorus pesticides
azinphos-methyl, 197-199
chlorpyrifos, 199
DDVP, 199-200
diazinon, 200-201
disulfoton, 201
ethion, 201-202
ethyl parathion, 209-210
fenitrothion, 202-203
fensulfothion, 203-204
formothion, 204-205
glyphosate, 205
isofenphos, 205-206
malathion, 206-207
methyl parathion, 208-210
parathion, 207-208
AOAC Official Methods of Analysis (1990)
Subject Index
I-37
sulprofos, 210-211
temefos, 211
TEPP, 211-212
Oxalic acid
in canned vegetables, 993-994
safe handling of, 651, 1225
Oxamyi
in grapes and potatoes, 292-294
Oxazepam, 622
Oxidation methods
1-monoglycerides and monoglyceride
concentrates, 983-984
thiourea in orange juice, 1161
Oxidation products
of heated vegetable oils, 967-968
Oxides (mixed)
in color additives, 1134
Oxidizers
safe handling of, 653, 1227
6,6'-Oxybis(2-naphthalenesulfonic
acid)
in FD&C Red No. 40, 1127-1128
in FD&C Yellow No. 6, 1130-1132
Oxygen
microchemical determination,
343_344
in water, 315-316
Oxygen flask combustion methods
bromine, chlorine, or iodine
determination, 338-339
sulfur determination, 346-347
Oxyquinoline sulfate, 551, 578
Oxytetracycline
in feeds, 126-127
Oxythioquinox
in formulations, 226-227
Oysters
frozen and thawed shucked,
detection of, 865-866
indole in, 877-878
poliovirus 1 in, 495-496
shell in canned products, 390
shucked, drained liquid from, 867
shucked, volume of, 867
Vibrio cholerae in, 492-494
P-4000
in food, 1172
Packaging materials
insect penetration, 406-407
Paint
lead in, 233
Palm kernel oil
in cocoa butter and in fat extracted
from milk chocolate, 772
silver number for detection of, 772
Pamaquine, 593, 606
Pancreatin digestion flotation method
filth in baked goods, 386
Pancreatin digestion methods
filth in baked goods, 386
filth in corn flour, 385
Pantothenic acid
assays, 1080-1082
in vitamin preparations, 1085-1086
Papain
filth in, 406
proteolytic activity, 1164-1165
Papaverine, 535, 546
Papaya leaves
filth in, 399, 400
Paper and paperboard
filth in rice paper, 384-385
PCBs in, 285-286
Paper chromatographic methods
acids (volatile) in bread, 792-793
anthocyanins in fruit juices, 923
ergotamine in drugs, 589-590
malvidin glucosides in grape juice,
923-924
organic acids (foreign) in fruit
juices, 921-922
organic acids in vanilla extract,
896-897
organochlorine and
organophosphorus pesticide
residues, 275, 282
sulfonamide mixtures in drugs, 567
vanilla resins in vanilla extract, 894
vanillin and ethyl vanillin in vanilla
extract, 891-892
Paper chromatographic-
spectrophotometric method
lysergic acid diethylamide in drug
powders, 621
Paprika
filth in, 399, 401, 404-405
Parabens, 524
Paraffin
in confectionery, 1025
Paraldehyde
in drugs, 563-564, 578
Paralytic shellfish poison, 881-882
Paraoxon
on apples and green beans, 286-287
Paraphenylenediamine
in hair dyes and rinses, 366
Paraquat
in formulations, 227-228
Parasites
in fish muscle, 882-883
Parathion
on apples, carrots, endive, kale,
lettuce, potatoes, and
strawberries, 287-289
on apples and green beans, 286-287
confirmatory method, 289-290
in formulations, 207-208
residues, 308
Paregoric, 581-582
Paris green
Bordeaux mixtures with, 149-150,
158
pesticide formulations, 153-154
Parsley
filth in, 399
Partition chromatographic methods
7-BHC (lindane) in formulations,
176-178
diatomaceous earth for drugs, 498
Pasta, see Macaroni products
Patulin
in apple juice, 1209-1210
PCBs
in fish, 283-284
in foods, 280
in paper and paperboard, 285-286
PCNB
in formulations, 195
Peaches
azinphos-methyl pesticide residues
on, 294
captan pesticide residues in, 296
dodine pesticide residues in, 299
thiourea in frozen fruit, 1161-1162
Peanut butter
examination of, 950
starch in, 950
Peanut oil
in oils and fats, 978
in olive, cottonseed, corn, and
soybean oils, 978-979
Peanuts and peanut products
aflatoxins in, 1187-1190, 1195
filth and extraneous materials in, 380
Pears
captan pesticide residues in, 296
dodine pesticide residues in, 299
fluorine on, 248-249
glyodin pesticide residues in, 302
lead on, 254-255
Peas
Alaska, piperonyl butoxide pesticide
residues in, 309
solids (alcohol-insoluble) in frozen
products, 996
solids in canned peas, 990
weevils in, 394
Peat
ash in, 37
cation exchange capacity for, 39
mechanical analysis of, 37
moisture in, 37
nitrogen in, 38
organic matter in, 38
particle size range of, 37
pH determination of, 37
sample preparation, 36
sampling of, 36
sand in, 37-38
water capacity and volumes for,
38-39
Pebulate
in herbicide formulations, 221
Pecans
dodine pesticide residues in, 299
filth in, 379
Pectic acid
in cacao products, 765-766
in fruit products, 917
Pectin gel methods
coliforms in dairy products, 434
microorganisms in foods, 433-434
Penicillin
in milk, 829-831
Pentabromoacetone method
citric acid in fruits and fruit
products, 918
Pentachlorophenol
in gelatin, 308-309
1-38
Subject Index
AOAC Official Methods of Analysis (1990)
Pentaerythrityl tetranitrite, 527,
528-529, 546
Pentane
safe handling of, 654, 1228
Pentobarbital, 537, 546, 559, 578
Pentosans
in feeds, 84
in wines, 747
Pentylenetetrazol, 538, 546
Pepper
filth in, 398-399, 401, 405
nitrogen in nonvolatile ether extract
of, 1000
piperine in preparations, 1002-1003
Salmonella in, 478-480
Pepper sauce
filth in, 405
Peppermint extract, 905
Peppermint leaves
filth in, 399, 400
Pepsin digestibility
of protein feeds, 78-79
Perchloric acid
safe handling of, 651, 1225
Perchloric acid methods
dye in color additives, 1125
potassium in plants, 48
Perchromic acids
safe handling of, 650, 1224
Performic acids
safe handling of, 650, 1224
Period ate oxidation method
propylene glycol in drugs, 499
Permanganate method
oxygen (dissolved) in water,
315-316
Permanganate oxidation number
for vinegars, 1009
Permanganates
safe handling of, 654, 1228
Permatone orange, 1121
Permethrin
in pesticide formulations, 167-168
Peronine, 534
Peroxidase
in frozen vegetables, 998
Peroxide value
of oils and fats, 956
Peroxides
safe handling of, 654, 1228
Perphenazine, 536, 546
Persulfate method
manganese in water, 331
Pesticide formulations
allethrin, 164-166
amitrole, 223-224
arsenic in, 147-149
arsenious oxide in, 153-154
Bordeaux mixtures, 158
brodifacoum, 224
calcium arsenate, 149, 154-155
calcium cyanide, 159
carbamates, 212-213, 214-216,
217-220, 221-222
chloramine T, 161
common and chemical names,
230-231
contamination by organochlorine
pesticides, 152-153
copper carbonate, 155
copper in, 149-150, 154
copper naphthenate, 155
cyhexatin, 225
cypermethrin in, 166-167
diquat, 225
dodine, 225-226
fentin in, 156-157
fentin-maneb, 157
fertilizer-pesticide mixtures, 147
fluorine in, 150-152
formaldehyde, 226
fumigant mixtures, 164
inorganic and organometallic
pesticides and adjuvants,
153-164
lead in, 149
lime sulfur, 159-160
mineral oil-soap emulsions, 162
moisture in, 147, 154, 158
a-naphthylthiourea in, 163
7V-octyJ bicycloheptene
dicarboximide, 172-173
organic thiocyanate sprays, 228
organohalogen pesticides, 174-197
organophosphorus pesticides,
197-212
oxythioquinox, 226-227
paraquat, 227-228
Paris green, 153-154
permethrin in, 167-168
piperonyl butoxide, 170, 172
potassium cyanate, 159
potassium cyanide, 159
pyrethrin, 170-172
quaternary ammonium compounds,
228
rotenone, 167-170
sabadilla alkaloids, 173
sample preparation, 147
sampling, 147
seed disinfectants, 162-163
soaps, 162
sodium cyanide, 159
sodium hypochlorite in, 160-161
substituted ureas, 216-217, 218, 220
sulfoxide, 230-231
thallous sulfate in, 163-164
triadimefon technical and
formulations, 228-229
triazines, 213-214, 219, 220-221,
222-223
warfarin in rodenticides, 229-230
zinc arsenite, 149, 155
zinc in, 150
Pesticide residues
azinphos-methyl, 294
benzene hexachloride, 294
biphenyl in citrus fruits, 295-296
2-(p-rm-butylphenoxy)- 1 -methyl
2-chloroethyl sulfite (aramite),
296
captan, 296
carbamate residues, 291-294
carbaryl, 296-298
/?-chlorophenyl phenyl sulfone, 298
common and chemical names of,
310-311
DDT, 298
dichlone, 298-299
dodine, 299
endosulfan and endosulfan sulfate,
284-285
ethylan (perthane), 298
ethylene dibromide, 299-300
ethylenethiourea, 300-302
glyodin, 302
hexachlorobenzene, 302-303
lindane, 294
malathion, 303
maleic hydrazide, 303-304
methoxychlor, 304
mirex, 302-303
monofluoroacetic acid, 305-306
multiresidue methods, 274-285
naphthyleneacetic acid, 306-307
nicotine, 307-308
organochlorine residues, 274-284
organophosphorus residues,
274-283, 286-290
parathion, 308
pentachlorophenoJ , 308-309
piperonyl butoxide, 309
poly chlorinated biphenyl s (PCBs),
285-286
tetradifon, 184-185
tetrasul, 284-285
thiram, 309-311
Pesticides
Certified Reference Materials, 647,
1221
safe handling of, 652, 1226
Pet foods
crude fat in, 79-80
Petroleum ether
extract of roasted coffee, 760
extract of tea, 762
safe handling of, 654, 1228
Petroleum ether extraction method
oil in cereal adjuncts, 730
pH
of acidified foods, 988-989
of baked products, 795
of beer, 712
of bread, 794
of brewing sugars and sirups, 734
of cacao products, 764
of flour, 780
of macaroni products, 797
of peat, 37
of water, 312-313
of wines, 744
Pharmaceutical Manufacturers
Association-Food Chemicals
Codex-AOAC method
papain proteolytic activity, 1165
Phenacetin
with acetanilid in drugs, 555
with aminopyrine in drugs, 556
with antihistamines in drugs, 515
with aspirin in drugs, 555-556
with caffeine in drugs, 555, 556
AOAC Official Methods of Analysis (1990)
Subject Index
I-39
chemical name, 546, 578, 606
microchemical tests, 538
with phenobarbital in drugs, 556
with phenyl salicylate in drugs, 556
Phenaglycodoi, 564, 578
Phenazopyridine HCI, 525, 538, 546
Phencyclidine HCi, 623, 624
Pheniramine maleate
in dosage forms, 515, 546
Phenmetrazine, 540, 546
Phenobarbital
with aminophylline in drugs, 560
with aminopyrine in drugs, 556
with aspirin in drugs, 557
with caffeine in drugs, 556
chemical name, 546, 578
with mannitol hexanitrate in drugs,
527-528
microchemical tests, 537, 539
with phenacetin in drugs, 556
with phenytoin in drugs, 560
with theobromine in drugs, 561
Phenol coefficient method
disinfectant testing, 133- 135, 137
Phenolic drugs
p-aminosalicylic acid and isoniazid
in drugs, 549
dinitrophenol in drugs, 550
guaiacol in drugs, 550
guaifenesin in drugs, 550
hexylresorcinol in drugs, 551
methyl salicylate in drugs, 551-552
oxyquinoline sulfate in drugs, 551
phenolphthalein in dosage forms,
552-553
phenol sulfonates in drugs, 552
thymol in dosage forms, 552-553
vasoconstrictors, 524
Phenolphthalein
in drugs, 552-553, 557, 578
Phenols
in hazardous substances, 236
salicylic acid in drugs in presence
of, 548
Phenolsulfonates
in deodorants, 364-365
in drugs, 552
Phenothiazine
chemical name, 578
drugs, 536
in drugs, 574-575
in feeds, 106
Phenprocoumon, 565-566, 578
Phenyl salicylate, 556, 578
Phenylalkanolamine salts, 519-520
Phenylephrine HCL, 516-519, 546
Phenylethylamines
amphetamines, 520-521
mephenterrnine sulfate, 521
Phenylhydrazine-/?-sulfonic acid
in FD&C Yellow No. 5, 1128-1129
Phenylmercuric chloride
mercury in, 509-510
Phenylmethylbarbituric acid, 537, 546
Phenylpropanolamine, 540
Phenylpropanolamine HCI, 519-520,
546
Phenytoin, 560-561, 578
Phenytoin sodium, 562
Phloxine B, 1121
Phosphatase
in butter, 840
in casein, 824-825
in cheese, 847-850
in cream, 833
in ice cream and frozen desserts,
852
in milk, 821-824
Phosphorus
in baking powders, 689
in beer, 713
with calcium and iron in vitamin
preparations, 504
in color additives, 1134-1135
in cordials and liqueurs, 706
in distilled liquors, 695
in eggs, 856
in feeds, 87-88
in fertilizers, 12—17
in flour, 779
in food dressings, 1005
in fruits and fruit products, 916-917
in gelatin, 929
in infant formulas, 1106-1107,
1112-1113
in liming materials, 4, 7
in meat, 933-935
in meat extracts and similar
products, 947
microchemical determination,
344-345
in plants, 40, 42, 56
in vinegars, 1007-1008
in water, 328-330
in wines, 743-744
Phosphorus pentoxide
in liming materials, 4
Phosphotungstic acid
safe handling of, 654, 1228
Photoelectric colorimetric method
chlorophyll in plants, 62
Photofluorometric methods
reserpine in feeds, 108
ethoxyquin residues in animal
tissues, 628-629
Photometric methods
benzoyl peroxide bleach in flour,
784
color of beer, 708
color of laboratory wort, 727
color of white wines, 739
coumarin in vanilla extract, 892-893
phosphorus in feeds, 88
phosphorus in water, 328-329
Phthalic acid derivatives
in color additives, 1132
Phthalocyaninato (2-) copper, 1122
Physostigmine, 535, 546, 591, 606
Physostigmine alkaloids, 590-592
Physostigmine salicylate, 580,
590-591, 606
Physostigmine sulfate, 580, 590-591,
606
Phytate
in foods, 800-801
Phytotoxins, 1213
Pickles
filth in, 405
Pickling spice
filth in, 398
Picloram
in formulations, 196
Picric acid
safe handling of, 651, 1225
Pigment
in flour, 785
in plants, 62-63
separation and identification, 1115
Pilocarpic acid, 598
Pilocarpine, 535, 546, 598, 606
Pilocarpine HCI, 580
Pilocarpine nitrate, 580
Pimiento extract, 905-906
Pineapple
fith in canned juices, 392-393
mold in juices, 418
Pinene
in lemon and orange oils, 903
Pinto beans
piperonyl butoxide pesticide residues
in, 309
Piperazine
in drugs, 533
in feeds, 106-107
Piperine
in pepper preparations, 1002-1003
Piperonyl butoxide
in Alaska peas, barley, hulled rice,
oats, pinto beans, and wheat,
309
in formulations, 170, 172
Pirimicarb
in formulations, 219-220
Pistachio nuts
aflatoxins in, 1195
Plant materials
carotenes in, 1048
in vanilla extract, 895-896
xanthophylls and carotenes in,
1048-1049
Plants
aluminum in, 40, 43
arsenic in, 50
ash of, 40
barium in, 40
boron in, 40, 42, 50
calcium in, 40, 42, 43
carotenes in, 63
Certified Reference Materials, 646,
1220
chloride in, 50-51
chlorophyll in, 62—63
cobalt in, 43-45
copper in, 40, 42, 45-46
ether extract of, 59
fiber (crude) in, 59
fluoride in, 51-56
fructose in, 59
glucose in, 59
iron in, 40, 42, 43, 46
lignin in, 60^62
1-40
Subject Index
AOAC Official Methods of Analysis (1990)
magnesium in, 40, 42, 46
manganese in, 42, 46-47
metals in, 40-50
moisture in, 40
molybdenum in, 40, 47
nitrogen in, 59-60
phosphorus in, 40, 42, 56
pigments in, 62-63
potassium in, 40, 42, 47-48
sample preparation, 40, 58-59
sampling of, 40
sand in, 40
selenium in, 57
silica in, 40
sodium in, 40, 47-48
starch in, 60
strontium in, 40
sucrose in, 59
sugars in, 58-59
sulfur in, 58
zinc in, 40, 42, 48-50
Plate counts
Bacillus cereus in foods, 464-466
Clostridium perfringens in foods,
462-464
microorganisms in eggs and Qgg
products, 427
Platinic chloride method
potassium in plants, 48
Platinum recovery procedure
for potassium in fertilizers, 23
Plato table
degrees Brix, specific gravity, and
degrees Baume of sugar
solutions, 1260-1265
Plums
azinphos-methyl pesticide residues
on, 294
captan pesticide residues in, 296
Podophyllum, 602
Poisons (natural)
aflatoxin M„ 1199-1207
aflatoxins, 1186-1207
marine toxins, 1213
mycotoxins, 1184-1185
ochratoxins, 1207-1209
patulin, 1209-1210
phytotoxins, 1213
sterigmatocystin, 1210-1211
zearalenone in, 1211-1213
Polarimetric methods
camphor in drugs, 602
glucose in sugars and sirups, 1016
lactose in milk, 810
raffinose in sugars and sirups,
1015-1016
sucrose in maple products, 1034
sucrose in molasses, 1022
sucrose in sugars and sirups,
1012-1016
RRR- or <2//-raoct-tocopherol in
drugs and food or feed
su pplements , 1 074- 1 075
Polarization
of honey, 1027
of maple products, 1034
of vinegars, 1008
Polarization methods
commercial glucose in wine, 741
malic acid in fruits and fruit
products, 919, 920-921
oils of lemon, lime, and orange in
oil-base flavors, 900
starch in flour, 783
Polarographic methods
fumaric acid in food, 1137
bismuth compounds in drugs, 502
guaifenesin in drugs, 550
lead in fish, 257-258
organophosphorus pesticide residues,
289-290
Polenske values
acids (volatile) in oils and fats, 958
Poliovirus 1
in oysters, 495-496
Polychlorinated biphenyls
in paper and paperboard, 285-286
Polycyclic aromatic hydrocarbons
in food, 1176-1178
Polymers
of heated vegetable oils, 967-968
Polysorbate 60
in shortening, oils, and dressings,
1163
Polythiazide, 573, 578
Polyvalent flagellar test tube test
Salmonella in foods, 476
Polyvalent somatic slide or plate test
Salmonella in foods, 475-476
Pomeroy-Kirshman-AJsterberg
method
oxygen (dissolved) in water,
315-316
Ponceau 3R, 1115
Ponceau SX, 1115, 1119
Popcorn
filth in, 393
Poppy seed
filth in, 398-399
foreign matter in, 398
Pork
filth in sausage, 390-391
Potassium
in baking powders, 688-689
in beer, 722-723
with calcium and sodium in
electrolyte replenishes,
503-504
in distilled liquors, 695
in fertilizers, 23-27
in fruits and fruit products, 915
in infant formulas, 1106-1107, 1110
in plants, 40, 42, 47-48
safe handling of, 651, 1225
in seafood, 870-871
in soaps, 162
in tobacco, 64-65
in water, 327-328
in wines, 744
Potassium bromate
with caffeine in drugs, 504
in cold wave neutralizers, 366
in elixir, 504-505
Potassium bromide-bromate, 643,
1217
Potassium cyanate formulations, 159
Potassium cyanide formulations, 159
Potassium dichromate, 643, 1217
Potassium hydroxide
safe handling of, 651, 1225
Potassium iodide
in feeds, 84
in ointments, 506
Potassium permanganate, 643, 1218
Potassium thiocyanates, 640, 1214
Potatoes
calcium in canned products,
991-992
ethylenethiourea pesticide residues
in, 300-302
filth in chips, 393
filth in dehydrated products, 397
N-methylcarbamate residues in,
292-294
moisture in frozen french fries, 998
naphthyleneacetic acid pesticide
residues in, 306-307
organophosphorus pesticide residues
on, 287-289
Potentiometric methods
acidity of beer, 711-712
chloride in cheese, 842
chloride in milk-based formula, 1113
chlorides in tobacco, 64
chlorides in water-soluble color
additives, 1135-1136
chlorine in feeds, 85
diastatic activity of honey, 1033
dicofol in formulations, 189
fentin in pesticide formulations,
156-157
fluoride in plants, 51-52
fluorides in hazardous substances,
232-233
liming materials, 1-2
monosodium glutamate in food,
1174-1175
nitrate in forages, 357-358
pH of cacao products, 764
pH of flour, 780
quaternary ammonium compounds in
formulations, 228
salt (chlorine as sodium chloride) in
seafood, 870
sodium chloride in canned
vegetables, 992-993
Poultry
adulteration of meat products, 948
calcium in mechanically separated
meat, 941
decoquinate residues in chicken
tissues, 628
ethoxyquin residues in chicken
tissues and eggs, 628-629
fat (crude) in poultry products, 932
filth and sand in chicken giblet
paste, 389
moisture in poultry products, 931
AOAC Official Methods of Analysis (1990)
Subject Index
1-41
nalidixic acid in chicken liver and
muscle, 631
Powdered milk
allatoxin M, in, 821
Power-Chesnut method
caffeine in roasted coffee, 758
caffeine in tea, 761
Precipitation method
mercury in organic mercurial seed
disinfectants, 162
Prednisolone, 616-617, 619
Prednisone, 616-618, 619
Premixes
arprinocid in, 94
furazolidone in, 99—101
roxarsone in, 108-110
Preservatives
benzoic acid in food, 1141-1144
borates in food, 1144-1145
boric acid in food, 1144-1147
boron in caviar, 1145
in butter, 840
in cream, 833
dehydroacetic acid in cheese,
1147-1148
diethylcarbonate in food, 1147
diethylcarbonate in wines, 750
in evaporated milk, 833
fluorides in food, 1148-1149
formaldehyde in food, 1149
formic acid in food, 1 149
in ground beef, 1144
hydrogen peroxide in milk, 1149
in milk, 820
monochloroacetic acid in liquids and
preservatives , 1 1 49- 1 1 50
monochloroacetic acid in
nonalcoholic beverages and
wines, 1150-1151
nitrites in curing preparations, 1 151
propionates in food, 1151
quaternary ammonium compounds in
food and beverages, 1151-1155
salicyclic acid in food and
beverages , 1 1 55- 1 156
sorbic acid in dairy products,
1156-1157
sorbic acid in food, 1143-1144
sorbic acid in wines, 749
sulfites in food, 1159-1160
sulfurous acid in food, 1157-1159,
1160
sulfurous acid in wines, 749
thiourea in fruits and fruit products,
1161-1163
in wines, 749-750
see also Food additives (direct); and
specific analyte
Press cake
filth in, 373-374
Primidone, 576, 578
Probarbital, 537, 546
Procainamide HC1, 524-525, 546
Procaine, 524, 535, 606
Procaine HCL, 524, 546, 580
Procaine penicillin
in feeds, 127
Process cheese
lactose in, 846
Progestational steroids, 612-613
Proline
in honey, 1026-1027
Promethazine, 536, 546
Propachlor
in formulations, 196
Prophenpyridamine, 579-580
Propionates
in food, 1151
Propionic acid
in bread, 791-793
in cake, 795
in seafood, 874
Propoxur
technical and formulations, 220
Propoxycaine, 524
Propoxycaine HC1, 524, 546
Propoxy!
on apples, cabbage, collards, corn
kernels, green beans, kale, and
turnip tops, 291-292
n-Propyl alcohol
in distilled liquors, 700-701
Propyl gailate
in foods, 1137-1138, 1140-1141
in oils and fats, 1138-1139
Propylene glycol
in cigarette filler and ground
tobacco, 65
in drugs, 499
in vanilla extract, 890-891
and water in cosmetics, 360
Propylthiouracil, 574, 578
Protein
in baked products, 795
in beer, 713
in bread, 793
in brewing sugars and sirups, 733
in cereal adjuncts, 731
in dried milk, 834
efficiency ratio, 1095-1098
in flour, 781-782
in fruit products, 917
in grains, 788
in ice cream and frozen desserts,
851
in macaroni products, 797
in malt, 727
in milk, 808-809, 816-818
in milk chocolate, 772
in sweetened condensed milk, 834
in tea, 762
in yeast, 737
Protein (crude)
in feeds, 70-76
in meat, 937
in nuts and nut products, 949
in plants, 59-60
Protein nitrogen unit precipitation
method
allergenic extracts in drugs, 604-605
Protein-reducing substances
in milk, 809-810
Proteolytic activity
of flour and malted wheat flour,
786-787
Proteolytic chsllproofing enzymes
in beer, 719
Proximate analysis
of milk -based formula, 1113
Prunes
moisture in, 912-914
Pseudoephedrine, 540
Pseudoephedrine HO, 587-588
Pseudomonas aeruginosa
disinfectant efficacy against, 135, 137
Psicose
in com sirups and sugars,
1042-1043
Pteroylglutamic acid
in vitamin preparations, 1083-1084
Pycnometer methods
alcohol by volume in distilled
liquors, 692
specific gravity of beer, 709
specific gravity of distilled liquors,
691-692
specific gravity of lemon and orange
oils, 898, 901
specific gravity of lemon, orange,
and lime extracts, 898
specific gravity of milk, 805
specific gravity of oils, 951-952
specific gravity of vanilla extract,
890
specific gravity of water, 312
specific gravity of wines, 739
weight (apparent) per unit volume of
fats and oils, 951-952
Pyoverdine
in eggs, 862-863
Pyranine concentrated, 1120
Pyrantel tartrate
in feeds, 107
Pyrethrin
in formulations, 170-172
Pyridine
safe handling of, 654, 1228
Pyrilamine maleate, 539, 546
Pyrilamine, 513, 579-580
Pyrogallol
in hair dyes, 366
Quaternary ammonium compounds
in aqueous solutions and milk,
1154-1155
in commercial preservatives,
1151-1153
in eggs, 863
in foods and beverages, 1153-1154
in formulations, 228
in milk, 1151
in nonalcoholic beverages, 756
Quinacrine HC1, 593, 606
Quinalizarin method
boron in plants, 50
Quinic acid
in cranberry juice cocktail and apple
juice, 920-921
1-42
Subject Index
AOAC Official Methods of Analysis (1 990)
Quinidine, 535, 546
Quinine
ethylcarbonate, 592, 606
with acetanilid and caffeine, 555
chemical name, 578
with diacetylmorphine in drug
powders, 620
with iron and strychnine in elixirs,
592-593
with morphine in drugs, 555
Quinizarin green SS, 1120
Quinoline yellow SS, 1122
Quinoline yellow WS, 1122
Quinolinium molybdophosphate
methods
phosphorus in fertilizers, 13-14, 15,
16-17
phosphorus in plants, 56
Quisumbing-Thomas methods
invert sugar in sugars and sirups,
1017
sugars in plants, 59
Racephedrine, 535, 540
Racephenicoi
in feeds, 108
Radioactive chemicals
safe handling of, 654, 1228
Radioactive tracer methods
7-BHC (lindane) in formulations,
178
Radioactivity
barium- 140 in milk, 353-355
cesium- 137 in milk and other foods,
353-355
iodine- 131 in milk and other foods,
353-355
radioactive contamination,
emergency level measures, 355
of solid substances, 349
strontium-89 and -90 in milk,
351-353
strontium-90 in water, 349-351
tritium in water, 349
Raffinose
in sugars and sirups, 1015-1016
Raffinose hydrate
refractive indices of solutions, 1281
Raisins
filth in, 393
moisture in, 912-914
Raney powder method
nitrogen in fertilizers, 19
Rapid method
potassium in plants, 48
Raspberries
concentrates, 907
insects in frozen products, 391-392
mold in, 418
Rat bioassay
protein efficiency ratio, 1095-1096
vitamin D in milk, vitamin
preparations, and feed
concentrates, 1091-1094
Rat hemoglobin repletion bioassay
iron bioavailability, 1098-1099
Rauwolfia alkaloids, 593-598
Rauwolfia serpentina drugs, 595-597
RDX
in wastewater and groundwater,
334-336
Red peppers
filth in, 398-401, 405
Reducing substances
in molasses, 1023-1024
Reduction method
nitroglycerin in drugs, 526
Reference standards
aflatoxins, 1186-1187
Certified Reference Materials,
645-648, 1219-1222
mycotoxins, 1185-1186
Refraction method
alcohol in wines, 739
Refractive index
of butterfat, 837
of lemon and orange oils, 901
Refractive index method
solids (soluble) in tomato products,
990-991
Refractometer methods
alcohol by volume in distilled
liquors, 693
alcohol in beer, 710-711
solids (soluble) in fruits and fruit
products, 915
Reichert-MeissI values
acids (volatile) in oils and fats, 958
of cacao fat, 771
Reineckate method
quaternary ammonium compounds in
commercial preservatives,
1152-1153
Renard test
peanut oil in oils and fats, 978
Rescinnamine
in drugs, 595-598, 606
Reserpine
in drugs, 593-597, 606
in feeds, 108
Resorcinol
in hair lotions, 366
Rhodamine B, 1121
Rhodamine B stearate, 1121
Riboflavin
assays, 1080-1082
in foods and vitamin preparations,
1052-1054
in ready-to-feed milk-based
formulas, 1107
in vitamin preparations, 1086-1087
Rice
breakfast cereals, antioxidants in,
791
filth in flours, products, and paper,
384-385
filth in rice cereals, 387-388
hulled, piperonyl butoxide pesticide
residues in, 309
Roberts copper method
dextran in raw cane sugar,
1020-1021
Robertson method
Jones modification of, 20
nitrogen in fertilizers, 20
Roese-Gottleib method
fat in cream, 832
fat in dried milk, 835
fat in evaporated milk, 833
fat in ice cream and frozen desserts,
851
fat in milk, 811
fat in sweetened condensed milk,
834
fat in whey cheese, 844
Romer minicolumn method
aflatoxins in foods and feeds,
1187-1188
Ronnel
in feeds, 108
Rosemary
extract, 905-906
filth in, 399
foreign matter in, 398
Rosin oil
in oils and fats, 977
Rot
in foods, 416-418
in tomato products, 422-423
Rot fragment count, 422-423
Rotenone
in derris and cube powder, 168-170
Roxarsone
in feeds and premixes, 108-110
Rubber articles
extractives from, 1 180
Rutin, 603, 606
Sabadilla alkaloids
in formulations, 173
Saccharides (major)
in corn sirups and sugars,
1042-1043
Saccharides (minor)
in corn sirups and sugars, 1044
Saccharin
in food, 1172-1174
in nonalcoholic beverages,
1167-1168, 1174
Saccharometers
temperature corrections for, 1271
Saffron
in macaroni products, 798-799
Safrole
in nonalcoholic beverages, 754-755
Sage
filth in, 399, 402-403
foreign matter in, 398
Salad dressings
filth in, 405
gums in, 1006-1007
Salad oils
cold test, 979
Salicylamide, 554-555, 578
Salicylates
cinchophen in presence of, 599
Salicylic acid
chemical name, 546, 578
in drugs in presence of other
phenols, 548
in food and beverages, 1155-1156
AOAC Official Methods of Analysis (1990)
Subject Index
I-43
in hair lotions, 366
microchemical tests, 539
Salmon
canned, ethanol in, 880-881
Salmonella
biochemical identification, 473-475
in candy and candy coatings,
476-478
in casein, 470-471
in cheese powders, 478-480
in chocolate, 470-471, 478-480
in coconut, 476-478
colorimetric monoclonal enzyme
immunoassay screening method,
480-484
colorimetric polyclonal enzyme
immunoassay screening method,
484-486
culture media and reagents, 467-470
DNA hybridization screening
method, 488-490
in eggs and egg products, 470-471,
476-480
in fish and other marine products,
476-478
fluorogenic monoclonal enzyme
immunoassay screening method,
486-488
in frog legs, 476-478
in garlic powder, 470-471
identification in foods, 471-473
in low-moisture foods, 484
in meats and meat products,
476-480
in milk, 470-471, 476-478
in milk powders, 478-480
motile, in foods, 490-492
in nonfat dry milk, 470-471,
476-478
in onion powder, 470-471
in pepper, 478-480
serological tests, 475-476
in yeast (dry inactive), 476-478
Salmonella choleraesuis
disinfectant efficacy against,
135-136
Salmonella typhi
disinfectant efficacy against,
133-135
Salt
calcium in, 333-334
chlorine as sodium chloride in
seafood, 870
in butter, 837
in cheese, 842
in meat and meat products, 933
iodine in iodized salt, 334
magnesium in, 334
matters insoluble in, 332-333
moisture in, 322
reporting results, 334
sample preparation, 332
sulfate in, 333
Sample collection
cheese, 840
cream, 831
milk, 804-805
Sample preparation
acids (dibasic) in nonalcoholic
beverages, 751
aflatoxins, 1185
arsenic in water, 331
baking powders and baking
chemicals, 685
beer, 708
boric acid in water, 331
bread, 790-791
bromine in water, 331
butter, 836-837
cacao products, 763
canned vegetables, 987
cheese, 840-841
confectionery, 1024
corn sirups and sugars, 1039
cream, 831
dried milk, 834
evaporated milk, 833
feeds, 69, 91
fertilizers, 10-11
fish and marine products, 864
food dressings, 1004
fruits, 911
grains, 788
honey, 1025-1026
hops, 732
ice cream and frozen desserts, 851
iodine in water, 331
liming materials, 1, 4, 6
macaroni products, 796
malt, 724
manganese in water, 331
maple products, 1034
meat and meat products, 931
meat extracts and similar products,
947
milk, 805
nuts and nut products, 949
oils and fats, 951
peat, 36
pesticide formulations, 147
phosphorus in fertilizers, 12, 15, 16
plants, 40, 58-59
prepared mustard, 1003
roasted coffee, 758
salicyclic acid in food and
beverages, 1155
salt, 332
spices, 999
for sugars and sirups, 1010
sweetened condensed milk, 833
tea, 761
vinegars, 1007
Sampling
aflatoxins, 1185
ammoniacal solutions, 9
anhydrous ammonia, 9-10
butter, 803-804, 836
cereal adjuncts, 730
cheese, 804
condensed milk, 803
dairy products, 802-803
dried milk and its products, 803
dried milk, 834
drugs, 498
eggs and egg products, 427, 853
evaporated milk, 803, 833
feeds, 69, 91
fertilizers (liquid), 9
fertilizers (solid), 9
flour, 777
fruits, 910
hops, 731-732
infant formula, 1107
liming materials, 1
milk and liquid milk products, 803
malt, 723-724
molasses, 1021
peat, 36
plants, 40
pesticide formulations, 147
sweetened condensed milk, 833
yeast, 735-736
Sand
in chicken giblet paste, 389
in ground pepper, 405
in peat, 37-38
in plants, 40
in spices and condiments, 398
Sanitizing activity
of disinfectants, 138-140
Santonica, 604
Santonin, 603-604, 606
Saponification methods
cacao fat, 772
menthol in drugs, 602
Saponification number
of oils and fats, 957
Sauerkraut
filth in, 397
Sausage
water (added) in, 931
Savory
filth in, 399
foreign matter in, 398
Scallops
shucked, volume of, 867
Schaeffer's salt
in FD&C Red No. 40, 1127-1128
in FD&C Yellow No. 6, 1130-
1132
Scintillation spectrometric method
tritium in water, 349
Scopolamine, 535, 546
Secobarbital, 537, 546
Secobarbital sodium, 559, 578
Sedatives, see Hypnotics and sedatives
Sediment test method
milk, 375-378
Sedimentation methods
chicken excrement and filth in eggs
and egg products
filth in capsicums, 401
filth in nuts, 379
filth in tea, 375
maggots in blueberries and cherries,
392
Sedimentation/flotation methods
filth in coffee and coffee sunstitutes,
374
filth in garlic powder and ground
onion, 402
1-44
Subject Index
AOAC Official Methods of Analysis (1990)
Sediments
Certified Reference Materials, 648,
1222
in milk, 821
Seed disinfectants
formaldehyde in, 226
organic mercurial, 162-163
Seeds
in berry fruits, 914
light filth in, 380
Selenium
in food, 237-239, 269-270
in plants, 57
Semiautomated methods
chlorpheniramine maleate in drug
tablets, 513-514
ferrous sulfate in drugs, 507-508
nitroglycerin in sublingual drug
tablets, 529
prednisolone or prednisone in drugs,
616-617
reserpine in drugs, 595
vitamin C in food, 1060-1061
Semimicro method
fat in fish meal, 872
Serological flagellar screening test
Salmonella identification in foods,
471-473
Serological test
Salmonella in foods, 475-476
Serum
copper in, 357
Sesame oil
in oils and fats, 979
Shaffer-Somogyi micro method
glucose in sugars and sirups, 1017
Shellac
in confectionery, 1025
Shellfish growing water
fecal coliforms and Escherichia coli
in, 436-437
Shortenings
glycerides in, 985-986
1-monoglycerides in, 984-985
polysorbate 60 in, 1163
trans isomers in, 969-970
Shrimp
filth in canned products, 390
frozen, drained weight of, 867
indole in, 877-879
in shrimp cocktail, 867 — 868
Sichert-Bleyer method
glucose in cacao products, 774-775
Sieving methods
filth in cinnamon, 402
filth in sauerkraut, 397
filth in starch, 385
foreign matter in spices and
condiments, 398
insect eggs in flour, 384
shell in cacao products, 764
sorting corn grits, 730
Sigma value method
foam collapse rate of beer, 714-715
Silica
in face powders, 365
in liming materials, 4
in plants, 40
in water, 322
Silicon
in liming materials, 6, 7-8
Silicon dioxide
in liming materials, 4
Silicotungstic acid method
nicotine in tobacco products,
173-174
Silver
in water, 324-325
Silver diethyidithiocarbamate method
arsenic in food, 245
Silver iodate
safe handling of, 654, 1228
Silver nitrate
safe handling of, 654, 1228
standard solutions of, 644, 1218
Silver number
for coconut and palm kernel oils,
772
Silver protein
in drugs, 512
Single-tablet assay
progestational steroids in drugs,
612-613
reserpine in drugs, 594-595
Sirups
filth in, 394
p -Sitosterol
in butter oil, 975-976
Slags
neutralizing value for, 3
sulfide sulfur in, 3-4
Snack foods
extraneous materials in, 393
Soaps
moisture in, 162
sodium and potassium in, 162
Soda beverages
benzoate, caffeine, and saccharin in,
752
Soda lye
carbonate and hydroxide in, 232
Sodium
in baking powders, 688-689
in beer, 723
with calcium and potassium in
electrolyte replenishers,
503-504
in chloramine T, 161
in distilled liquors, 695
in fertilizers, 33-34
in foods for special dietary use, 1099
in fruits and fruit products, 915-916
in infant formulas, 1106-1107, 1110
in plants, 40, 47-48
safe handling of, 651, 1225
in seafood, 870-871
in soaps, 162
in water, 328
in wines, 744
Sodium acetate
in halogenated fluorescein colors,
1136
Sodium acid pyrophosphate,
685-686
Sodium aluminum phosphate
neutralizing value, 686
Sodium benzoate
in soda beverages, 752
Sodium bicarbonate
cinchophen in presence of, 599
Sodium biphenyl
safe handling of, 651, 1225
Sodium biphenyl reduction method
chlorine in organohalogen pesticides,
175
Sodium bromide
in elixir, 504-505
Sodium cacodylate, 501, 546
Sodium carbonate
in soda lye, 232
Sodium chloride
in acid dyes, 1136
in ash of bread, 793
in canned vegetables, 992-993
in nuts and nut products, 950
Sodium cyanide formulations, 159
Sodium cyclamate
in canned fruit, 1168-1169
Sodium ethySate
safe handling of, 651, 1225
Sodium fluosilicate
in pesticide formulations, 152
Sodium halides
in halogenated fluorescein colors,
1136
Sodium hydroxide
safe handling of, 651, 1225
in soda lye, 232
in sodium hypochlorite, 161
solution strengths, 658
standard solutions, 644-645,
1218-1219
Sodium hypochlorite
in pesticide formulations, 160-161
Sodium lauryl sulfate
in egg white, 1163-1164
Sodium methylate
safe handling of, 651, 1225
Sodium perborate
in cold wave neutral izers, 366
Sodium peroxide
safe handling of, 651, 1225
Sodium peroxide method
sulfur in plants, 58
Sodium saccharin
in nonalcoholic beverages,
1167-1168
in soda beverages, 752
Sodium salicylate, 555, 578
Sodium sulfate
in acid dyes, 1136
Sodium TCA
in formulations, 197
Sodium tetraphenylboron method
potassium in fertilizers, 26-27
Sodium thiosuifate, 645, 1219
Soft curd cheese
lactose in, 846-847
Soil
in frozen fruits and vegetables, 393
in spinach (frozen), 396
AOAC Official Methods of Analysis (1990)
Subject Index
I-45
Soil acidifiers
aluminum in, 36
Solids
in baked products, 795
in bread, 791
in canned peas, 990
in canned vegetables, 989, 990
in cordials and liqueurs, 705-706
in cream, 831
in eggs, 853
in evaporated milk, 833
in flour, 777
in food dressings, 1004
in frozen peas, 996
in frozen spinach, 995
in fruits and fruit products, 914, 915
in ginger extract, 905
in ice cream and frozen desserts,
851
in lemon, orange, and lime extracts,
901
in macaroni products, 796
in maple products, 1034
in milk, 807, 816-818, 1296-1297
in nonalcoholic beverages, 755
in prepared mustard, 1003
in processed tomato products, 990
in roasted coffee, 760
in seafood, 868
in sirups, 1011-1012
in sweetened condensed milk, 834
in tomato products, 990-991
in vanilla extract, 894
in vinegars, 1007, 1008-1009
in water, 317
in wines, 741
in yeast, 736
see also Moisture; Water
Soluble acids
in oils and fats, 957
Soluble matter
in color additives, 1136
Solvents
in drugs, 498-499
Somatic cells
in milk, 496-497, 805
Somatic grouping
Salmonella in foods, 476
Sorbic acid
in cheese, 1156-1157
in cottage cheese, 847
in dairy products, 1156-1157
in food, 1143-1144
in wines, 749
Sorbitol
in food, 1167
Sorensen method
nitrogen (amino) in meat, 941
Sound spectrographic method
voice print identification, 639
Sour cream
filth in, 378-379
Soxhlet extraction method
fat in cacao products, 770-771
Soy protein
in raw and heat-processed meat
products, 942-945
Soybean flour
ash in, 791
fiber (crude) in, 791
filth in, 385
in meat, 942
moisture in, 791
nitrogen in, 791
in uncooked cereal products, 788
Soybean oil
peanut oil in, 978-979
Soybeans
aflatoxins in, 1195
Sparteine, 535, 546
Spearmint extract, 905
Spearmint leaves
filth in, 399, 400
Species identification
beef and poultry adulteration of meat
products, 948
canned Pacific salmon, 887-889
Specific conductance
of water, 312
Specific gravity
of beer, 709
of canned vegetables, 991
of cordials and liqueurs, 705
of distilled liquors, 691
of lemon and orange oils, 901
of lemon, orange, and lime extracts,
898
of milk, 805
of molasses, 1022
of nonalcoholic beverages, 755
of oils, 951-952
of vanilla extract, 890
of water, 312
of wines, 739
Specific gravity methods
alcohol in beer, 710
alcohol in lemon, orange, and lime
extracts, 898
fat (crude) in meat, 932
Spectinomycin
in feeds, 127-128
Spectrophotofiuorometric methods
estradiol valerate in drugs, 607
lasalocid in feeds, 131
nalidixic acid in chicken liver and
muscle, 631
nequinate in feeds, 103
reserpine-rescinnamine alkaloids in
Rauwolfia serpentina drugs,
595-597
Spectrophotometry absorbance
characteristics
lemon and orange oils, 901-902
Spectrophotometric methods
acetaminophen and salicylamide in
drugs, 554
acetaminophen in drugs, 553-554
2-acetylamino-5-nitrothiazole in
feeds, 91
acids in hops, 732-733
adulteration of processed Florida
orange juice, 929-930
alkomide in feeds, 91
p-aminobenzoic acid in feeds, 92
2-amino-5-nitrothiazole in feeds, 92
/^-aminosalicylic acid and isoniazid
in drugs, 549
amphetamine drugs, 520
amprolium in feeds, 92-93
amyl p-dimethylaminobenzoate in
suntan preparations, 367-368
ANOT in animal tissues, 625
antihistamines in drugs in presence
of aspirin, phenacetin, and
caffeine, 515
antimony in food, 242-243
arprinocid in premixes, 94
arsanilic acid in premixes, 94
arsenic in animal tissues, 626
arsenic in feeds, 356
aspirin and phenobarbital in drugs,
557
azinphos-methyl pesticide residues,
294
bendroflumethiazide in drugs, 570
benzocaine and antipyrene in drugs,
522-523
benzoic acid in nonsolid food and
beverages, 1141-1142
benztropine mesylate in drugs,
583-584
betaine in orange juice, 917-918
bithionol in feeds, 95
biuret in fertilizers, 22-23
boric acid in food, 1146-1147
boron in fertilizers, 29-30
butabarbital sodium in drugs,
561-562
cadmium in feeds, 95
caffeine in nonalcoholic beverages,
752-754
calcium pantothenate in vitamin
preparations, 1079
captan pesticide residues, 296
carbadox in feeds, 95-96
carotenes and xanthophylls in dried
plant materials and mixed feeds,
1048
carotenes in fresh plant materials and
silages, 1048
chloral hydrate in drugs, 562
chloramben in formulations, 182
chlorogenic acid in green coffee, 757
p-chlorophenyl phenyl sulfone
pesticide residues, 298
chlorophyll in plants, 62-63
codeine and terpin hydrate in elixirs,
580-581
color in spices, 999
color of beer, 708
color of distilled liquors, 690
color of laboratory wort, 727
color of raw cane sugars, 1010
coloring matter in distilled liquors,
690-691
colors (synthetic) in oils and fats, 982
conjugated estrogens in drugs, 607
dehydroacetic acid in cheese,
1147-1148
dexamethasone phosphate in drugs,
614
1-46
Subject Index
AOAC Official Methods of Analysis (1990)
diacetyl morphine and quinine in
drug powders, 620
dichlone pesticide residues, 298-299
dichlorophene in drugs, 529-530
dienestrol in drugs, 609-610
diethylstilbestrol in drugs, 609
diethylstilbestrol in feeds, 97
digitoxin in drugs, 599-600
digoxin and total digitoxosides in
drugs, 601
dimetridazole in feeds, 97-98
diquat in formulations, 225
dodine pesticide residues in fruits,
299
dyes in color additives, 1125
ephedrine in solid dosage drugs,
586-587
esters in distilled liquors, 697-698
ethinyl estradiol in drugs, 607-608
formaldehyde in maple sirup,
1037-1038
fusel oil in distilled liquors, 699-700
glyodin pesticide residues in fruits,
302
griseofulvin, 130-131
hexachJorocyclopentadiene in
technical chlordane, 183
hexachlorophene in deodorants,
363-364
hexestrol in drugs, 610
hydroxymethylfurfural in honey,
1031
intermediates in FD&C No. 1, 1126
iron in drugs, 506-507
iron in flour, 778-779
lactic acid in fruits and fruit
products, 921
lactic acid in milk and milk
products, 806-807
maleic hydrazide pesticide residues,
303-304
mannitol hexanitrate and
phenobarbitol in drugs, 527-528
menadione sodium bisulfite in drugs,
567
mephentermine sulfate in drugs,
512-513
mestranol in drugs, 611
mestranol with ethynodiol di acetate
in drugs, 611-612
methapyrilene in expectorants, 513
cis, ds-methylene interrupted
polyunsaturated fatty acids in
oils, 969
methyl salicylate in drugs, 551-552
mixed color, zinc in plants, 48-49
molybdovanadophosphate, for
phosphorus in fertilizers, 12-13,
16, 17
naphthyleneacetic acid pesticide
residues, 306-307
neostigmine methylsulfate in drugs,
591-592
niacinamide in multivitamin
preparations, 1057-1058
nicarbazin in feeds, 103
nicotine in feeds, 103-104
nicotine residues, 307-308
nifursol in feeds, 104
nitarsone in feeds, 105
nithiazide in feeds, 105
nitrodan in feeds, 106
nitromide in feeds, 106
nitrophenide in feeds, 106
norepinephrine in epinephrine
preparations, 515-516
papain proteolytic activity, 1165
paraquat in formulations, 227-228
pentaerythrityl tetranitrate in drugs,
528
phenobarbital and aminophylline in
drugs, 560
phenobarbital and phenytoin in
drugs, 560-561
phenobarbital and theobromine in
drugs, 561
phenol sulfonates in deodorants,
364-365
phenothiazine in feeds, 106
phenylalkanolamine salts in elixirs
and sirups, 519-520
phenylethylamines in drugs, 521
phenylpropanolamine HC1 in drugs,
519
phenytoin sodium in drug capsules,
562
phosphatase in casein, 824—825
phosphorus in color additives,
1134-1135
phosphorus in milk-based infant
formula, 1112-1113
pigment in flour, 785
piperazine in feeds, 106-107
piperine in pepper preparations,
1002-1003
piperonyl butoxide pesticide
residues, 309
poly cyclic aromatic hydrocarbons
and benzo[a]pyrene in food,
1176-1178
poly thiazide in drugs, 573
polyunsaturated acids in oils and
fats, 960-963
preservatives in ground beef, 1 144
procainamide HO in drugs, 524-525
progestational steroids in drugs, 612
propylthiouracil in drugs, 574
protein-reducing substances in milk,
809-810
pyrantel tartrate in feeds, 107
pyrilamine in cough sirup, 513
quinine in drugs, 592
rescinnamine in drugs, 597-598
reserpine in drugs, 593-594
reserpine-rescinnamine alkaloids in
Rauwolfia serpentina drugs, 597
ronnel in feeds, 108
roxarsone in feeds and premixes,
108-109
rutin in drugs, 603
single color, zinc in plants, 49-50
sorbic acid in dairy products, 1156
sorbic acid in wines, 949
subsidiary dyes in FD&C Yellow
No. 5, 1132
subsidiary dyes in FD&C Yellow
No. 6, 1132
sulfadiazine and sulfamerazine,
568-569
suifaguanidine in feeds, 111
sulfanitran in feeds, 111-112
sulfaquinox aline in feeds, 112
sulfonamides in feeds, 112-115
sulfoxides in formulations, 230-231
tannin in distilled liquors, 703
thiazide drugs, 572-573
thiourea in frozen fruit, 1161-1162
thiram pesticide residues, 309-311
titanium in cheese, 271
4-toluene-azo-2-naphthol-
3-carboxylic acid, 1132
trisulfapyrimidines in drugs,
567-568
uric acid in flour, 415
vitamin A in margarine, 1045
warfarin in rodenticides, 229-230
zoalene in feeds, 115
zoalene residues in animal tissues,
635
see also Atomic absorption
spectrophoto metric methods;
Infrared spectrophotometric
methods
Spectrophotometric
molybdovanadate method
phosphorus in fruits and fruit
products, 916-917
Spectroscopic methods
strontium in water, 324
see also Infrared spectroscopic
methods
Spicer-Edwards flagellar test tube
test
Salmonella in foods, 476
Spices and other condiments
acidity (total) of food dressings,
1005
acidity of prepared mustard, 1003
adulterants in spices, 1002
alcohol extract of spices, 1000
alginates in food dressings, 1007
ash of spices, 1000
chlorides in prepared mustard, 1003
color in spices, 999
copper-reducing substances in spices,
1000
ether extract of prepared mustard,
1003
ethylene dichloride and
trichloroethylene in spice
oleoresins, 1175
fat (total) in food dressings, 1005
fiber (crude) in prepared mustard,
1004
fiber (crude) in spices, 1000
filth in spices, 397-407
food dressings, 1004
foreign matter in, 398
AOAC Official Methods of Analysis (1990)
Subject Index
I-47
gums in mayonnaise and French
dressing, 1005-1006
gums in salad dressing, 1006-1007
methylene chloride extract of spices,
1000
moisture in spices, 999
mold in, 420
nitrogen (total) in food dressings,
1005
nitrogen in nonvolatile ether extract
of pepper, 1000
nitrogen in prepared mustard, 1003
nitrogen in spices, 1000
oil (volatile) in mustard seed,
1001-1002
oil (volatile) in spices, 1001
oil in food dressings, 1005
permanganate oxidation number, 1009
phosphorus (total) in food dressings,
1005
piperine in pepper preparations,
1002-1003
prepared mustard, 1003
sample preparation, 999
solids (total soluble) in vinegar,
1008-1009
solids (total) in food dressings, 1004
solids in prepared mustard, 1003
starch in food dressings, 1007
starch in prepared mustard, 1004
starch in spices, 1000
sucrose in food dressings, 1005
sugars (reducing) after inversion in
food dressings, 1005
sugars (reducing) before inversion in
food dressings, 1005
tannin in cloves and allspice, 1000
vinegars, 1007-1008
Spinach
carbaryl pesticide residues in,
297-298
ethylenethiourea pesticide residues
in, 300-302
nicotine residues in, 307-308
soil in frozen products, 396
solids (total) in frozen products, 995
Spiral plate method
bacteria in foods and cosmetics, 431
Spiral vessel count
of cacao products, 766-767
Spirits, 690-704
Sporeforming organisms
in low-acid canned foods, 458-459
Spores
in sugars, 458
in tomato products, 423
Sporicidal activity
of disinfectants, 141-142
Spot test identification
gums in drugs, 604
Squalene
in oils and fats, 972
Standard reference color method
beer, 708
Standard solutions
of ammonium and potassium
thiocyanates, 640, 1214
of arsenious oxide, 640, 1214
buffer solutions for calibration of pH
equipment, 640-641,
1214-1215
buffers and indicators for
colorimetric pH comparisons,
641-642, 1215-1216
of hydrochloric acid, 642-643,
1216-1217
of iodine, 643, 1217
and materials, 640, 1214
of metals, 41
mixed element, 41
of potassium bromide-bromate, 643,
1217
of potassium dichromate, 643, 1217
of potassium permanganate, 643,
1218
of silver nitrate, 644, 1218
of sodium hydroxide, 644-645,
1218-1219
of sodium thiosulfate, 645, 1219
of sulfuric acid, 645, 1219
of titanium trichloride, 645, 1219
Staphylococcal enterotoxin
in foods, 451-455
Staphylococci
hemolytic, in eggs and egg products,
428
Staphylococcus
in chilled, frozen, precooked, or
prepared foods, 429-430
Staphylococcus aureus
disinfectant efficacy against, 135,
136-137
in foods, 449-451
Starch
in baking powders, 687
in beer, 713
in brewing sugars and sirups, 734
in cacao products, 775
in cereals, 789
in confectionery, 1024
dessert powders, 930
in face powders, 365
in feeds, 83-84
filth in, 385
in flour, 783
in food dressings, 1007
in fruits and fruit products, 922-923
in meat, 946-947
in peanut butter, 950
in plants, 60
in prepared mustard, 1004
in roasted coffee, 760
in spices, 1000
Starch gel-zone electrophoresis
method
identification of fish species, 883
Starchy flour
in meat, 941
Steam distillation method
acidity of fruit products, 918
Steam distillation-
spectrophotometric method
furfural in distilled liquors, 701
Stearic acid
in face powders, 365
Steinhoff methods
glucose in corn sirups and sugars,
1042
Stereochemical composition
hydralazine HC1, 545
Sterigmatocystin
in barley and wheat, 1210-1211
Sterility (commercial)
of canned, low-acid foods, 455-459
Steroids
adrenocortico, 613-618
optical crystallographic properties of,
664-665
progestational, 612-613
refractive indices for, 670
Sterol acetate melting point method
vegetable fats in butterfat, 973-974
Sterols
in baked products, 795
in bread, 793
in macaroni products, 797-798
Stone cell and group count
of cacao products, 767-770
Stramonium alkaloids, 583
Strawberries
dodine pesticide residues in, 299
mold in frozen products, 419
organochlorine and
organophosphorus pesticide
residues in, 282-283
organophosphorus pesticide residues
on, 287-289
thiram pesticide residues in,
309-311
Streptococci
hemolytic, in eggs and egg products,
428
Streptomycin
in feeds, 128
in milk, 829-831
Strontium
in plants, 40
in water, 324
Strontium-89
in milk, 351-353
Strontium-90
in milk, 351-353
in water, 349-351
Strychnine
chemical name, 547, 606
in drugs, 592, 598-599
microchemical tests, 535
nitrate, 580, 606
sulfate, 580, 606
titration factors, 580
Sublimation method
saccharin in food, 1174
Substituted urea pesticides
chlorotoluron, 216-217
chloroxuron, 216-217
fluometuron, 218
metoxuron, 216-217
propoxur, 220
1-48
Subject Index
AOAC Official Methods of Analysis (1990)
Succinic acid
in eggs, 858-859, 861-862
Sucrose
in cacao products, 773-774
in confectionery, 1024
in cordials and liqueurs, 706
density of solutions, 1295
in eggs, 856-857
in feeds, 83
in food dressings, 1005
in fruits and fruit products, 922
in gelatin, 930
in honey, 1028-1030
in lemon, orange, and lime extracts,
901
in maple products, 1034
in milk chocolate, 773
in molasses, 1022
in nonalcoholic beverages and
concentrates, 756
in nuts and nut products, 950
per cent in sugar solutions, 1280
in plants, 59
in presweetened cereals, 789-790
refractive indices of solutions,
1273-1279
in sugar beets, 1039
in sugars and sirups, 1012-1016
in sweetened condensed milk, 834
temperature corrections for per cent
in sugar solutions, 1283
in vanilla extract, 894
volume factors for thermal
expansion, 1295
in wines, 741
Sugar beets
sucrose in, 1039
Sugar inversion methods
glucose and sucrose in eggs,
856-857
nonsugar solids in wines, 741
Sugar solutions
degrees Brix, specific gravity, and
degrees Baume, 1260-1265
specific gravity by per cent extract
by weight, 1266-1270
Sugars and sugar products
arabinose in sugars and sirups, 1019
ash of sugars and sirups, 1012
in baked products, 795
in bread, 794
brewing sugars and sirups, 733-735
in canned vegetables, 993
color of raw cane sugars, 1010
confectionary, 1024-1025
corn sirups and sugars, 1039-1044
dextran in raw cane sugar,
1020-1021
extraneous materials in, 393-394
in feeds, 83
in flour, 780-781
in food dressings, 1005
fructose in sugars and sirups,
1018-1019
in fruits and fruit products, 922
galactose in sugars and sirups, 1019
glucose in sugars and sirups, 1016,
1017-1018
honey, 1025-1033
in honey, 1027-1028
invert sugar in sugars and sirups,
1016-1017
lactose in sugars and sirups, 1019
in licorice extracts, 909
maltose in sugars and sirups, 1019
maple, sap, maple sirup, and maple
sirup products, 1034-1039
moisture in sugars, 1010-1011
molasses and molasses products,
1021-1024
nitrogen in sugars and sirups, 1012
in nonalcoholic beverages, 756
in nuts and nut products, 950
in plants, 58-59
purity of lactose in sugars and
sirups, 1019-1020
raffinose in sugars and sirups,
1015-1016
reducing sugars in wines, 741
reducing sugars required for reduction
of Soxhlet solution, 1284-1285
reducing, in beer, 712
in roasted coffee, 760
sample preparation for sugars and
sirups, 1010
solids in sirups, 1011-1012
sucrose in sugars and sirups,
1012-1016
sugar beets, 1039
sugars and sirups, 1010-1021
thermophilic bacterial spores in, 458
in vinegars, 1008
xylose in sugars and sirups, 1019
see also specific sugars
Sulfabromomethazine, 634-635, 636
Sulfadiazine, 539, 547, 567-569, 578
Sulfadimethoxine, 634-635, 636
Sulfaguanidine
in feeds, 111
Sulfamerazine
chemical name, 578
in drugs, 567-569
in feeds, 112-114
Sulfamethazine
in animal tissues, 634-635
chemical name, 578, 636
in drugs, 567-568
in feeds, 111, 112-114
in milk, 829-831
in swine, 631-634
Sulfamethoxazole, 569, 578
Sulfamethoxine
in feeds, 110-111
Sulfanilamide, 539, 547, 569, 578
Sulfanilic acid
in FD&C Yellow No. 5, 1128-1129
in FD&C Yellow No. 6, 1130-1132
Suifanitran
in feeds, 111-112
Sulfapyridine, 539, 634-635, 636
Sulfapyridine sodium monohydrate,
539, 547
Sulfaquinoxaline
chemical name, 636
in drugs, 634-635
in feeds, 92, 112-114
Sulfates
in baking powders, 688-689
in salt, 333
in vinegars, 1008
in water, 330-331
in wines, 744
Sulfated ash
in color additives, 1134
Sulfathiazole
in animal tissues, 634-635
chemical name, 636
in drugs, 539, 547
in feeds, 112-114
Sulfide sulfur
in calcium silicate slags, 3-4
Sulfides
in deodorants, 363
in depilatory powders, 365
in lime sulfur formulations, 160
Sulfisoxazole, 569-570, 578
Sulfites
in food, 1159-1160
in meat, 1 159
Sulfonamides
in animal tissues, 634-635
in drugs, 567-570
in feeds, 112-114
optical crystallographic properties of,
665-666
refractive indices for, 670-671
Sulfonmethane/sulfonethyimethane,
563, 578
Sulfoxides
in formulations, 230-231
Sulfur
in ash of fruits and fruit products,
917
in color additives, 1125
in fertilizers, 34
in fruit products, 917
in lime sulfur, 159-160
microchemical methods, 345-347
in plants, 58
Sulfur amino acids
in food and feed ingredients,
1102-1103
Sulfur dioxide
in beer, 718
in food, 1157-1158, 1160
in meat and meat products, 947
safe handling of, 654, 1228
Sulfur-containing drugs
allopurinol in drug tablets, 575-576
diethylpropion HC1 in drug substance
and tablets, 576-577
disulfram in drug tablets, 574
methimazole in drugs, 573-574
phenothiazine in drugs, 574-575
primidone in drug tablets, 576
propylthiouracil in drugs, 574
thiouracil in drugs, 574
see also Sulfonamides
AOAC Official Methods of Analysis (1 990)
Subject Index
I-49
Sulfuric acid
safe handling of, 651, 1225
solution strengths, 658
standard solutions of, 645, 1219
Sulfurous acid
in dried fruit, 1157-1159
in food, 1157-1159
in meats, 1160
in wines, 749
Sulprofos
in formulations, 210-211
Sunset yellow FCF, 1115, 1119
Suntan preparations, 367-368
Surface plating method
Staphylococcus aureus in foods,
440-451
Sweep codistillation method
organophosphorus pesticide residues
on fruits and vegetables,
287-289
Sweetened condensed milk
ash of, 834
fat in, 834
lactic acid in, 834
lactose in, 834
protein in, 834
sample preparation, 833
sampling, 833
solids (total) in, 834
sucrose in, 834
Swimming pool disinfectants
efficacy, 145-146
Swine
sulfamethazine residues in, 631-634
Sympathomimetic drugs
microchemical tests, 536, 540
optical crystallographic properties of,
666
refractive indices for, 671
Synthetic drugs
amitriptyline in tablets and
injectables, 525-526
dichlorophene in, 529-530
fluorescein sodium in, 532-533
mannitol hexanitrate in, 527-528
meprobamate with pentaerythrityl
tetranitrite in, 528-529
metheneamine in tablets, 530-532
metheneamine mandelate with
metheneamine in, 531-532
methocarbamol in tablets and
injectables, 526
methylene blue in, 531-532
microchemical tests, 536, 538-541
nitrate esters in, 527
nitroglycerin in dosage forms,
526-527, 529
pentaerythrityl tetranitrite in,
528-529
phenazopyridine HC1 in drugs, 525
phenobarbitol with mannitol
hexanitrate in, 527-528
piperazine in, 533
2,4,5-T
in formulations, 196-197
Talbutal, 537, 547
Tamarind pulp
filth in, 406
Tannin
in cloves and allspice, 1000
in distilled liquors, 703
in wines, 746
Tarragon
filth in, 399
Tartaric acid
in baking powders, 687
in cheese, 844-845
in cordials and liqueurs, 706
in fruits and fruit products, 918
in nonalcoholic beverages, 751
in tartrate powders, 686-687
in wines, 745
Tartrate powders
tartaric acid in, 686-687
Tartrazine
in macaroni products, 799
in foods , 1115
methods for, 1119
TDE
multiresidue methods, 279
Tea
ash in, 761
caffeine in, 761-762
copper and nickel in, 242
copper and nickel, 762
fiber (crude) in, 762
filth in, 375
moisture, 762
nickel in, 269
petroleum ether, 762
protein in, 762
sample preparation, 761
water extract of, 762
Tea seed oil
in oilve oil, 979
Temefos
in formulations, 211
Temperature correction
specific gravity of oils, 952
TEPP
in formulations , 211-212
Terbuthylazine
in formulations, 220-221
Terpin hydrate, 580-581, 606
Tertiary butyl alcohol
in distilled liquors, 701-702
Tetrabromofluorescein, 1121
Tetrachloroethylene
in drugs, 499
Tetracycline
in milk, 829-831
Tetradifon
in apples and cucumbers, 284-285
in formulations, 197
Tetrasul
in apples and cucumbers, 284-285
Thallous sulfate
in rodenticides, 163-164
Theobromine, 561, 578, 584, 606, 776
Theobromine-calcium salicylate
drugs, 584
Theophylline, 541, 547, 584, 606
Thermistor method
water (added) in milk, 819-820
Thermoelectric-vapor pressure
method
molecular weight, 337
Thermophilic bacterial spores
in sugars, 458
Thiabendazole
in feeds, supplements, and premixes,
114-115
Thiamine
in bread, 1051-1052
in foods, 1049-1051
in grain products, 1051
in milk-based formula, 1113-1114
in milk-based infant formula,
1113-1114
in vitamin preparations, 1091
Thiazides, 570-573
Thiethylperazine, 536, 547
Thin layer chromatographic methods
aflatoxin h { in eggs, 1195-1197
aflatoxin Mj in cheese, 843
aflatoxin M] in dairy products,
1199-1201
aflatoxin M, in milk, 821
aflatoxin Mj in milk and cheese,
1200-1201
aflatoxins B, and M { in liver,
1201-1203
aflatoxins in coconut, 1191
aflatoxins in corn, 1191
anatoxins in cottonseed products,
1192-1193
anatoxins in green coffee, 1195
anatoxins in pistachio nuts, 1195
anatoxins in soybeans, 1195
alkomide in feeds, 91-92
benzoic acid in nonsolid food and
beverages, 1141-1143
chlorotoluron, chloroxuron, or
metoxuron in formulations,
216-217
deoxynivalenol in wheat, 1205-1207
dexamethasone in drug substance
and elixirs, 614-615
erucic acid in oils and fats, 965-967
excrement on foods and containers,
414-415
FD&C color additives in foods,
1117-1118
navoring additives in vanilla extract,
896
high fructose corn sirup in honey,
1031-1032
hydrocortisone in drugs, 613-614
identification of anatoxin Bj, 1197
mammalian feces in heatrprocessed
materials, 413-414
nonnutritive sweeteners in
nonalcoholic beverages,
1167-1168
ochratoxin A in green coffee, 1208-
1209
ochratoxins in barley, 1207-1208
1-50
Subject Index
AOAC Official Methods of Analysis (1990)
organochlorine and
organophosphorus pesticide
residues, 275, 280-282
organochlorine pesticide
contamination of pesticide
formulations, 152-153
patulin in apple juice, 1209-1210
standards for aflatoxins, 1186-1187
sterigmatocystin in barley and wheat,
1210-1211
sulfonamide residues in animal
tissues, 634-635
sulfonamides in feeds , 113-114
urine on foods and containers,
411-412
zearalenone in corn, 1211-1212
Thin layer chromatographic-
spectrophotometric methods
biphenyl pesticide residues in citrus
fruits, 295-296
preparation of standards for
mycotoxins, 1185-1186
Thin layer polyacrylamide gel
isoelectric focusing method
generic identification of cooked and
frozen crabmeat, 886, 889
identification of fish species,
883-884
Thiocarbamates
in herbicide formulations, 221
organic sprays, 228
Thiocyanogen number
of oils and fats, 957
Thiolgiycolate solutions
in cold permament waves, 366
Thiosulfate
in lime sulfur formulations, 160
Thiouracil, 574, 578
Thiourea
in frozen peaches, 1161-1162
in orange juice, 1160-1161
in orange peel, 1162-1163
Thiram
in apples, celery, corn, strawberries,
and tomatoes, 309-311
in formulations, 222
Thonzylamine HC1
in dosage forms, 515, 547
Thrips
in frozen blackberries and
raspberries, 391-392
Thujone
in cordials and liqueurs, 707
Thyme
filth in, 399
foreign matter in, 398
oil, 905-906
Thymol, 552-553, 578
Thyroid drugs, 618-619
Tin
in food, 270-271
Titanium
in cheese, 271, 842
in face powders, 365
in liming materials, 7
Titanium dioxide
in liming materials, 4
Titanium oxides
in face powders, 365
in liming materials, 4, 7
Titanium trichloride, 645, 1219
Titer test
for oils and fats, 954-955
Titrimetric methods
acid value of butterfat, 837
acidity of brewing sugars and sirups,
734
acidity of cheese, 842-843
acidity of food dressings, 1005
acidity of honey, 1033
acidity of milk, 805
acidity of prepared mustard, 1003
acidity of water, 313
acids (volatile) in oils and fats, 958
aldehydes as acetaldehyde in frozen
vegetables, 996-997
aldehydes in distilled liquors,
698-699
alkalinity of ash of cacao products, 7
alkalinity of ash of dry skim milk,
835
alkalinity of ash of wines, 742
alkalinity of water, 313-314
alkoxyl groups, determination,
347-348
allethrin, 164-166
aluminum and iron in plants, 43
amitrole in formulations, 223-224
amphetamine drugs, 520
ct-amyJase of malt, 729
arsenic in pesticide formulations,
149
arsenic in plants, 50
arsenic in sodium cacodylate, 501
barium in water, 327
benzoic acid in food, 1141-1142
benzoic and salicylic acids in drugs,
548
bicarbonate in water, 322
boric acid in food, 1146
boron in fertilizers, 28-29
calcium and magnesium in drugs,
502-503
calcium in beer, 715, 716
calcium in canned vegetables,
991-992
calcium in fertilizers, 28-29
calcium in flour, 779
calcium in mechanically separated
poultry and beef, 941
calcium in plants, 43
calcium silicate slags, 3-4
calcium, phosphorus and iron in
vitamin preparations, 504
calomel in dosage forms, 511
carbon dioxide in wines, 748-749
carbonate and hydroxide in soda lye,
232
carbonate in water, 322
carbromal in drugs, 558-559
catalase in frozen vegetables,
997-998
cation exchange capacity for peat,
39
chemical oxygen demand of water,
316-317
chenopodium oil in drugs, 602
chloride in distilled liquors, 696-697
chloride in plants, 50-51
chlorides in prepared mustard, 1003
chlorine in calcium hypochlorite and
bleaching powder, 161
chlorine in feeds, 85
cholesterol in eggs, 855-856
cobalamin in vitamin preparations.
1082-1083
copper in copper naphthenate
formulations, 155
copper in fertilizers, 31
dalapon (sodium salt) in
formulations, 185
diacetylmorphine in drug tablets,
620
diastatic power of malt, 727-729
dithiodiglycolic acid in cold
permament waves, 366-367
dodine in formulations, 225-226
dyes in color additives, 1123-1124
esters and aldehydes in distilled
liquors, 697
esters in lemon oil, 903
fat acidity of grains, 788-789
fatty acids (free) in crude and
refined oils, 957
fatty acids and butyric acid in butter,
837
fertilizers, 1.9
fluorine determination, 341-342
folic acid in vitamin preparations,
1083-1084
guaiacol in drugs, 550
hardness of water, 323
hexylresorcinol in drugs, 551
hydrocyanic acid in beans, 1213
iodine in dosage forms, 506
iodine in iodized salt, 334
iron in fertilizers, 31
iron in plants, 46
iron in water, 323
isopropanol in lemon and orange
flavors, 899
isopropanol in lemon extracts, 899
Koettstorfer number of oils and fats,
957
lactose in bread, 794
lead number (Wichmann) of vanilla
extract, 893-894
liming materials, 1-2, 4-5
lipid phosphorus in flour, 782-783
magnesium in fertilizers, 32, 33
malic acid in fruits and fruit
products, 918
martius yellow in color additives,
1132
mercurous iodide in tablets, 511
mercury in organic mercurial seed
disinfectants, 162-163
methanol in vanilla extract, 894
methenamine in deodorants, 364
methenamine in tablets, 530-531
methylene blue in drugs, 531-532
AOAC Official Methods of Analysis (1990)
Subject Index
1-51
moisture in liquid molasses,
1021-1022
monochloroacetic acid in,
1150-1151
neutralizing values of baking
chemicals, 685-686
niacin and niacinamide in vitamin
preparations, 1084-1085
nitrites in curing preparations, 1 151
nitrogen (ammonia) in water, 320
nitrogen (total) in water, 319
opium alkaloids, 579
oxygen (dissolved) in water,
315-316
oxyquinoline sulfate in drugs, 551
pamaquine in drugs, 593
pantothenic acid in vitamin
preparations, 1085-1087
papain proteolytic activity,
1164-1165
parathion in formulations, 208
peroxidase in frozen vegetables, 998
peroxide value of oils and fats, 956
phenazopyridine HCL in drugs, 525
phosphorus in fruits and fruit
products, 916
potassium in fertilizers, 26-27
propylene glycol in vanilla extract,
890-891
protein in feeds, 74
quinacrine HCL in drugs, 593
quinine ethyl carbonate in drugs, 592
recoverable oils in fruits and fruit
products, 923
reducing substances in molasses,
1023-1024
salt in butter, 837
salt in meat, 933
salt in seafood, 870
selenium in food, 270
silver protein in drugs, 512
sodium chloride in nuts and nut
products, 950
sodium hypochlorite in pesticide
formulations , 1 60- 1 6 1
sodium TCA in formulations, 197
squalene in oils and fats, 972
starch in meat, 946-947
starch in plants, 60
subsidiary dyes in D&C Red Nos. 6
and 7, 1132
sugars in flour, 780-781
sulfides in depilatory powders, 365
sulfur determination, 345, 347
sulfurous acid in meats, 1160
tannin in cloves and allspice, 1000
TEPP in formulations, 211-212
thymol in dosage forms, 552-553
tin in food, 270
tragacanth drugs, 604
urea in deodorants, 365
vitamin assays, 1081-1082
TNT
in wastewater and groundwater,
334-336
Tobacco
alkaloids in, 65-66
chlorides in, 64
glycerol in, 65
moisture in, 64
nicotine in, 65-66, 173-174
nitrogen in, 64
potassium in, 64-65
propylene glycol in, 65
triethylene glycol in, 65
4-Toluene-azo-2-naphthol-
3-carboxylic acid
in D&C Red Nos. 6 and 7, 1132
Tomatoes
benzoic acid in products, 1142
calcium in canned products,
991-992
filth in canned and comminuted
products, 397
mold in processed products,
419-420
organochlorine and
organophosphorus pesticide
residues in, 282-283
rot in communited products,
422-423
solids (soluble) in processed
products, 990
solids (total) in processed products,
990
specific gravity of canned products,
991
thiram pesticide residues in,
309-311
yeasts and spores in processed
products, 423
Toney Red, 1121
Total chloride method
tetrachloroethylene in drugs, 499
Toxic dusts
safe handling of, 654, 1228
Toxic solvents
safe handling of, 652, 1226
Toxicity
of aflatoxin B,, 1199
ot-Toxin estimation method
Clostridium perfringens in foods,
463-464
TPTZ colorimetric method
iron in distilled liquors, 696
Tragacanth drugs, 604
Tranquilizers
optical crystallographic properties of,
667
refractive indices for, 671
Trans isomers
in margarines and shortenings,
969-970
Travers method (modified)
fluorine in pesticide formulations,
151
Triadimefon
technical and formulations, 228-229
Triamino-s-triazine
in fertilizer mixes, 22
Triazine pesticides
anilazine, 213-214
metribuzin, 219
terbuthylazine, 220-221
triazines, 222-223
Trichloroacetic acid
safe handling of, 654, 1228
Trichioroethylene
in spice oleoresins, 1175
in wheat and corn grain, 290-291
Trichophyton mentagrophytes
disinfectant efficacy against, 138
Triethylamine
safe handling of, 653, 1227
Triethylene glycol
in cigarette filler and ground
tobacco, 65
Trifluoroacetic acid
safe handling of, 654, 1228
Triflupromazine, 536, 547
Trifluralin
in formulations, 179-180
Triglycerides
in oils and fats, 972-973
2,4,5-Trihydroxybutyrophenone
in oils and fats, 1138-1139
Trimethobenzamide HC1, 542-543,
547
Trimethylamine nitrogen
in seafood, 869-870
Tripelennamine HO, 539, 547
Triprolidine HC1, 587-588, 606
Tristearin
in lard, 979-980
Trisulfapyrimidines, 567-568
Tritium
in water, 349
Trolamine, 539, 547
Tropane alkaloids, 583-584
Tryptophan
in foods and food and feed
ingredients, 1101-1102
Tuberculocidal activity
of disinfectants, 142-143
filth in, 398-399
Tumeric
foreign matter in, 398
Turbidimetric methods
chlortetracycline HC1 in feeds,
119-120
cobalamin in ready-to-feed milk-
based formulas, 1110-1112
cobalamin in vitamin preparations,
1083
critical temperature of dissolution of
oil from butter or margarine,
838
fat in milk, 815-816
folic acid in vitamin preparations,
1083-1084
monensin in feeds, 123-124
niacin and niacinamide in ready-to-
feed milk-based formulas, 1109
niacin and niacinamide in vitamin
preparations, 1084-1085
pantothenic acid in vitamin
preparations, 1086, 1087
sulfate in water, 330-331
vitamin assays, 1082
Turnip greens
filth in, 395-396
1-52
Subject Index
AOAC Official Methods of Analysis (1990)
Turnip tops
JV-methylcarbamate residues on,
291-292
Tylosin
in feeds, 90, 128-129
Ultraviolet light examination
urine on foods and containers, 407
Ultraviolet screening method
vanillin in vanilla extract, 891
Ultraviolet spectrophotometric
methods
benfluralin in formulations, 179-180
benzaldehyde in almond extract, 904
caffeine in instant tea, 761-762
santonin in drug mixtures, 603-604
trifluralin in formulations, 179-180
vanillin in vanilla extract, 891
7-Undecalactone
in cordials and liqueurs, 707
in nonalcoholic beverages, 756
Unsaponifiahle matter
in cocoa butter, 771
in eggs, separation of, 855
Unsaponifiable residue
of flour, 783
of macaroni products, 797
of oils and fats, 971-972
Uranine, 1122
Uranyl acetate
safe handling of, 654, 1228
Uranyl acetate method
sodium in plants, 48
Urea
in deodorants, 365
in feeds, 76-77
in fertilizers, 21-22
2-isopropyl-4-oentenoyl, in drugs,
563
urease test, 407-409
xanthydrol test, 409
Urease methods
urea and ammoniacal nitrogen in
feeds, 76
urea in fertilizers, 21
Urease test
Salmonella identification in foods,
471-473
urea on foods and containers,
407-409
Urease- bromothymol blue-agar test
urine on grain, 410-411
Uric acid
in flour, 415, 788
on foods and containers, 414
Urine
on foods and containers, 407-409,
411-412
on grain, 410-411
Use-dilution methods
disinfectant testing, 135-137
Vacuum oven methods
moisture and volatile matter in oils
and fats, 951
moisture in roasted coffee, 760
solids and moisture in flour, 777
soilds and moisture in macaroni
products, 796
solids in yeast, 736
solids (total) in eggs, 853
Vacuum-desiccation method
water in fertilizers, 11-12
Valeric acid
in bread, 792-793
in seafood, 874
Van Slyke method
nitrogen (amino) in meat, 941
Vanilla extract and its substitutes
alcohol in, 890
ash of, 894
color in, 894-895
coumarin in, 892-893
ethyl vanillin in, 891-893
flavoring additives in, 896
glycerol in, 890
lead number (Wichmann) of,
893-894
methanol in, 894
nonvanillin vanilla volatiles in, 898
organic acids in, 896-898
plant material (foreign) in, 895-896
propylene glycol in, 890-891
solids (total) in, 894
specific gravity of, 890
sucrose in, 894
vanilla resins in, 894
vanillin in, 891-893
Vanilla resins
in vanilla extract, 894
Vanillin
in vanilla extract, 891-893
Vanishing cream, 368
Vapor pressure osmometric method
water (added) in milk, 820
Vegetable fats
in butterfat, 973-975
Vegetable oils
animal fats in, 976-977
in nonalcoholic beverages,
1166-1167
polymers and oxidation products of
heated oils, 967-968
Vegetable products (canned)
acids (total) in, 993
ash of, 991
calcium in, 991-992
drained weight of, 987
field corn in mixtures of field and
sweet corn, 994
lactic acid in, 994
oxalic acid in, 993-994
pH of acidified foods, 988-989
sample preparation of, 987
sodium chloride in, 992-993
solids (alcohol-insoluble) in canned
peas, 990
solids (insoluble) in, 990
solids (soluble) in, 990
solids (soluble) in tomato products,
990-991
solids (total) in, 989
solids (total) in processed tomato
products, 990
specific gravity of, 991
sugars in, 993
water activity of, 987-988
Vegetable products (dried)
water in, 994-995
Vegetable products (frozen)
aldehydes as acetaldehyde in,
996-997
catalase in, 997-998
fibrous material in green beans, 996
moisture (loss of mass on drying) in
french-fried potatoes, 998
net contents of food containers, 995
peroxidase in, 998
solids (alcohol-insoluble) in peas,
996
solids (total) in spinach, 995
thawing, 995
Vegetable tissues .
in feeds, identification of, 89
Vegetables and vegetable products
extraneous materials in, 394-397
filth in flakes, 399
filth in green leafy vegetables,
395-396
mold and rot in, 419-422
soil in frozen products, 393
see also Green vegetables; and
specific vegetables
Vernolate
in herbicide formulations, 221
Veterinary analytical toxicology
arsenic in feeds, 356
copper in liver, 356-357
copper in serum, 357
nitrate in forages, 357-358
Vibrio cholerae
in oysters, 492-494
Villavecchia test
sesame oil in oils and fats, 979
Vinbarbitai, 537, 547
Vinegars, 1007-1009
Virus
in beef, 494-495
Viscometer methods
viscosity of beer, 710
Viscosity
of acidulated flour- water suspension,
787-788
of beer, 710
of fruit products, 912
Vitamin A
in margarine, 1045
in mixed feeds, premixes, and foods,
1045-1047
Vitamin AD concentrates
vitamin D in, 1067-1068
Vitamin B 1? see Thiamine
Vitamin B 2 , see Riboflavin
Vitamin B 6
in food extracts, 1089-1091
in ready-to-feed milk-based
formulas, 1107-1108
Vitamin B 12
activity in vitamin preparations,
1082-1083
Vitamin C
AOAC Official Methods of Analysis (1990)
Subject Index
I-53
in food, 1060-1061
in juices, 1058-1059
in ready-to-feed milk-based
formulas, 1108-1109
in vitamin preparations, 1058-1060
Vitamin D
in fortified milk and milkpowder,
1068-1069
in milk, vitamin preparations, and
feed concentrates, 1091-1094
in mixed feeds, premixes, and pet
foods, 1069-1070
in multivitamin preparations,
1066-1067
in vitamin AD concentrates,
1067-1068
in vitamin preparations, 1061-1065
Vitamin D 3
in poultry feed supplements,
1094-1095
Vitamin E
in drugs, 1078-1079
nomenclature rules, 1070-1071
tocopherol isomers in mixed
tocopherol concentrate, 1076
a-tocopherol and a-tocopherol
acetate in foods and feeds,
1071-1074
RRR- or all-rac-a-tocopheroi in
drugs and food or feed
supplements, 1074-1075
tocopheryl acetate in supplemental
vitamin E concentrates,
1077-1078
a-tocopheryl acetate (supplemental)
in foods and feeds, 1075-1076
Vitamin K
menadione sodium bisulfite in feed
premixes, 1079-1080
Vitamin preparations
amino acids in, 1087-1089
calcium pantothenate in, 1079
cobalamin in, 1082-1083
folic acid in, 1083-1084
niacin and niacinamide in,
1084-1085
pantothenic acid in, 1085-1086
pteroylglutamic acid in, 1083-1084
riboflavin in, 1086-1087
thiamine HC1 in 1091
vitamin B 12 activity in, 1082-1083
vitamin C in, 1058-1060
vitamin D in, 1061-1065,
1091-1094
Vitamins and other nutrients
bioassay methods, 1091-1099
calcium, phosphorus and iron in
vitamin preparations, 504
chemical methods, 1045-1080
in enriched bread, 793
in infant formula, 1 106-1 1 14
microbiological methods, 1080-1091
nutritionally related components,
1049-1106
see also specific vitamins and
nutrients
Voice print identification, 639
Volatile esters
in cordials and liqueurs, 706-707
Volatile matter
in color additives, 1125
in oils and fats, 951
Volatility
of herbicides, 153
Volatilization method
mercury in organic mercurial seed
disinfectants, 162
Volhard titrimetric method
chloroform or carbon tetrachloride in
drugs, 499
effervescent potassium bromide with
caffeine in drugs, 504
Volume
of peat, 38-39
Volumetric thiosulfate methods
copper in Bordeaux mixtures, 158,
159
copper in copper carbonate
formulations, 155
copper in Paris green pesticide
formulations, 154
Warfarin
in rodentieides, 229-230
Warfarin potassium, 565-566, 578
Warfarin sodium, 565-566, 578
Wastewater
TNT, RDX, HMX, and 2,4-DNT in,
334-336
Water
acidity, 313
activity of canned vegetables,
987-988
alkalinity, 313-314
aluminum in, 322, 324
in anhydrous ammonia, 9-10
arsenic in, 331, 332
barium in, 327
bicarbonate in, 322
biochemical oxygen demand,
314-315
boric acid in, 331, 332
bromide in, 331-332
bromine in, 331
cadmium in, 324-325
calcium in, 324
capacity of peat, 38, 39
carbonate in, 322
certified reference materials for, 648,
1222
chemical oxygen demand, 316-317
chloride in, 320-321
chromium in, 324-325
copper in, 324-325
in dried vegetables, 994-995
and ethyl alcohol in cosmetics, 359
extract of tea, 762
in fertilizers, 11-12
fluoride in, 321-322
hardness, 323
hydrogen sulfide in, 322
iodide in, 331-332
iodine in, 331
iron in, 322-323, 324-325
lead in, 324-325
magnesium in, 324-325
manganese in, 331
mercury in, 326-327
nitrogen in, 318-320
organic carbon in, 317-318
oxygen (dissolved) in, 315-316
pH, 312-313
phosphorus in, 328-330
potassium in, 327-328
reporting results, 332
saline, 317
silica in, 322
silver in, 324-325
solids in, 317
specific conductance, 312
specific gravity, 312
strontium in, 324
strontium-90 in, 349-351
sulfate in, 330-331
tritium in, 349
zinc in, 324-325
see also Wastewater; Groundwater
Water (added)
in cream, 832
in milk, 818-820
in sausage, 931
Weevils
in beans and peas, 394
Weight (apparent) per unit volume
of fats and oils, 952
Wet ashing methods
calcium in feeds, 84-85
lipids and lipid phosphorus in eggs,
854-855
Wet digestion method
potassium in fertilizers, 23
Wet sieving
liming materials, 1
Wheat
deoxynivalenol in, 1205-1207
filth in, 381-382
filth in whole wheat cereals, 388
fumigant residues in grain, 290-291
piperonyl butoxide pesticide residues
in, 309
sterigmatocystin in, 1210-1211
Wheat flour
a-amylase in flour, meal, and malted
cereals, 786
ash of, 777-778
benzoyl peroxide bleach (benzoic
acid) in, 784
bromates and iodates in white and
whole wheat flour, 784-785
calcium in, 779
carbon dioxide (total) in self-rising
flour, 779-780
chlorine in fat of, 783
diastatic activity of, 785-786
extract (cold-water soluble) of, 777
fat (crude) of ether extract in, 780
fat acidity, 780
fat in, 780
fiber (crude) in, 780
fiber (total dietary) in, 780
1-54
Subject Index
AOAC Official Methods of Analysis (1990)
inorganic material (added) in
phosphated flour, 778
iron in, 778-779
lipid phosphorus in, 782-783
lipids in, 782
moisture in, 777
nitrogen (nitrite) in, 783-784
pH of, 780
phosphated and self-rising, ash of,
778
phosphorus in, 779
pigment in, 785
protein in, 781-782
proteolytic activity of flour and
malted wheat flour, 786-787
sampling of, 777
solids (total) in, 777
starch in, 783
sugars in, 780-781
unsaponifiable residue of, 783
uric acid in, 788
viscosity of acidulated flour-water
suspension, 787-788
Wheat germ
filth in, 383-384
Wheat gluten
filth in, 385
Whey cheese
fat in, 844
WHO-AOAC method
cadmium and lead in cookware, 241
Wijs method
iodine absorption number of oils and
fats, 956
Wiley method
melting point of fats and fatty acids,
953-954
WiHard-Winter distillation method
fluoride in plants, 52
Williams field test
alcohol by volume in distilled
liquors, 693-694
Wines
acidity of, 744-745
alcohol in, 739-741
aldehydes in, 747
alkalinity of ash of, 742
p-asarone in, 750
ash of, 742
caloric content of, 742
carbohydrate content of, 742
carbon dioxide in, 747-749
carmel in, 747
chlorides in, 743
citric acid in, 746
copper in, 743
coumarin in, 750
cyanide in, 749
diethylcarbonate in, 750
dry, glycerol in, 741
extract of, 741
flavors in, 750
fructose and glucose in, 741-742
glucose in, 741
iron in, 743
lactic acid in, 746
malic and citric acids in, 746
monochloroacetic acid in,
1150-1151
nitrogen in, 746
nonsugar solids in, 741
pentosans in, 747
pH of, 744
phosphorus in, 743-744
physical examination, 739
potassium and sodium in, 744
preservatives in, 749-750
sodium in, 744
sorbic acid in, 749
specific gravity, 739
sucrose in, 741
sugars (reducing) in, 741
sulfates in, 744
sulfurous acid in, 749
sweet, glycerol in, 741
tannin in, 746
tartaric acid in, 745
white, color of, 739
Winter green extract, 905
Winton lead number
of maple products, 1035-1036
Wort
characteristics, 734-735
extract from beer, 710
laboratory, color of, 727
specific gravity of, 709-710
Xanthine alkaloids, 541, 584
Xanthophylls
in dried plant materials and mixed
feeds, 1048-1049
Xanthoproteic test
protein in feeds, 70
Xanthydrol test
urea, 409
Xylose
in sugars and sirups, 1019
Yeast
count for maple sirup, 1038
protein in, 737
Salmonella in, 476-478
sampling of, 735-736
solids (total) in, 736
in tomato products, 423
Yellow AB, 798-799, 1115
Yellow OB, 798-799, 1115
Yohimbine, 535, 547
ZDBT colorimetric method
copper in distilled liquors, 695
a-Zearalenol
in corn, 1212-1213
Zearalenone
in corn, 1211-1213
Zerban-Sattler method
glucose in cacao products, 774
Zinc
in deodorants, 361-362
in face powders, 365
in feeds, 84
in fertilizers, 27-28, 34-35
in food, 237-239, 272-273
in infant formulas, 1106-1107, 1110
in pesticide formulations, 150
in plants, 40, 42, 48-50
in water, 324-325
Zinc arsenite
Bordeaux mixtures with, 149-150
formulations, 149, 155
Zincon ion exchange method
zinc in fertilizers, 35-36
Zirconium
in antiperspirants, 362
Zoalene
in animal tissues, 635, 636
in feeds, 90, 101, 115
Index of Method Numbers
871.01,
978
920.03
, 19
920.64, 741
920.126
, 845
920.187, 1034
890.01,
645, 1219
920.04
, 19
920.65, 741
920.127
, 890
920.188, 1034
892.01,
19
920.05
, 19
920.66, 741
920.128
, 890
920.189, 1034
892.02
1160
920.06
20
920.67, 742
920.129
, 890
920.190, 1035
893.01
979
920.07
> 21
920.68, 742
920.130
, 892
920.191, 1035
896.01,
810
920.08
,40
920.69, 745
920.131
, 894
920.192, 1035
896.02,
1016
920.09
,46
920.70, 746
920.132
, 894
920.193, 317
897.01
226
920.10
58
920.71, 747
920.133
, 894
920.194, 322
897.02
977
920.11
147
920.72, 770
920.134
, 898
920.195, 322
898.01,
226
920.12
, 147
920.73, 770
920.135
, 898
920.196, 322
898.02,
902
920.13
, 153
920.75, 771
920.136
, 898
920.197, 323
898.03,
1000
920.14
, 153
920.76, 771
920.137
, 901
920.198, 324
899.01
686
920.15
154
920.77, 771
920.138
, 901
920.199, 324
900.01
821
920.16
154
920.78, 771
920.139
, 901
920.200, 324
900.02,
1012
920.17
, 154
920.79, 771
920.140
, 901
920.201, 327
900.03
1271
920.18
, 154
920.80, 771
920.141
, 901
920.202, 331
902.01
894
920.19
154
920.81, 772
920.142
, 901
920.203, 331
902.02
894
920.20
154
920.82, 773
920.143
, 903
920.204, 331
905.01
702
920.21
, 154
920.83, 775
920.144
, 904
920.205, 332
905.02,
811
920.22
, 155
920.84, 775
920.145
905
920.206, 332
905.03
1148
920.23
, 155
920.85, 780
920.146
, 905
920.207, 1115
906.01
59
920.24
155
920.86, 780
920.147
,905
920.208, 1115
906.02
900
920.25
158
920.87, 781
920.148
,905
920.209, 1118
906.03,
1017
920.26
158
920.88, 757
920.149
911
920.210, 530
908.01
1250
920.27
, 158
920.89, 757
920.150
912
920.211, 598
909.01
899
920.28
, 158
920.90, 757
920.151
914
920.212, 951
909.02
904
920.29
158
920.91, 758
920.152
917
920.213, 952
909.03
1257
920.30
159
920.92, 758
920.153
932
920.214, 685
909.04,
1258
920.31
159
920.93, 758
920.154
941
921.01, 43
910.01
43
920.32
, 160
920.94, 758
920.155
947
921.02, 46
910.02
114.1.
920.33
160
920.95, 759
920.156
953
921.03, 59
910.03
918
920.34
160
920.96, 759
920.157
954
921.04, 151
911.01
232
920.35
173
920.97, 760
920.158
955
921.05, 154
911.02
990
920.36
69
920.98, 760
920.159
956
921.06, 155
911.03
324
920.37
77
920.99, 761
920.160
957
921.07, 902
912.01
244
920.38
77
920.100, 761
920.161
957
921.08, 952
912.02
270
920.39
79
920.101, 760
920.162
, 957
921.09, 955
912.03
604
920.40
83
920.102, 762
920.163
979
921.10, 977
913.01
, 942
920.41
84
920.103, 762
920.164
999
921.11, 1004
913.02
1231
920.42
84
920.104, 762
920.165
1000
921.12, 1035
915.01
50
920.43
84
920.105, 807
920.166
, 1000
921.13, 620
915.02
270
920.44
987
920.106, 820
920.167
, 1000
921.14, 1298
915.03
, 1213
920.45
987
920.107, 831
920.168
1000
922.01, 40
916.01
, 1002
920.46
688
920.108, 832
920.169
1000
922.02, 40
916.02
555
920.47
694
920.109, 832
920.170
1003
922.03, 147
916.03
555
920.48
694
920.110, 832
920.171
1003
922.04, 149
916.04
, 555
920.49
708
920.111, 832
920.172
1003
922.05, 149
916.05
, 555
920.50
709
920.112, 832
920.173
1003
922.06, 780
916.06
555
920.51
712
920.113, 833
920.174
1003
922.07, 819
916.07
555
920.52
712
920.114, 833
920.175
1010
922.08, 820
916.08
556
920.53
713
920.115, 833
920.176
1012
922.09, 835
916.09
, 556
920.54
713
920.116, 837
920.177
1024
922.10, 914
917.01
, 4
920.55
713
920.117, 837
920.178
1025
922.11, 960
917.02
4
920.56
739
920.118, 837
920.179,
1025
922.12, 332
917.03
11
920.57
739
920.119, 838
920.180,
1025
922.13, 557
917.04
87
920.58
739
920.120, 840
920.181,
1026
922.14, 602
918.01
150
920.59
739
920.121, 840
920.182,
1027
923.01, 58
918.02
901
920.60
741
920.122, 840
920.183,
1027
923.02, 685
918.03
902
920.61
741
920.123, 842
920.184,
1028
923.03, 777
919.01
5
920.62
741
920.124, 842
920.185,
1034
923.04, 782
920.01,
17
920.63
741
920.125, 844
920.186,
1034
923.05, 782
1-55
1-56
Number Index
923.06
, 782
925.51
, 991
923.07
, 854
925.52
, 993
923.08
, 1004
925.53
, 993
923.09
, 1016
925.54
, 331
923.10
, 366
925.55
, 334
923.11
, 531
925.56
, 335
923.12
, 552
925.57
, 335
923.13
, 557
925.58
,512
924.01
, 1
925.59
, 559
924.02
, 1
926.01
, 162
924.03
, 1
926.02
, 162
924.04
, 148
926.03
693
924.06
, 917
926.04
, 790
924.07
, 917
926.05
, 791
924.08
, 917
926.06
, 796
924.09
, 917
926.07
796
924.10
, 1256
926.08
841
925.01
,47
926.09
, 894
925.02
, 149
926.10
> 900
925.03
, 159
926.11
900
925.04
, 69
926.12
951
925.05
, 83
926.13
1015
925.06
, 987
926.14
1016
925.07
, 770
926.15
, 322
925.08
, 777
926.16
501
925.09
, 777
926.17
526
925.10
, 777
926.18
602
925.11
, 796
927.01
161
925.12
,796
927.02,
84
925.13
, 798
927.03,
809
925.14
, 759
927.04,
832
925.15
,760
927.05
834
925.16
, 761
927.06
858
925.17
, 761
927.07
945
925.18
, 762
927.08
986
925.19
, 762
927.09
498
925.20
, 804
927.10
501
925.21
, 805
927.11
511
925.22
, 805
927.12
584
925.23
, 807
928.01
, 2
925.24
809
928.02
30
925.25
831
928.03
43
925.26
, 831
928.04
50
925.27
, 843
928.05
844
925.28
, 852
928.06
917
925.29
, 853
928.07
, 931
925.30
, 853
928.08
, 935
925.31
, 854
928.09
, 994
925.32
, 854
928.10
, 499
925.33
, 899
928.11
, 501
925.34
,918
929.01
, 9
925.35
922
929.02
, 10
925.36
922
929.03
, 48
925.37
, 922
929.04
, 150
925.38
, 922
929.05
, 915
925.39
, 941
929.06
, 915
925.40
949
929.07
, 916
925.41
958
929.08
, 979
925.42
1005
929.09
, 1017
925.43
1005
929.10
, 499
925.44
1005
929.11
, 511
925.45
1010
929.12
, 552
925.46,
1012
929.13
, 586
925.47,
1014
929.14
602
925.48
1014
930.01
20
925.49,
1024
930.02
20
925.50,
1024
930.03
20
AOAC Official Methods of Analysis (1990)
930.04
» 40
930.05
, 40
930.06
, 50
930.07
, 59
930.08
, 758
930.09
, 59
930.10
> 59
930.11
, 236
930.12
, 159
930.13
, 162
930.14
, 162
930.15
, 69
930.16
, 987
930.17
, 691
930.18
, 711
930.19
, 739
930.20
, 764
930.21
, 772
930.22
, 793
930.23
, 793
930.24
, 796
930.25
, 797
930.26
, 797
930.27
, 797
930.28
, 810
930.29
, 834
930.30
, 835
930.31
, 836
930.32
, 846
930.33
, 851
930.34
262
930.35
, 1007
930.36
, 1016
930.37
, 1016
930.38
, 1115
930.39
349
930.40
, 533
930.41
, 548
930.42
, 552
930.43
553
930.44
1284
930.45
1285
931.01
, 56
931.02
, 58
931.03
226
931.04
763
931.05
770
931.06
, 856
931.07
856
931.08
1149
931.09
916
931.10
916
931.11
500
931.12
511
931.13
586
931.14,
602
932.01
60
932.02
80
932.03,
779
932.04,
785
932.05,
805
932.06,
835
932.07,
835
932.08,
854
932.09,
857
932.10,
905
932.11,
905
932.12
, 915
932.13
, 919
932.14
, 1011
932.15
, 1018
932.16
, 1094
932.17
, 500
932.18
, 501
932.19
502
932.20
503
932.21
, 506
932.22
, 563
932.23
, 583
932.24
, 598
932.25
602
932.26
, 603
932.27
, 604
932.28
604
932.29
1272
933.01
56
933.02
59
933.03
152
933.04
772
933.05
844
933.06
856
933.07
919
933.08
971
933.09
349
933.10
551
933.11
602
933.12
1280
934.01
69
934.02
87
934.03
788
934.04
1174
934.05
910
934.06
912
934.07
258
934.08
499
934.09
510
935.01
20
935.02
23
935.03
48
935.04
, 48
935.05
51
935.06
147
935.07
160
935.08
160
935.09
, 161
935.10
, 161
935.11
, 70
935.12
84
935.13
84
935.14
87
935.15
, 690
935.16
, 701
935.17
, 704
935.18
705
935.19
705
935.20
,710
935.21
, 710
935.22
710
935.23
712
935.24
713
935.25
723
935.26
724
935.27,
724
935.28
724
935.29
725
935.30
, 725
935.31
, 727
935.32
772
935.33
772
935.34
784
935.35
, 787
935.36
791
935.37
791
935.38
793
935.39
795
935.40
821
935.41
834
935.42
842
935.43
842
935.44
1164
935.45
916
935.46
929
935.47
933
935.48
937
935.49
941
935.50
253
935.51
254
935.52
949
935.53
949
935.54
950
935.55
950
935.56
1004
935.57
1005
935.58
1005
935.59,
1005
935.60
1005
935.61
1006
935.62
1017
935.63
1018
935.64
1019
935.65
1019
935.66
505
935.67
511
935.68
540
935.69
550
935.70
658
936.01
36
936.02
46
936.03
» 48
936.04
f 57
936.05
> 170
936.06
774
936.07
778
936.08
778
936.09
, 929
936.10
, 904
936.11
1213
936.12
979
936.13
991
936.14
, 1091
936.15
, 642, 1216
936.16
644, 1218
936.17
499
936.18
584
936.19
1283
937.01,
32
937.02,
33
937.03,
46
937.04,
746
937.05,
806
937.06,
846
AOAC Official Methods of Analysis (1990)
Number Index
I-57
937.07
864
940.22,
780
942.28
498
937.08
867
940.23,
797
942.29
552
937.09
870
940.24
847
942.30
556
937.10
921
940.25
868
942.31
591
937.11
978
940.26
915
942.32
592
937.12
1005
940.27
957
942.33
1260
937.13
420
940.28
957
943.01
85
937.14
584
940.29
1000
943.02
780
937.15
, 604
940.30
1004
943.03
918
937.16
, 840
940.31
990
943.04
972
938.01
168
940.32
365
943.05
1040
938.02
774
940.33
1086
943.06
384
938.03
111
940.34
384
943.07
558
938.04
799
940.35
644, 1218
943.08
559
938.05
836
940.36
427
944.01
3
938.06
837
940.37
428
944.02
778
938.07
860
940.38
556
944.03
779
938.08
868
940.39
1286
944.04
799
938.09
872
941.01
31
944.05
859
938.10,
990
941.02
33
944.06
923
938.11,
361
941.03
48
944.07
248
938.12,
1091
941.04
76
944.08
250
938.13,
504
941.05
797
944.09
272
938.14
525
941.06
835
944.10
1009
938.15
550
941.07
835
944.11
361
938.16
557
941.08
,851
944.12
1083
938.17
599
941.09
, 855
944.13
1084
938.18
774
941.10
, 1172
944.14
563
939.01
163
941.11
, 999
944.15
593
939.02
772
941.12
, 1000
944.16
1281
939.03
780
941.13
, 1003
945.01
, 20
939.04
783
941.14
, 1039
945.02
, 23
939.05
788
941.15
, 1048
945.03
, 30
939.06
906
941.16
, 383
945.04
, 30
939.07
907
941.17
, 641, 1215
945.05
, 152
939.08
907
941.18
, 644, 1218
945.06
, 691
939.09
270
941.19
, 504
945.07
, 694
939.10
992
941.20
, 505
945.08
, 697
939.11
321
941.21
, 506
945.09
710
939.12
, 640, 1214
941.22
, 556
945.10
712
939.13
, 643, 1217
942.01
, 31
945.11
718
939.14
, 427
942.02
, 34
945.12
727
939.15
> 499
942.03
, 34
945.13
, 727
939.16
, 548
942.04
, 62
945.14
, 730
939.17
, 569
942.05
> 70
945.15
730
939.18
, 602
942.06
, 692
945.16
, 730
940.01
, 33
942.07
, 899
945.17
, 730
940.02
33
942.08
,906
945.18
, 731
940.03
, 62
942.09
, 1149
945.19
, 731
940.04
, 169
942.10
, 1150
945.20
,732
940.05
, 686
942.11
, 1151
945.21
, 732
940.06
, 705
942.12
, 1151
945.22
, 733
940.07
, 705
942.13
, 1153
945.23
, 733
940.08
, 705
942.14
,916
945.24
, 733
940.09
, 705
942.15
,918
945.25
, 734
940.10
, 706
942.16
,945
945.26
, 734
940.11
, 706
942.17
, 244
945.27
734
940.12
, 706
942.18
,954
945.28
, 734
940.13
, 706
942.19
,982
945.29
, 734
940.14
, 706
942.20
, 1039
945.30
, 734
940.15
, 706
942.21
, 366
945.31
, 736
940.16
, 706
942.22
, 368
945.32
, 937
940.17
, 713
942.23
, 1049
945.33
, 746
940.18
, 718
942.24
, 407
945.34
, 772
940.19
, 744
942.25
, 640, 1214
945.35
, 776
940.20
, 749
942.26
640, 1214
945.36
, 783
940.21
, 764
942.27
645, 1.21.9
945.37
, 783
945.38,
788
945.39,
790
945.40,
793
945.41,
793
945.42,
794
945.43,
795
945.44,
795
945.45
797
945.46
807
945.47
831
945.48
, 833
945.49
835
945.50
838
945.51
852
945.52
874
945.55
930
945.56
, 930
945.57
945
945.58
, 246
945.59
, 1017
945.60
, 1017
945.61
, 1039
945.62
, 1039
945.63
, 1040
945.64
, 1040
945.65
, 1042
945.66
, 1042
945.67
, 1042
945.68
, 987
945.69
, 51.2
945.99
, 994
945.70
, 366
945.71
, 366
945.72
, 366
945.73
, 1079
945.74
, 1085
945.75
, 369
945.76
, 391
945.77
, 391
945.78
, 392
945.79
, 394
945.80
, 394
945.81
, 394
945.82
, 395
945.83
, 397
945.84
, 402
945.85
, 405
945.86
, 405
945.87
, 406
945.88
, 407
945.89
, 419
945.90
, 419
945.91
, 419
945.92
,419
945.93
, 419
945.94
, 420
945.95
, 420
945.96
, 423
945.97
, 551
945.98
, 548
945.10<
ft, 602
945.10
1, 1296
945.10.
2,980
946.01
, 821
946.02
, 840
946.03
, 847
946.04
, 852
946.05
947.01
947.02
947.03
947.04
947.05
947.06
947.07
947.08
947.09
947.10
947.11
947.12
947.13
947.14
947.15
947.16
948.01
948.02
948.03
948.04
948.05
948.06
948.07
948.08
948.09
948.10
948.11
948.12
948.13
948.14
948.15
948.16
948.17
948.18
948.19
948.20
948.21
948.22
948.23
948.24
948.25
948.26
948.27
948.28
948.29
948.30
948.31
949.01
949.02
949.03
949.04
949.05
949.06
949.07
949.08
949.09
949.10
949.11
949.12
949.13
949.14
949.15
949.16
950.01
950.02
950.03
607
179
186
86
786
805
831
838
862
890
1 174
997
1132
643,
586
592
593
3
60
163
80
685
685
747
778
779
818
831
841
857
861
871
871
877
1160
1161
1161
929
949
1023
1133
1134
1075
378
645,
552
574
591
23
28
29
61
176
211
772
914
305
305
1025
339
643,
504
523
561
11
69, 91
685
1217
1219
1217
1-58
Number Index
AOAC Official Methods of Analysis (1990)
950.04
950.05
950.06
950.07
950.08
950.09
950.10
950.11
950.12
950.13
950.14
950.15
950.16
950.17
950.18
950.19
950.20
950.21
950.22
950.23
950.24
950.25
950.26
950.27
950.28
950.29
950.30
950.31
950.32
950.33
950.34
950.35
950.36
950.37
950.38
950.39
950.40
950.41
950.42
950.43
950.44
950.45
950.46
950.47
950.48
950.49
950.50
950.51
950.52
950.53
950.54
950.55
950.56
950.57
950.58
950.59
950.60
950.61
950.62
950.63
950.64
950.65
950.66
950.67
950.68
950.69
950.70
693
697
711
711
724
727
727
735
751
751
751
751
751
751
751
751
751
751
751
751
752
755
755
755
755
756
756
756
756
756
785
791
793
793
795
796
758
833
838
840
903
905
931
949
949
949
950
950
950
950
1005
1007
1017
1019
332
332
1118
1123
1124
1125
1125
1125
1125
1125
1126
1126
1126
950.71
950.72
950.73
950.74
950.75,
950.76
950.77,
950.78
950.79
950.80
950.81
950.82
950.83
950,84
950.85
950.86
950.87
950.88
950.89
950.90
950.91
950.92
950.93
950.94
951.01
951.02
951.03
951.04
951.05
951.06
951.07
952.01
952.02
952.03
952.04
952.05
952.06
952.07
952.08
952.09
952.10
952.11
952.12,
952.13
952.14
952.15
952.16
952.17
952.18
952.19
952.20
952.21
952.22
952.23
952.24.
952.25
952.26
952.27
952.28
952.29,
953.01
953.02
953.03
953.04
953.05
953.06
953.07,
1 1 32
1132
1132
1132
1132
1132
1134
1134
1135
1136
1136
1136
1136
362
365
380
389
389
392
393
393
394
586
386
43
228
783
363
363
365
111
159
85
703
765
794
851
899
868
1140
1151
1152
1172
245
264
362
364
364
366
366
1082
375
419
422
337
345
347
574
603
106
40
45
45
49
164
178
69
953.08
953.09
953.10
953.11
953.12
953.13
953.14.
953.15
953.16
953.17.
953.18
953.19
954.01
954.02
954.03
954.04
954.05
954.06
954.07
954.08
954.09
954.10
954.11
954.12,
954.13
954.14
954.15.
954.16
954.17,
955.01
955.02
955.03
955.04
955.05
955.06
955.07,
955.08
955.09
955.10
955.11
955.12,
955.13
955.14,
955.15,
955.16
955.17
955.18
955.19
955.20
955.21
955.22
955.23
955.24
955.25
955.26
955.27
955.28
955.29
955.30
955.31
955.32
955.33
955.34
955.35,
955.36
955.37
955.38
809
901
902
867
1147
1147
910
911
911
1051
499
92
70
79
797
875
1148
1154
918
950
304
1010
1028
364
520
520
568
599
94
1
3
11
17
21
26
30
41
41
63
133
135
135
135
136
137
138
690
695
708
717
729
733
745
746
744
752
793
793
840
892
902
903
973
1000
1017
312
902
955.39,
1125
955.40
1127
955.41
1127
955.42
381
955.43
381
955.44
393
955.45
397
955.46
397
955.47
418
955.48
345
955.49
347
955.50
355
955.51
512
955.52
557
955.53
559
955.54
560
955.55
, 607
955.56
, 620
955.57
, 659
955.58
> 668
956.01
, 47
956.02
, 690
956.03
, 784
956.04
, 958
956.05
, 294
956.06
, 996
956.07
, 347
956.08
, 515
956.09
, 597
956.10
, 97
956.11
, 103
957.01
, 11
957.02
, 12
957.03
, 692
957.04
, 757
957.05
, 759
957.06
, 831
957.07
> 875
957.08
, 1149
957.09
, 1168
957.10
, 1168
957.11
1171
957.12
919
957.13
960
957.14
296
957.15
303
957.16
1036
957.17
1051
957.18
344
957.19
509
957.20
511
957.21
521
957.22
94
957.23
115
957.24
118
958.01
12
958.02
26
958.03
50
958.04
702
958.05
853
958.06
946
958.07
294
958.08
298
958.09
1033
958.10
515
958.11
519
958.12
519
958.13,
527
958.14,
527
958.15,
549
958.16,
583
958.17,
593
959.01,
9
959.02,
9
959.03,
21
959.04,
64
959.05,
699
959.06,
775
959.07,
862
959.08,
881
959.09
1145
959.10
296
959.11
1017
959.12
1031
959.13
1036
959.14
409
959.15
> 513
959.16
, 513
959.17
, 601
959.18
, 106
960.01
> 16
960.02
, 16
960.03
, 16
960.04
, 22
960.05
f 47
960.06
, 59
960.07
, 65
960.08
> 66
960.09
, 138
960.10
, 153
960.11
, 170
960.12
, 173
960.13
, 186
960.14
, 228
960.15
, 229
960.16
, 701
960.17
,716
960.18
, 736
960.19
, 744
960.20
, 747
960.21
, 748
960.22
, 752
960.23
756
960.24
, 793
960.25
, 758
960.26
, 814
960.27
, 820
960.28
, 821
960.29
837
960.30
, 839
960.31
, 840
960.32
, 851
960.33
, 851
960.34
, 852
960.35
863
960.36
, 894
960.37
, 895
960.38
, 1142
960.39
, 931
960.40
> 248
960.41
, 298
960.42
, 308
960.43
, 309
960.44
, 1026
AOAC Official Methods
of Analysis (1990)
Number Index
960.45, 1045
962.20, 414
964.24, 640, 1214
966.13, 891
968.10, 766
960.46,
1080
962.21
,536
964.25
, 528
966.14, 904
968.11,
760
960.47
1087
962.22
, 592
964.26
, 567
966.15, 904
968.12,
802
960.48
1095
962.23
, 603
964.27
, 569
966.16, 915
968.13
824
960.49,
378
962.24
, 621
964.28
, 92
966.17, 980
968.14,
850
960.50
388
962.25
, 125
964.29
> 98
966.18, 983
968.15
893
960.51,
398
962.26
, 128
965.01
» 6
966.19, 985
968.16
1137
960.52
342
962.37
, 1295
965.02
f 6
966.20, 1021
968.17
1139
960.53,
498
962.38
, 1295
965.03
, 6
966.21, 1116
968.18
1163
960.54
527
963.01
, 4
965.04
, 7
966.22, 359
968.19
, 919
960.55,
536
963.02
, 4
965.05
, 7
966.23, 429
968.20
923
960.56,
541
963.03
, 16
965.06
, 7
966.24, 430
968.21
, 924
960.57,
541
963,05
, 64
965.07
, 7
966.25, 533
968.22
1188
960.58,
551
963.06
, 149
965.08
, 11
966.26, 635
968.23
, 981
960.59,
555
963.07
,90
965.09
, 27
966.27, 108
968.24
, 287
960.60,
589
963.08
, 695
965.10
, 30
966.28, 114
968.25
, 295
960.61
609
963.09
,695
965.11
, 30
966.29, 114
968.26
, 297
960.62,
95
963,10
,699
965.12
, 142
966.30, 130
968.27
, 1021
960.63,
100
963.11
,718
965.13
, 145
967.01, 31
968.28
, 1022
960.64,
101
963.12
, 732
965.14
, 183
967.02, 36
968.29
, 1038
960.65,
105
963.13
, 744
965.15
, 217
967.03, 37
968.30
, 987
960.66,
108
963.15
, 770
965.16
, 69, 91
967.04, 37
968.31
, 991
960.67,
121
963.16
, 896
965.17
, 88
967.05, 38
968.32
, 1057
961.02,
140
963.17
,906
965.18
, 689
967.06, 223
968.33
, 379
961.03,
169
963.18
, 864
965.19
i 714
967.07, 76
968.34
, 379
961.04,
175
963.19
, 1141
965.20
, 715
967.08, 695
968.35
, 380
961.05,
175
963.20
1158
965.21
, 719
967.09, 695
968.36
, 389
961.06
736
963.21
243
965.22
, 730
967.10, 747
968.37
, 390
961.07,
819
963.22
964
965.23
, 731
967.11, 753
968.38
, 401
961.08
824
963.23
302
965.24
, 792
967.12, 808
968.39
, 406
961.09,
1159
963.24
303
965.25
, 757
967.13, 867
968.40
, 521
961.10
1162
963.25
1007
965.26
, 823
967.14, 883
968.41
, 522
961.11,
981
963.26
995
965.27
, 833
967.15, 1142
968.42
, 522
961.12
294
963.27
998
965.28
, 1137
967.16, 912
968.43
, 558
961.13,
1034
963.28
410
965.29
, 910
967.17, 923
968.44
, 562
961.14
1054
963.29
343
965.30
915
967.18, 975
968.45
, 564
961.15
1089
963.30
499
965.31
, 925
967.19, 994
968.46
, 564
961.16
341
963.31
613
965.32
955
967.20, 1126
968.47
, 574
961.17
533
963.32
91
965.33
, 956
967.21, 1058
968.48
, 628
961.18
579
963,33
95
965.34
969
967.22, 1059
968.49
, 130
961.19
579
963.34
111
965.35
982
967.23, 374
968.50
, 126
961.20
589
963.35
112
965.36
298
967.24, 396
969.01
, 9
961.21
597
963.36
1282
965.37
332
967.25, 467
969.02
, 13
961.22,
607
963.37
672
965.38
373
967.26, 470
969.04
» 27
961.23
625
964.01,
32
965.39
385
967.27, 471
969.05
, 38
961.24
92
964.02
137
965.40
398
967.28, 475
969.06
, 57
961.25
105
964.03
155
965.41
419
967.29, 499
969.07
, 187
961.26
115
964.04
199
965.42
516
967.30, 502
969.08
, 225
962.01
5
964.05,
210
965.43
560
967.31, 548
969.09
, 227
962.02
13
964.06
87
965.44
580
967.32, 573
969.10
, 85
962.03
15
964.07,
88
965.45
604
967.33, 581
969.11
, 719
962.04
15
964.08
744
965.46
610
967.34, 95
969.12
739
962.05
182
964.09,
748
965.47
93
967.35, 103
969.13
, 754
962.06
185
964.10
891
965.48
119
967.36, 106
969.14
, 798
962.07
193
964.11,
896
965.49
963
967.37, 106
969.15
, 761
962.08
197
964.12
871
966.01
56
967.38, 106
969.16
, 815
962.09
, 80
964.13,
1151
966.02
64
967.39, 119
969.17
, 837
962.10
937
964.14
1165
966.03
64
967.40, 122
969.18
, 837
962.11
, 743
964.15
1178
966.04
141
967.41, 127
969.19
, 841
962.12
, 744
964.16,
242
966.05
164
968.02, 67
969.20
, 851
962.13
, 753
964.17
290
966.06
183
968.03, 67
969.21
, 851
962.14
, 829
964.18,
296
966.07
200
968.04, 193
969.22
, 897
962.15
, 883
964.19,
299
966.08,
222
968.05, 230
969.23
870
962.16
, 1157
964.20,
307
966.09
696
968.06, 71
969.24
, 872
962.17
1001
964.21,
1037
966.10,
743
968.07, 77
969.25
, 1145
962.18
, 1026
964.22,
989
966.11,
794
968.08, 84
969.26
, 1146
962.19
1033
964.23,
392
966.12,
891
968.09, 700
969.27
, 1167
1-59
-60
Number Index
AOAC Official Methods of Analysis (1990)
969.28
969.29
969.30
969.31
969.32
969.33
969.34
969.35
969.36
969.37
969.38
969.39
969.40
969.41
969.42
969.43
969.44
969.45
969.46
969.47
969.48
969.49
969.50
969.51
969.52
969.53
969.54
969.55
969.56
969.57
970.01
970.02
970.03
970.04
970.05
970.06
970.07
970.08
970.09
970.10
970.11
970.12,
970.13
970.14
970.15
970.16
970.17
970.18
970.19
970.20
970.21
970.22
970.23
970.24
970.25
970.26
970.27
970.28
970.29
970.30
970.31
970.32
970.33
970.34
970.35
970.36
970.37
1168
1175
921
933
272
963
984
1021
1022
1022
1026
1042
1078
387
393
402
404
406
415
337
349
516
532
594
621
91
91
96
108
111
16
18
19
20
186
186
225
89
89
90
90
696
696
708
710
718
732
743
743
763
764
764
767
788
790
803
803
803
803
804
858
886
1144
1146
1149
1151
1174
970.38
970.39
970.40
970.41
970.42
970.43
970.44
970.45
970.46
970.47,
970.48
970.49
970.50
970.51
970.52
970.53
970.54
970.55
970.56
970.57
970.58
970.59
970.60
970.61
970.62
970.63
970.64
970.65
970.66
970.67
970.68
970.69
970.70
970.71
970.72
970.73
970.74
970.75
970.76
970.77
970.78,
970.79
970.80
970.81
970.82
970.83
970.84
970.85
970.86
970.87
970.88
970.89
970.90
970.91
971.01
971.02
971.03
971.04
971.05
971.06
971.07
971.08
971.09
971.10
971.11
971.12.
971.13
1180
916
917
917
935
1184
1185
1190
1195
1197
1199
1213
974
976
274
289
306
1001
1022
1022
1023
990
867
360
366
367
1048
1052
372
375
375
383
386
387
392
396
406
418
418
425
518
528
553
563
581
593
631
97
105
108
110
124
1266
1273
26
65
233
162
181
182
187
222
78
711
860
898
864
971.14
869
971.15
, 1156
971.16
1165
971.17
, 1169
971.18
922
971.19
933
971.20
242
971.21
262
971.22
1186
971.23
1190
971.24
, 1191
971.25
949
971.26
, 999
971.27
992
971.28
, 995
971.29
996
971.30
, 1071
971.31
381
971.32
, 388
971.33
393
971.34
, 393
971.35
404
971.36
, 406
971.37
, 517
971.38
, 567
971.39
, 571
971.40
, 585
971.41
, 591
971.42
, 611
971.43
> 612
971.44
, 101
971.45
, 103
971.46
, 104
971.47
, 108
971.48
, 120
971.49
, 128
972.01
> 12
972.02
, 33
972.03
, 33
972.04
, 143
972.05
, 152
972.06
, 987
972.07
, 697
972.08
, 698
972.09
, 698
972.10
700
972.11
702
972.12
717
972.13
727
972.14
750
972.15
763
972.16
816
972.17
823
972.18,
907
972.19
1147
972.20,
912
972.21,
929
972.22,
933
972.23,
257
972.24,
257
972.25,
258
972.26,
1191
972.27,
1195
972.28,
957
972.29,
309
972.30,
1130
972.31,
1070
972.32,
384
972.33
385
972.34,
385
972.35
385
972.36,
385
972.37
386
972.38,
390
972.39,
397
972.40,
405
972.41,
410
972.42
419
972.43,
341
972.44
455
972.45,
458
972.46
502
972.47,
520
972.48,
529
972.49,
554
972.50,
561
972.51,
562
972.52,
563
972.53.
581
972.54,
594
972.55
602
972.56,
123
972.57
128
972.58,
405
972.59
405
973.01,
35
973.02
36
973.03
37
973.04
37
973.05
37
973.06
38
973.07
38
973.08
, 39
973.09
39
973.10
, 232
973.11
163
973.12
, 165
973.13
179
973.14
, 180
973.15
183
973.16
184
973.17
184
973.18
82
973.19
, 703
973.20
749
973.21
, 760
973.22
816
973.23
890
973.24
865
973.25
869
973.26
875
973.27
1166
973.28
1167
973.29
1173
973.30
1176
973.31
938
973.32
241
973.33
245
973.34
247
973.35
255
973.36
271
973.37
1207
973.38
1210
973.39
298
973.40
, 312
973.41
, 312
973.42
, 313
973.43
, 313
973.44
, 314
973.45
, 315
973.46
, 316
973.47
,317
973.48
, 318
973.49
, 319
973.50
, 320
973.51
, 320
973.52
, 323
973.53
, 327
973.54
328
973.55
, 328
973.56
329
973.57
, 330
973.58
1127
973.59
360
973.60
, 390
973.61
395
973.62
, 405
973.63
406
973.64
412
973.65
637
973.66
349
973.67
353
973.68
496
973.69
498
973.70
519
973.71
531
973.72
565
973.73
567
973.74
572
973.75
583
973.76
607
973.77
609
973.78
626
973.79
628
973.80
101
973.81,
127
974.01
33
974.02,
233
974.03
204
974.04,
212
974.05
221
974.06
83
974.07,
710
974.08
749
974.09,
844
974.10,
1156
974.11,
1163
974.12,
911
974.13,
256
974.14,
264
974.15,
269
974.16,
1195
974.17,
1199
974.18,
1209
974.19,
978
974.20,
980
974.21,
285
974.22,
286
974.23,
1038
974.24,
993
974.25,
995
AOAC Official Methods of Analysis (1990)
Number Index
974.26, 996
975.46, 387
977.15, 263
979.14, 826
981.17, 1068
974.27,
324
975,47
396
977.16
, 1185
979.15
, 889
981.18
375
974.28,
363
975.48
398
977.17
, 967
979.16
, 242
981.19
382
974.29,
1045
975.49
398
977.18
, 290
979.17
, 256
981.20
391
974.30
1079
975.50
402
977.19
, 321
979.18
, 1188
981.21
400
974.31
1098
975.51
, 418
977.20
, 1030
979.19
, 969
981.22
412
974.32
390
975.52
, 639
977.21
, 1039
979.20
, 1026
981.23
638
974.33
395
975.53
, 346
977.22
, 326
979.21
, 1029
981.24
513
974.34
,421
975.54
,476
977.23
, 1129
979.22
, 1031
981.25
574
974.35
, 637
975.55
, 450
977.24
,401
979.23
, 1042
981.26
587
974.36
338
975.56
, 494
977.25
, 404
979.24
1064
981.27
93
974.37
, 351
975.57
, 524
977.26
, 459
979.25
402
982.01
29
974.38
, 463
975.58
, 600
977.27
, 431
979.26
403
982.02
172
974.39
, 512
975.59
, 611
977.28
, 501
979.27
, 623
982.03
194
974.40
, 550
975.60
,121
977.29
, 503
980.01
» 31
982.04
195
974.41
, 573
975.61
,131
977.30
, 506
980.02
34
982.06
200
974.42
, 591
976.01
, 23
977.31
, 595
980.03
41
982.07
211
974.43
, 607
976.02
, 189
977.32
,612
980.04
172
982.08
219
974.44
, 614
976.03
, 196
977.33
, 616
980.05
180
982.09
690
974.45
, 626
976.04
, 215
977.34
, 621
980.06
181
982.10
692
974.46
, 112
976.05
, 72
977.35
, 95
980.07
194
982.11
719
974.47
, 113
976.06
, 72
977.36
> 97
980.08
196
982.12
720
974.48
, 125
976.07
, 686
977.37
, 119
980.09
197
982.13
723
974.49
, 126
976.08
, 708
977.38
, 657
980.10,
201
982.14
789
975.01
> 31
976.09
, 715
978.01
, 14
980.11,
209
982.15
825
975.02
, 35
976.10
, 716
978.02
, 18
980.12
210
982.16
825
975.03
, 42
976.11
, 739
978.03
, 52
980.13,
773
982.17
827
975.04
, 51
976.12
, 750
978.04
, 59
980.14
776
982.18
828
975.05
, 232
976.13
786
978.05
184
980.15,
819
982.19
908
975.06
, 232
976.14
, 843
978.06
, 207
980.16,
885
982.20
878
975.08
, 86
976.15
, 845
978.07
, 208
980.17,
1144
982.21
926
975.09
90
976.16
, 865
978.08
, 208
980.18,
1173
982.22
938
975.10
, 749
976.17
, 867
978.09
, 208
980.19
270
982.23
239
975.11
, 763
976.18
870
978.10
81
980.20,
1192
982.24,
1201
975.12
, 764
976.19
870
978.11
712
980.21,
1200
982.25,
1202
975.13
, 771
976.20
,915
978.12
745
980.22,
303
982.26,
1203
975.14
794
976.21
, 932
978.13
820
980.23
1031
982.27,
968
975.15
762
976.22
, 1211
978.14
849
980.24
1130
982.28
1128
975.16
, 805
976.23
,284
978.15
1195
980.25,
1135
982.29
1069
975.17
, 808
976.24
,362
978.16
300
980.26,
1066
982.30,
1096
975.18
, 809
976.25
, 1099
978.17
1032
980.27
388
982.31
380
975.19
810
976.26
, 1103
978.18,
987
980.28
411
982.32
384
975.20
842
976.27
, 389
978.19,
379
980.29
420
982.33
418
975.21
, 842
976.28
, 637
978.20
401
980.30
422
982.34
422
975.22
, 847
976.29
, 346
978.21
401
980.31
464
982.35
645,
975.23
, 853
976.30
,462
978.22
401
980.32
, 454
982.36
,439
975.24
898
976.31
, 451
978.23,
436
980.33
497
982.37
590
975.25
905
976.32
, 524
978.24
473
980.34
529
982.38
604
975.26
1147
976.33
, 570
978.25
496
980.35
586
982.39
618
975.27
, 1147
976.34
, 595
978.26
496
980.36
, 101
982.40
, 631
975.28
, 1148
976.35
,599
978.27
507
980.37
, 449
982.41
, 633
975.29
1155
976.36
,629
978.28,
512
981.01
156
982.42
94
975.30
1155
976.37
, 123
978.29,
620
981.02
197
982.43
, 118
975.31
1155
977.01
, 15
978.30,
107
981.03
, 199
982.44
, 129
975.32
1157
977.02
, 59
978.31,
122
981.04
, 220
983.01
, 21
975.33
, 935
977.03
, 192
979.01,
66
981.05
, 723
983.02
, 23
975.34
269
977.04
, 208
979.02,
156
981.06
824
983.03
, 31
975.35
1187
977.05
,209
979.03,
188
981.07
, 879
983.04
, 34
975.36
1187
977.06
, 216
979.04,
201
981.08
, 886
983.05
, 36
975.37
1197
977.07
, 218
979.05,
206
981.09
, 926
983.06
, 169
975.38
1208
977.08
, 713
979.06,
712
981.10
, 937
983.07
, 190
975.39
967
977.09
, 750
979.07,
742
981.11
, 951
983.08
» 191
975.40
291
977.10
,763
979.08,
752
981.12
, 988
983.09
, 204
975.41
1055
977.11
, 842
979.09,
788
981.13
, 1127
983.10
, 205
975.42,
1061
977.12
, 868
979.10,
789
981.14
, 1134
983.11
, 224
975.43,
1074
977.13
, 876
979.12,
760
981.15
, 1053
983.12
, 705
975.44,
1100
977.14
,937
979.13,
823
981.16
, 1056
983.13
> 741
1-61
1219
1-62 Number Index
AOAC Official Methods of Analysis (1990)
983.14, 842
984.24, 1041
985.26, 990
986.15, 237
988.04, 213
983.15, 1178
984.25
, 998
985.27
, 1067
986.16
1203
988.05, 70
983.16, 1143
984.26
, 1060
985.28
, 1102
986.17
1205
988.06, 709
983.17, 915
984.27
, 1106
985.29
, 1105
986.18
1206
988.07, 748
983.18, 931
984.28
, 393
985.30
, 1107
986.19
972
988.08, 829
983.19, 941
984.29
, 416
985.31
, 1107
986.20
299
988.09, 868
983.20, 266
984.30
, 420
985.32
, 1107
986.21
, 999
988.10, 942
983.21, 283
984.32
, 357
985.33
, 1108
986.22
332
988.11, 268
983.22, 1044
984.33
, 357
985.34
, 1 109
986.23
1110
988.12, 1020
983.23, 1100
984.34
, 441
985.35
> 1110
986.24
1112
988.13, 1117
983.24, 357
984.35
448
985.36
, 381
986.25
1113
988.14, 1076
983.25, 437
984.36
, 456
985.37
,400
986.26
1113
988.15, 1101
983.26, 466
984.37
, 505
985.38
,403
986.27
1113
988.16, 374
983.27, 508
984.38
, 526
985.39
,403
986.28
382
988.17, 413
983.28, 525
984.39
, 598
985.40
, 356
986.29
414
988.18, 433
983.29, 569
984.40
613
985.41
,458
986.30
356
988.19, 438
983.30, 622
985.01
42
985.42
,478
986.31
357
988.20, 492
983.31, 634
985.02
212
985.43
,495
986.32
431
988.21, 543
984.01, 32
985.03
166
985.44
, 536
986.33
435
988.22, 544
984.02, 52
985.04
174
985.45
, 542
986.34
444
988.23, 576
984.03, 147
985.05
184
985.46
570
986.35
480
988.24, 566
984.04, 157
985.06,
195
985.47
575
986.36
576
988.25, 608
984.05, 178
985.07,
202
985.48
569
986.37
567
988.26, 616
984.06, 185
985.08,
228
985.49
588
986.38
617
988.27, 614
984.07, 188
985.09,
741
985.50
620
986.39
109
988.28, 622
984.08, 192
985.10,
742
985.51
99
987.01
205
989.01, 198
984.09, 214
985.11,
746
986.01
234
987.02
722
989.02, 203
984.10, 218
985.12,
882
986.02
166
987.03
723
989.03, 74
984.11, 219
985.13,
1181
986.03
167
987.04
1159
989.04, 812
984.12, 220
985.14,
931
986.04,
181
987.05
1182
989.05, 811
984.13, 74
985.15,
932
986.05,
196
987.06
948
989.06, 1193
984.14, 711
985.16,
270
986.06
190
987.07
1002
989.07, 545
984.15, 810
985.17,
1197
986.07
203
987.08
1007
989.08, 582
984.16, 835
985.18,
1212
986.08
226
987.09
449
989.09, 1077
984.17, 909
985.19,
952
986.09
214
987.10
488
989.10, 435
984.18, 940
985.20,
965
986.10,
215
987.11
484
989.11, 434
984.19, 241
985.21,
970
986.11,
800
987.12
554
989.12, 474
984.20, 951
985.22,
282
986.12,
880
988.01
22
989.13, 490
984.21, 284
985.23,
292
986.13,
920
988.02
225
989.14, 484
984.22, 1019
985.24,
308
986.14,
927
988.03
174
989.15, 486
984.23, 1035
985.25,
1026
Live Music
Archive
Librivox
Free Audio
Metropolitan Museum
Cleveland
Museum of Art
Internet
Arcade
Console Living Room
Open Library
American
Libraries
TV News
Understanding
9/11